Acanthopanax water extract for preventing or treating cerebral arterial thrombosis

Abstract

The invention relates to the field of biological medicine, in particular to an acanthopanax water extract for preventing or treating cerebral arterial thrombosis. The invention provides a preparation method of an acanthopanax senticosus water extract. The preparation method comprises the following steps: grinding dried acanthopanax senticosus roots into powder; mixing the dried acanthopanax root powder with a 70-90% ethanol water solution, performing continuous ultrasonic treatment at room temperature for 25-35 minutes, performing suction filtration, and collecting filtrate; carrying out rotary evaporation on the obtained filtrate to remove the ethanol aqueous solution to obtain a traditional Chinese medicine crude extract; adding pure water into the traditional Chinese medicine crude extract, and carrying out ultrasonic redissolution to obtain a traditional Chinese medicine crude extract; petroleum ether, ethyl acetate and n-butyl alcohol are sequentially used for extracting the traditional Chinese medicine crude extracting solution, and a water-phase extracting solution is obtained; freeze-drying the water-phase extracting solution to obtain the acanthopanax water extract. The eleutherococcus senticosus water extract prepared by the method shows an obvious protection effect on SH-SY5Y cells damaged by OGD / R, and can be used for preventing or treating cerebral arterial thrombosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a Acanthopanax senticosus water extract for preventing or treating ischemic stroke. BACKGROUND

[0002] Stroke, commonly known as apoplexy, is a group of diseases caused by intracranial blood circulation disorders resulting in brain tissue damage. Because of the characteristics of high morbidity, high mortality, high disability rate, high recurrence rate, slow recovery, and many complications, and the serious reduction of the quality of life of patients, stroke is considered as one of the three major diseases threatening human health. Stroke is generally divided into ischemic and hemorrhagic types, of which ischemic stroke accounts for more than 80% of the total number of stroke patients. Ischemic stroke refers to the brain tissue necrosis caused by stenosis or occlusion of the cerebral blood supply artery (carotid artery and vertebral artery) and insufficient blood supply to the brain. Ischemic stroke can be induced by various factors, such as bad living habits such as staying up late and alcoholism, age factors, and underlying diseases such as hypertension and hyperlipidemia. At present, there is a lack of effective treatment drugs for ischemic stroke. It is a long way to go to find a reasonable drug target for preventing and treating ischemic stroke and to develop corresponding treatment drugs.

[0003] Acanthopanax senticosus (Rupr. Maxim.) Harms is a plant of the Araliaceae family and Acanthopanax genus, which is produced in China, Japan, Korea, and Russia, and mainly distributed in Northeast China, Hebei, and Shanxi. Acanthopanax senticosus has been widely used as a traditional Chinese medicinal material in China for more than 2,000 years. Acanthopanax senticosus is listed as a superior product in Shennong's Herbal Classic, which believes that it has the effect of "benefiting qi and treating spasticity", and records that it can be used to treat heart and abdominal hernia, abdominal pain, and qi deficiency. It is pointed out in the Book of Differentiation of Materia Medica that Acanthopanax senticosus has the effect of "strengthening bones and tendons, strengthening willpower, and long-term use of light body and endurance".

[0004] Acanthopanax senticosus glycoside B can inhibit the production of ROS in the SD rat cortical neuron cell under hypoxia / reoxygenation conditions, increase the SOD activity of the cell, and reduce the MDA level, thereby playing a neuroprotective role by inhibiting oxidative stress damage. Studies have also shown that Acanthopanax senticosus glycoside B can play a protective role on ischemia-reperfusion injury brain by reducing the inflammatory response during cerebral ischemia, and its mechanism is related to the FOXO3a / NF-κB pathway. The main component of Acanthopanax senticosus injection is total flavonoids of Acanthopanax senticosus, which is a treatment drug for transient cerebral ischemic attack caused by liver and kidney deficiency, can improve cerebral circulation, resist oxidative stress to reduce secondary damage after cerebral ischemia, and can also regulate blood glucose and blood lipids, and has a certain effect on the prevention of cerebral ischemia. Acanthopanax senticosus polysaccharide is one of the main active ingredients of Acanthopanax senticosus for protecting the nervous system, and studies have shown that Acanthopanax senticosus polysaccharide can reduce the volume of cerebral infarction and brain water content in rats.

[0005] Exploring the effective extract of acanthopanax senticosus for treating ischemic stroke is still a technical problem that needs further research and solution. SUMMARY

[0006] The purpose of the present application is to provide an acanthopanax senticosus water extract capable of effectively preventing or treating ischemic stroke.

