阅读说明：本技术 一种治疗缺血性脑卒中的中药提取物及其制备方法和应用 (Traditional Chinese medicine extract for treating cerebral arterial thrombosis and preparation method and application thereof ) 是由 颜丙春 夏子豪 沈萌萌 盛鹏 赵林 于 2021-10-29 设计创作，主要内容包括：本发明涉及一种治疗缺血性脑卒中的中药提取物及其制备方法和应用,中药提取物为崧筋藤中乙酸乙酯部位；该中药提取物的制备方法：取干燥的崧筋藤药材,打粉机粉碎研磨成粗粉；用药材重量8倍量的95％的乙醇对崧筋藤粉末进行85℃下回流提取,将提取液过滤,得到滤液和滤渣；加入生药材重量5倍量95％的乙醇对滤渣进行提取过滤,混合滤液用蒸发器进行减压浓缩至无醇味并干燥,得到浸膏；依次用等体积石油醚、乙酸乙酯、正丁醇进行萃取,得到石油醚萃取液、乙酸乙酯萃取液、正丁醇萃取液；乙酸乙酯萃取液进行减压浓缩并干燥,得到崧筋藤中乙酸乙酯部位。通过本发明,能够减少脑梗死体积、增加脑血流量,增强缺血性脑卒中空间学习记忆能力。(The invention relates to a traditional Chinese medicine extract for treating ischemic stroke and a preparation method and application thereof, the traditional Chinese medicine extract is an ethyl acetate part in stem of cabbage; the preparation method of the traditional Chinese medicine extract comprises the following steps: pulverizing dry stem of cabbage into coarse powder; performing reflux extraction of caulis Seu folium Brassicae Capitatae powder with 95% ethanol 8 times the weight of the medicinal materials at 85 deg.C, and filtering the extractive solution to obtain filtrate and residue; adding 95% ethanol 5 times the weight of the crude drugs to extract and filter the filter residue, concentrating the mixed filtrate under reduced pressure by using an evaporator until no alcohol smell exists, and drying to obtain an extract; sequentially extracting with petroleum ether, ethyl acetate and n-butanol of equal volume to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract; the ethyl acetate extract is concentrated under reduced pressure and dried to obtain the ethyl acetate fraction of the stem of cabbage. The cerebral infarction volume can be reduced, the cerebral blood flow can be increased, and the learning and memory capacity of the cerebral arterial thrombosis space can be enhanced.)

1. A traditional Chinese medicine extract for treating ischemic stroke is characterized in that: the Chinese medicinal extract is the stem of cabbage and the stem of cabbage is the ethyl acetate part of stem of cabbage.

2. A preparation method of a traditional Chinese medicine extract for treating ischemic stroke is characterized by comprising the following steps: the method comprises the following steps:

(1) pulverizing dry stem of cabbage with a traditional Chinese medicine pulverizer, and grinding into coarse powder;

(2) extracting the stem of cabbage with 95% ethanol in an amount of 8 times the weight of the medicinal materials at 85 deg.C under reflux for 3 hr, and filtering the obtained extractive solution to obtain filtrate and residue;

(