[0007] The present application provides a preparation method of acanthopanax senticosus water extract, comprising the following steps:

[0008] a) grinding the dried root of acanthopanax senticosus into powder;

[0009] b) mixing the dried root powder of acanthopanax senticosus with 70% to 90% ethanol aqueous solution, continuously ultrasonic treating at room temperature for 25 to 35 min, then filtering, and collecting the filtrate;

[0010] c) rotary evaporating the obtained filtrate to remove the ethanol aqueous solution, to obtain a traditional Chinese medicine crude extract infusion;

[0011] d) adding pure water to the traditional Chinese medicine crude extract infusion and ultrasonic redissolving, to obtain a traditional Chinese medicine crude extract solution;

[0012] e) sequentially extracting the traditional Chinese medicine crude extract solution with petroleum ether, ethyl acetate and n-butanol, to obtain a water phase extract solution;

[0013] f) freeze-drying the water phase extract solution, to obtain the acanthopanax senticosus water extract.

[0014] Preferably, in step b), the dried root powder of acanthopanax senticosus is mixed with 70% to 90% ethanol aqueous solution at a mass-volume ratio of 1g:10 to 20mL.

[0015] Preferably, step b) further comprises at least one repeated extraction, which comprises: mixing the obtained filter residue with 70% to 90% ethanol aqueous solution, continuously ultrasonic treating at room temperature for 25 to 35 min, then filtering, and collecting the filtrate; and combining all the filtrates.

[0016] Preferably, the obtained filtrate is rotary evaporated at 35 to 45℃ until all the ethanol aqueous solution is removed.

[0017] Preferably, in step d), the mass-volume ratio of the traditional Chinese medicine crude extract infusion to pure water is 1g:65 to 75mL.

[0018] Preferably, in step e), the amount of each organic solvent is the same as that of pure water in step d).

[0019] Preferably, in step e), each organic solvent is extracted for three times.

[0020] The present application also provides an acanthopanax senticosus water extract obtained by the above preparation method.

[0021] The medicine containing the Acanthopanax senticosus water extract also belongs to the protection scope of the present application.

[0022] The use of the Acanthopanax senticosus water extract in the preparation of the medicine for treating ischemic stroke also belongs to the protection scope of the present application.

[0023] The cell experiment proves that the Acanthopanax senticosus water extract provided by the present application shows obvious protective effect on SH-SY5Y cells after OGD / R injury, can significantly reduce the apoptosis rate of SH-SY5Y cells and the expression of pro-apoptotic proteins in the OGD / R experiment, and the protective effect of 500 μg / mL and 1000 μg / mL Acanthopanax senticosus water extract is the best. Through the detection of related physiological and biochemical indexes, it is found that the Acanthopanax senticosus water extract can reduce the ROS level in SH-SY5Y cells, improve the SOD enzyme activity, reduce the MDA content, maintain the ATP level, inhibit SH-SY5Y cell apoptosis by inhibiting oxidative stress and protecting mitochondrial function, thereby confirming that the Acanthopanax senticosus water extract has a preventive and therapeutic effect on ischemic stroke. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The figure shows the cell viability of SH-SY5Y cells after oxygen-glucose deprivation and reperfusion (OGD / R) model at different times of oxygen-glucose deprivation; NC is a blank control group (SH-SY5Y cells cultured under normal conditions); 3, 6, 9, and 12 represent SH-SY5Y cells after 3, 6, 9, and 12 hours of oxygen-glucose deprivation, respectively.

[0025] Figure 2 The figure shows the cell viability of SH-SY5Y cells treated with different concentrations of Acanthopanax senticosus water extract; NC is a blank control group (SH-SY5Y cells without Acanthopanax senticosus water extract treatment); ns represents p>0.05 compared with NC; ** represents p<0.01 compared with NC.

[0026] Figure 3 The figure shows the effect of Acanthopanax senticosus water extract on the viability of SH-SY5Y cells under OGD / R injury; NC is a blank control group (SH-SY5Y cells cultured under normal conditions); ## represents p<0.01 compared with NC; ns represents p>0.05 compared with the OGD / R model group; * represents 0.01<p<0.05 compared with the OGD / R model group; ** represents p<0.01 compared with the OGD / R model group.

[0027] Figure 4The general optical microscope observation results of SH-SY5Y cells treated by different treatments are shown; a is a blank control group; b is an OGD / R model group; c is a positive drug control group (80 μM Edaravone); d is a 500 μg / mL Acanthopanax senticosus water extract treatment group; e is a 1000 μg / mL Acanthopanax senticosus water extract treatment group.

[0028] Figure 5 The Annexin V-PI apoptosis flow detection results of SH-SY5Y cells treated by different treatments are shown; a is a blank control group (NC); b is an OGD / R model group; c is a positive drug control group (80 μM Edaravone); d is a 500 μg / mL Acanthopanax senticosus water extract treatment group; e is a 1000 μg / mL Acanthopanax senticosus water extract treatment; f is the statistics of the apoptosis rate of each group, wherein NC represents a blank control group, ## represents p<0.01 compared with NC, and ** represents p<0.01 compared with the OGD / R model group.

[0029] Figure 6 The ROS level detection results of SH-SY5Y cells treated by different treatments are shown; a is a blank control group (NC); b is an OGD / R model group; c is a positive drug control group (80 μM Edaravone); d is a 500 μg / mL Acanthopanax senticosus water extract treatment group; e is a 1000 μg / mL Acanthopanax senticosus water extract treatment; f is the statistics of the ROS fluorescence intensity of each group, wherein NC represents a blank control group, ## represents p<0.01 compared with NC, and ** represents p<0.01 compared with the OGD / R model group.

[0030] Figure 7 The ATP content determination results of SH-SY5Y cells treated by different treatments are shown; wherein NC represents a blank control group, # represents 0.01<p<0.05 compared with NC, ns represents p>0.05 compared with the OGD / R model group, and * represents 0.01<p<0.05 compared with the OGD / R model group.

[0031] Figure 8 The effects of Acanthopanax senticosus extracts on the viability of SH-SY5Y cells under OGD / R damage in the comparative examples are shown; NC is a blank control group (SH-SY5Y cells cultured under normal conditions); "Acanthopanax senticosus-stone", "Acanthopanax senticosus-ethyl", "Acanthopanax senticosus-n-butanol", and "Acanthopanax senticosus-water" respectively represent Acanthopanax senticosus petroleum ether extract, Acanthopanax senticosus ethyl acetate extract, Acanthopanax senticosus n-butanol extract, and Acanthopanax senticosus water extract. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, the implementation of the present invention is not limited to the following embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0033] Example 1: Component analysis of Acanthopanax senticosus water extract

[0034] 1. Reagents and Instruments

[0035] Anhydrous ethanol, petroleum ether, ethyl acetate, and n-butanol were all purchased from Tianjin Fuyu. Pure water was purchased from Wahaha. The equipment included a water bath (Jiangyin Baoli), a grinder (Joyoung), a rotary evaporator (Swiss Buqi), an ultrasonic cleaner (Ningbo Xinzhi), a freeze dryer (Ningbo Xinzhi), and an ultra-low temperature freezer (Panasonic, Japan).

[0036] 2. Preparation of Acanthopanax senticosus aqueous extract

[0037] The dried roots of Acanthopanax senticosus (Rupr. Et Maxim.) Harms were purchased from Heilongjiang Deshunchang Traditional Chinese Medicine Pieces Co., Ltd. Aqueous extracts of Acanthopanax senticosus were prepared according to the following method:

[0038] (1) Weigh 100g of dried Acanthopanax senticosus root and grind it into powder using a grinder;

[0039] (2) Mix the obtained Acanthopanax senticosus dried root powder with 1500 mL of 80% ethanol aqueous solution, and treat it with continuous ultrasonic treatment (power of 360 W, frequency of 40 KHz) at room temperature for 30 min. Then filter it with a sand core funnel and collect the filtrate.

[0040] (3) Mix the filter residue with 1500 mL of 80% ethanol aqueous solution, and treat it with continuous ultrasonication (power 360 W, frequency 40 KHz) at room temperature for 30 min. Then filter it with a sand core funnel and collect the filtrate. Repeat this step twice.

[0041] (4) Combine the filtrates obtained from the three filtrations and dry them in a rotary evaporator at 37°C to obtain approximately 3g of crude extract of traditional Chinese medicine.

[0042] (5) Add 200 mL of pure water to the crude extract of Chinese medicine and sonicate (power 360 W, frequency 40 KHz) to redissolve it to obtain crude extract of Chinese medicine.

[0043] (6) The crude extract of traditional Chinese medicine was extracted with petroleum ether, ethyl acetate and n-butanol in sequence. Each organic solvent was used for extraction three times, and the amount of organic solvent used for each extraction was 200 mL, to obtain an aqueous extract.

[0044] (7) The aqueous extract is dried in a freeze dryer to obtain an aqueous extract solid or extract, namely Acanthopanax senticosus aqueous extract.

[0045] The prepared Acanthopanax senticosus water extract was stored in a -80℃ freezer for later use.

[0046] 3. Monomer component analysis of Acanthopanax senticosus water extract

[0047] 3.1 Reagents and Instruments

[0048] Sciex TripleTOF 6600 LC-MS / MS system (SCIEX, USA), chromatographic grade formic acid (Aladdin), chromatographic grade acetonitrile (Merck, Germany).

[0049] 3.2 Establishment of a Monomeric Component Library from Acanthopanax senticosus Water Extract

[0050] 3.2.1 Establishment of a chemical component library of Acanthopanax senticosus

[0051] Literature was searched using "Acanthopanax senticosus" and "chemical components of Acanthopanax senticosus" as keywords. Information on monomeric compounds in Acanthopanax senticosus (Chinese and English names, molecular formulas, molecular weights, and chemical structures) was recorded and compiled into a table to obtain a database of the chemical components of Acanthopanax senticosus.

[0052] 3.2.2 Detection of monomeric components in Acanthopanax senticosus water extract by liquid chromatography / mass spectrometry (LC / MS)

[0053] Weigh 10 mg of the prepared Acanthopanax senticosus water extract, dissolve it completely in 1 mL of ddH2O, filter it through a 0.22 μm sterile filter, and transfer it to a clean liquid chromatography vial. Sonicate the vial containing the prepared mobile phase for 15 min to remove air bubbles.

[0054] The operating conditions and detection methods for the ultra-high performance liquid chromatography-mass spectrometry (UHPLC) instrument are set as follows:

[0055] Chromatographic column: C18 ultra-high performance liquid chromatography column;

[0056] Mobile phase: Acetonitrile / water — 0.1% formic acid;

[0057] Sample loading volume: 1 μL;

[0058] Flow rate: 0.6 mL / min;

[0059] Elute with a gradient of 95% water and 5% acetonitrile to 100% acetonitrile for 16 min.

[0060] 3.2.3 Non-targeted analysis of LC / MS detection results based on a self-built Acanthopanax senticosus chemical component library

[0061] Open the LC / MS results with Peak View software, import the Acanthopanax senticosus chemical component library established through literature collection into Peak View software for retrieval, and analyze and compare the results list using indicators such as molecular formula, molecular weight, and error to determine the monomer components in the prepared Acanthopanax senticosus water extract, thus obtaining the Acanthopanax senticosus water extract monomer component library.

[0062] Use chemBioDraw software to draw the structural formulas of the monomeric compounds collected from the literature.

[0063] 3.3 Establishment of a target library of monomeric components from Acanthopanax senticosus water extract

[0064] The structural formulas of compounds from the obtained Acanthopanax senticosus water extract monomer component library were imported into the SwissTargetPrediction database. After setting the predicted species as "Homo sapiens", prediction was performed to obtain the target information (name, family, probability, etc.) that each compound may bind to in the human body.

[0065] Results and conclusions:

[0066] Based on literature review, we identified 130 monomeric compounds contained in Acanthopanax senticosus and established a chemical component library. Using this library as a foundation, we conducted a database search and compared the results with the liquid chromatography-mass spectrometry (LC-MS) analysis of the prepared Acanthopanax senticosus water extract, identifying 20 monomeric components in the extract (as shown in Table 1).

[0067] Table 1. Monomer components in Acanthopanax senticosus aqueous extract

[0068]

[0069]

[0070] Example 2: Cellular Experiment of Acanthopanax senticosus Water Extract

[0071] 1. Experimental materials, reagents and instruments

[0072] The SH-SY5Y human neuroblastoma cell line was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0073] Fetal bovine serum was purchased from Gibco. DMEM sugar-free medium was purchased from Procell. DMEM / F12 medium, penicillin-streptomycin mixture (100×), trypsin, and PBS were all purchased from HyClone. Thiazoline blue (MTT), dimethyl sulfoxide (DMSO), and BCA quantitative kits were all purchased from Solarbio. Reactive oxygen species (ROS), superoxide dismutase (SOD), and methane (MDA) assay kits were all purchased from Nanjing Jiancheng. Apoptosis assay kits were purchased from Jiangsu Kaiji. ATP assay kits were purchased from Beyotime.

[0074] 96-well plate (CORNING), multi-functional microplate reader (TECAN, Switzerland), CO2 incubator (Pentax, Germany), clean bench (Shanghai Boxun), flow cytometer (Beckman), inverted optical microscope (Tianjin Weiyi), inverted fluorescence microscope (Olympus).

[0075] 2. Experimental Methods

[0076] 2.1 Preparation of the solution to be used

[0077] Complete culture medium: 10% fetal bovine serum, 1% penicillin and antibiotics, 89% DMEM / F12 medium.

[0078] MTT solution: Dissolve 20 mg of thiazoline in 40 mL of PBS to prepare a 0.5 mg / mL solution, filter through a 0.22 μm sterile filter membrane, protect from light, and set aside for use.

[0079] Incubator conditions: 37℃, 5% CO2, 90% humidity.

[0080] The Acanthopanax senticosus water extract prepared in Example 1 was dissolved and diluted with complete culture medium to prepare different concentrations (4000 μg / ml, 2000 μg / ml, 1000 μg / ml, 500 μg / ml, 250 μg / ml, 125 μg / ml). The solutions were then filtered through a 0.22 μm sterile filter in a laminar flow hood to obtain the Acanthopanax senticosus solution to be tested. The solution was then aliquoted into sterile centrifuge tubes, sealed, and stored in a -20°C refrigerator for later use.

[0081] 2.2 Types of 96-well plates

[0082] SH-SY5Y cells grown to the logarithmic growth phase (when cells reach 80% confluence with the bottom of the flask) were placed in a clean bench, the original culture medium was discarded, and the cells were rinsed twice with PBS. They were then digested with 1 mL of 0.25% trypsin until the adherent cells became rounded and detached in a flowing, sand-like manner. 1 mL of complete culture medium was quickly added to stop the digestion. The cells were pipetted until single-celled and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 1 mL of complete culture medium and counted. 100 μL of PBS was added to the outer edge of each well of a 96-well plate, and the remaining wells were seeded with SH-SY5Y cells at a density of 10,000 cells / well. The plates were then incubated in an incubator for 24 h.

[0083] 2.3 Establishment of an oxygen-glucose deprivation-reperfusion injury model in SH-SY5Y cells

[0084] In in vitro studies, the oxygen-glucose deprivation / reperfusion (OGD / R) injury model is often used to simulate the injury mechanism during ischemic stroke to study and evaluate the protective effects of drugs on nerve cells in vitro. To confirm the conditions for establishing this classic model, we investigated the oxygen-glucose deprivation time. Specifically, SH-SY5Y cells grown to the logarithmic growth phase were seeded into 96-well plates according to the method described in section 2.2 above. After culturing for 24 hours, the cells were washed twice with DMEM sugar-free medium, and then 100 μL of DMEM sugar-free medium was added to each well. The cells were then placed in a hypoxic chamber (95% N2 + 5% CO2) and cultured under hypoxic conditions for 3, 6, 9, and 12 hours, respectively. The medium in the wells was then replaced with normal DMEM medium and cultured under normal conditions for 12 hours. Cell viability was then measured and calculated using the MTT assay. The hypoxic duration corresponding to the treatment group with cell viability approximately 50%–60% of the blank control group (SH-SY5Y cells without any treatment) was selected as the hypoxic duration for establishing the SH-SY5Y cell oxygen-glucose deprivation / reperfusion injury model.

[0085] MTT method: Add 100 μL of DMEM medium containing 0.5 mg / mL MTT to each well and incubate in the dark for 4 hours. Then add 150 μL of DMSO to each well and shake on a decolorizing shaker for 10 min to completely dissolve the crystals. Measure the absorbance (OD) of each well at 490 nm using a microplate reader. 490 ).

[0086] Cell viability (%) = OD of treatment group 490 / Blank control group OD 490 ×100%

[0087] The results showed that the oxygen-glucose deprivation time required to establish the SH-SY5Y cell OGD / R model was 9 hours. Figure 1 ).

[0088] 2.4 Determination of Safe Concentration Range

[0089] To determine a reasonable sample concentration range for the protective activity evaluation experiment and to eliminate the influence of Acanthopanax senticosus extract on cytotoxicity, we conducted a cytotoxicity experiment on SH-SY5Y cells. Specifically, SH-SY5Y cells grown to the logarithmic growth phase were seeded into 96-well plates according to the method described in section 2.2 above. After culturing for 24 hours, the original culture medium in the treatment group was discarded, and 100 μL of different concentrations (4000 μg / ml, 2000 μg / ml, 1000 μg / ml, 500 μg / ml, 250 μg / ml, and 125 μg / ml) of the test Acanthopanax senticosus extract (prepared as described in section 2.1) was added to each well. The control group cells were not treated and were cultured under normal conditions for another 24 hours. Cell viability was then determined and calculated using the MTT assay described above. The concentration of the extract corresponding to a cell viability in the treatment group greater than 70% of that in the control group was selected as the safe concentration.

[0090] Cytotoxicity assays showed that the cell viability (%) of the control group and the groups treated with 125, 250, 500, 1000, 2000, and 4000 μg / mL of Acanthopanax senticosus water extract were 100.00±3.97, 109.12±2.36, 110.20±4.31, 114.45±3.44, 113.81±2.36, 108.50±3.08, and 106.45±5.90, respectively. Cytotoxicity was determined according to the international standard for biocompatibility (ISO 10993-5), and the results are as follows: Figure 2 As shown, the aqueous extracts of Acanthopanax senticosus at concentrations of 125, 250, 500, 1000, 2000, and 4000 μg / mL were not toxic to SH-SY5Y cells (cell viability in each treatment group was 70% higher than that in the control group). Furthermore, compared with the control group, the viability of SH-SY5Y cells in each Acanthopanax senticosus aqueous extract treatment group was increased, and the 500 μg / mL and 1000 μg / mL Acanthopanax senticosus aqueous extract treatment groups showed highly significant differences. Figure 2 ).

[0091] 2.5 Protective effect of Acanthopanax senticosus aqueous extract on SH-SY5Y cells under ischemia-reperfusion injury

[0092] 2.5.1 Effect of Acanthopanax senticosus water extract on the viability of SH-SY5Y cells under OGD / R damage

[0093] Based on the results of the cytotoxicity assay, an aqueous extract of Acanthopanax senticosus at a concentration not exceeding 4000 μg / mL was used for the OGD / R experiment in SH-SY5Y cells. Details are as follows:

[0094] Following the method described in section 2.2 above, SH-SY5Y cells that have grown to the logarithmic growth phase were seeded into 96-well plates. The cells were cultured under normal conditions for 12 hours. A blank control group, an OGD / R model group, a positive drug control group, and a group treated with Acanthopanax senticosus water extract were set up.

[0095] The cells in the blank control group were cultured for another 33 hours under normal conditions without any treatment.

[0096] After the cells in the OGD / R model group were cultured for another 12 hours, they were washed twice with DMEM sugar-free medium. Then, 100 μL of DMEM sugar-free medium was added to each well, and the cells were placed in an anoxic chamber (95% N2 + 5% CO2) and cultured for another 9 hours under anoxic conditions. After that, the medium in the wells was replaced with normal DMEM medium and cultured for another 12 hours under normal conditions.

[0097] Cells in the positive control group were incubated with edaravone (80 μM) in culture wells for 12 hours, washed twice with DMEM sugar-free medium, and 100 μL of DMEM sugar-free medium was added to each well. The cells were then placed in an anoxic chamber (95% N2 + 5% CO2) and cultured for another 9 hours under anoxic conditions. The culture medium in the wells was then replaced with normal DMEM medium and cultured for another 12 hours under normal conditions.

[0098] For the Acanthopanax senticosus water extract treatment group, different concentrations of the test Acanthopanax senticosus drug solution (prepared in section 2.1 above) were added to the cell culture wells. After incubation for 12 hours, the cells were washed twice with DMEM sugar-free medium. 100 μL of DMEM sugar-free medium was added to each well, and the cells were placed in an anoxic chamber (95% N2 + 5% CO2) and cultured under anoxic conditions for another 9 hours. The medium in the wells was then replaced with normal DMEM medium and cultured under normal conditions for another 12 hours.

[0099] At the end of the experiment, cell viability in each group was measured and calculated using the MTT assay.

[0100] The results are as follows Figure 3 and Figure 4 As shown, the cell viability of the OGD / R model group was 52% of that of the blank control group, and cell shrinkage, reduced or even absent dendritic structures could be observed under a regular optical microscope, proving that the modeling was successful. Figure 4(a and b). The cell viability (%) of the positive drug control group and the groups treated with Acanthopanax senticosus water extract at concentrations of 125, 250, 500, 1000, 2000, and 4000 μg / mL were 68.10±0.58, 56.11±0.58, 67.18±3.69, 68±3.80, 71.91±7.19, 67.31±3.14, and 62.99±0.81, respectively. In the Acanthopanax senticosus water extract treatment groups, when the concentration of Acanthopanax senticosus water extract was below 1000 μg / mL, cell viability increased with increasing concentration of Acanthopanax senticosus water extract, showing a concentration-dependent effect, and cell morphology was somewhat restored. Figure 4 (d and e) Among them, the cell viability of the 1000 μg / mL Acanthopanax senticosus water extract treatment group increased by 20% compared with the OGD / R model group, which was better than the positive drug control group (80 μM edaravone); when the concentration of Acanthopanax senticosus water extract was higher than 1000 μg / mL, cell viability no longer increased and even decreased, indicating that 1000 μg / mL was the optimal concentration to reduce damage. Therefore, 500 μg / mL and 1000 μg / mL were selected as the drug concentrations for subsequent experiments.

[0101] 2.5.2 Apoptosis Detection

[0102] Apoptosis is a recognized important mechanism of cerebral ischemia-reperfusion injury. To investigate the relationship between Acanthopanax senticosus water extract and apoptosis, we used flow cytometry to detect early and late apoptosis markers. Specifically: at 2×10⁻⁶... 5 SH-SY5Y cells grown to the logarithmic growth phase were seeded into 6-well plates at a density of cells / well. After culturing the cells under normal conditions for 12 hours, blank control group, OGD / R model group, positive drug control group, and Acanthopanax senticosus water extract treatment group were set up. The treatment method for each group was the same as described in 2.5.1 above. After treatment, the cells in each group were digested with trypsin without EDTA and collected separately. The cells were washed with PBS 2-3 times. The cells were stained with Annexin V-PI apoptosis kit according to the instructions and then detected by flow cytometry.

[0103] Annexin V-PI apoptosis detection results for each group are as follows: Figure 5 As shown, the upper left, upper right, lower left, and lower right portions of the scatter plot represent cells in necrotic, late-apoptotic, normal, and early-apoptotic states, respectively. Experimental results showed that the apoptosis rates (%) of the blank control group, OGD / R model group, positive drug control group, and the groups treated with 500 and 1000 μg / mL of Acanthopanax senticosus water extract were 0.95±0.15, 10.45±1.34, 1.46±0.57, 2.2±0.51, and 1.47±0.34, respectively. The OGD / R model group had a significantly increased number of cells in the apoptotic phase (early-apoptotic + late-apoptotic) and dead cells compared to the blank group. Figure 5b). The number of apoptotic cells (early apoptosis + late apoptosis) and dead cells in the Acanthopanax senticosus water extract treatment groups (500 μg / mL and 1000 μg / mL) was significantly reduced compared to the model group. Figure 5 The results (d and e) showed extremely significant differences, indicating that the Acanthopanax senticosus water extract of the present invention can significantly reduce OGD / R-induced apoptosis in SH-SY5Y cells.

[0104] 2.5.3 Determination of the content of oxidative stress-related indicators

[0105] (1) Determination of reactive oxygen species (ROS) content

[0106] Press 2×10 5 SH-SY5Y cells grown to the logarithmic growth phase were seeded into 6-well plates at a density of cells / well. After culturing the cells under normal conditions for 12 hours, blank control group, OGD / R model group, positive drug control group, and Acanthopanax senticosus water extract treatment group were set up. The treatment method for each group was the same as described in 2.5.1 above. After treatment, the culture medium in the wells was aspirated, the cells were washed with PBS, and then the reactive oxygen species detection kit was used according to the instructions of the manufacturer to detect the reactive oxygen species using 10 μM DCFH-DA.

[0107] (2,7-Dichlorodihydrofluoresceindiacetate, reactive oxygen species probe) was incubated for 30 min, and then observed and quantified using a fluorescence microscope and an enzyme-linked immunosorbent assay (ELISA) reader, respectively.

[0108] The results are as follows Figure 6 As shown, the fluorescence intensity (%) of the OGD / R model group, the positive drug control group, and the groups treated with 500 and 1000 μg / mL Acanthopanax senticosus water extract relative to the blank control group were 285.98±4.36, 184.44±1.21, 200.18±12.29, and 185.96±4.12, respectively. This indicates that the fluorescence intensity of the OGD / R model group was significantly higher than that of the blank control group, and oxygen-glucose deprivation damage significantly increased the intracellular reactive oxygen species level (p<0.01). In contrast, the fluorescence intensity of the positive drug control group (edaravone treatment) and the Acanthopanax senticosus water extract treatment group was significantly lower than that of the OGD / R model group, and the effect of 1000 μg / mL Acanthopanax senticosus water extract was superior to that of 500 μg / mL Acanthopanax senticosus water extract.

[0109] (2) Determination of superoxide dismutase (SOD) and malondialdehyde (MDA) content

[0110] Press 2×10 5SH-SY5Y cells grown to the logarithmic growth phase were seeded into 6-well plates at a density of cells / well. After culturing the cells under normal conditions for 12 hours, blank control group, OGD / R model group, positive drug control group, and Acanthopanax senticosus water extract treatment group were set up. The treatment method for each group was the same as described in 2.5.1 above. After treatment, the original culture medium in the wells was aspirated, the cells were washed twice with PBS, digested with trypsin and centrifuged to remove the culture medium. The cells were resuspended in PBS and centrifuged, and the process was repeated twice. After lysing the cells with an ultrasonic cell disruptor, the SOD and MDA contents were measured according to the instructions using SOD and MDA detection kits, respectively. The results were read using a multi-mode microplate reader and calculated.

[0111] The results are shown in Table 2. The OGD / R model group showed decreased SOD activity, indicating a decline in cellular antioxidant capacity, while increased MDA levels indicated a heightened degree of oxygen free radical attack. In contrast, the Acanthopanax senticosus water extract treatment groups (500 μg / mL and 1000 μg / mL) showed significantly increased SOD activity and decreased intracellular MDA content compared to the OGD / R model group, indicating that the Acanthopanax senticosus water extract can enhance cellular antioxidant capacity and reduce the degree of oxygen free radical attack. Therefore, the Acanthopanax senticosus water extract of this invention can alleviate oxidative stress induced by OGD / R.

[0112] Table 2. SOD activity and MDA content in each treatment group

[0113] Cell processing groups SOD (U / mg prot) MDA (nmol / mg prot) Blank control group 128.02±3.32 2.20±0.15 OGD / R model group 102.84±2.98 5.57±0.13 Positive drug control group 127.97±1.46 3.83±0.16 500 μg / mL acanthopanax senticosus water extract treatment group 125.66±2.27 3.62±0.10 1000 μg / mL acanthopanax senticosus water extract treatment group 127.84±3.13 3.20±0.18

[0114] 2.5.4 Mitochondrial function assay

[0115] Adenosine triphosphate (ATP) is the energy source for brain tissue and an important substance for maintaining brain cell function. Mitochondrial aerobic respiration is the ATP production pathway; therefore, changes in mitochondrial function lead to changes in ATP content, and ATP levels reflect the normality of mitochondrial function. To investigate the effect of Acanthopanax senticosus water extract on mitochondrial function in SH-SY5Y cells under OGD / R injury, we measured ATP content. Specifically: at 2×10... 5 SH-SY5Y cells grown to the logarithmic growth phase were seeded into 6-well plates at a density of cells / well. After culturing the cells under normal conditions for 12 hours, blank control group, OGD / R model group, positive drug control group, and Acanthopanax senticosus water extract treatment group were set up. The treatment method for each group was the same as described in 2.5.1 above. After treatment, the original culture medium in the wells was aspirated, and the cells were washed twice with PBS. Cell lysis buffer was added to the wells on ice for 10 minutes for lysis. The cells were then centrifuged using a low-temperature high-speed centrifuge (4℃, 10000×g, 10min). The supernatant was collected, and the protein of each group was quantified by BCA method. Then, ATP was detected and calculated using an ATP detection kit according to the instructions.

[0116] The results are as follows Figure 7 As shown, the intracellular ATP contents of the blank control group, OGD / R model group, positive drug control group, and the groups treated with 500 and 1000 μg / mL Acanthopanax senticosus water extract were 16.27±0.18, 13.75±0.10, 15.72±0.35, 15.02±0.39, and 15.50±0.81 μmol / mg prot, respectively. The ATP content in the OGD / R model group was significantly lower than that in the blank control group, indicating mitochondrial dysfunction in the OGD / R model group. In contrast, the ATP content in the Acanthopanax senticosus water extract treatment group (1000 μg / mL) was significantly higher than that in the model group, indicating that 1000 μg / mL Acanthopanax senticosus water extract can alleviate the inhibitory effect of oxygen-glucose deprivation damage on mitochondrial function in SH-SY5Y cells.

[0117] In summary, the Acanthopanax senticosus water extract of the present invention can reduce ROS levels, increase SOD activity, reduce MDA content, and maintain ATP levels in SH-SY5Y cells, thereby inhibiting SH-SY5Y cell apoptosis by protecting mitochondrial function through inhibiting oxidative stress.

[0118] Comparative example:

[0119] The crude extract of traditional Chinese medicine was obtained by following the preparation steps (1) to (5) of the Acanthopanax senticosus water extract in Example 1.

[0120] The crude extract of the Chinese herbal medicine was extracted three times with 200 mL of petroleum ether each time. The aqueous extract was then dried in a freeze dryer to obtain the petroleum ether extract of Acanthopanax senticosus.

[0121] The crude extract of the Chinese herbal medicine was extracted three times with 200 mL of ethyl acetate each time. The aqueous extract was then dried in a freeze dryer to obtain the ethyl acetate extract of Acanthopanax senticosus.

[0122] The crude extract of traditional Chinese medicine was extracted three times with n-butanol, using 200 mL of n-butanol each time. The aqueous extract was then dried in a freeze dryer to obtain the n-butanol extract of Acanthopanax senticosus.

[0123] Following the method described in Example 2, Section 2.4, the safe concentration ranges of the above-mentioned Acanthopanax senticosus petroleum ether extract, Acanthopanax senticosus ethyl acetate extract, and Acanthopanax senticosus n-butanol extract were determined. The results showed that the safe concentrations of the Acanthopanax senticosus petroleum ether extract, Acanthopanax senticosus ethyl acetate extract, and Acanthopanax senticosus n-butanol extract were ≤1000 μg / mL.

[0124] Petroleum ether extract, ethyl acetate extract, and n-butanol extract of Acanthopanax senticosus were selected at concentrations of 1000 μg / mL, 500 μg / mL, and 250 μg / mL for OGD / R experiments in SH-SY5Y cells. A blank control group, an OGD / R model group, a positive drug control group, and a 1000 μg / mL aqueous extract treatment group were also set up. The experimental method was the same as in 2.5.1 of Example 2. The results are as follows: Figure 8 As shown, the petroleum ether extract, ethyl acetate extract, and n-butanol extract of Acanthopanax senticosus did not exhibit any protective activity against OGD / R damage, while the 1000 μg / mL aqueous extract of Acanthopanax senticosus significantly inhibited the decrease in SH-SY5Y cell survival (viability) caused by OGD / R, and its effect was better than that of the positive drug control group.

Claims

1. A method for preparing a water extract of Acanthopanax senticosus, comprising the following steps: a) grinding dried roots of Acanthopanax senticosus into powder; b) mixing the dried root powder with 70%-90% ethanol aqueous solution, continuously ultrasonic treating at room temperature for 25-35 min, and then filtering to collect the filtrate; c) rotary evaporating the filtrate to remove the ethanol aqueous solution to obtain a traditional Chinese medicine (TCM) extract; d) adding pure water to the TCM extract and ultrasonic redissolving to obtain a TCM extract solution; e) sequentially extracting the TCM extract solution with petroleum ether, ethyl acetate, and n-butanol to obtain an aqueous extract; and f) freeze-drying the aqueous extract to obtain the water extract of Acanthopanax senticosus. In step b), the dried root powder is mixed with 70%-90% ethanol aqueous solution at a mass-volume ratio of 1 g:10-20 mL. Step b) further comprises at least one repeated extraction, which comprises: mixing the obtained residue with 70%-90% ethanol aqueous solution, continuously ultrasonic treating at room temperature for 25-35 min, filtering to collect the filtrate, and combining all the filtrates. The obtained filtrate is rotary evaporated at 35-45℃ until all the ethanol aqueous solution is removed. In step d), the TCM extract and the pure water are mixed at a mass-volume ratio of 1 g:65-75 mL. In step e), the amounts of petroleum ether, ethyl acetate, and n-butanol are the same as the amount of pure water in step d). In step e), each organic solvent is extracted three times.

2. The production method according to claim 1, characterized by, 8.The water extract of Acanthopanax senticosus obtained by the method of any one of claims 1-7.

3. The preparation method according to claim 1, characterized in that, 9.A medicament comprising the water extract of Acanthopanax senticosus of claim 8.

4. The production method according to claim 1, characterized by, 10.Use of the water extract of Acanthopanax senticosus of claim 8 in the preparation of a medicament for treating ischemic stroke.

5. The preparation method according to claim 1, characterized in that, ​ 6. The method of claim 1, wherein, ​ 7. The preparation method according to claim 1, characterized in that, ​ ​ ​ ​

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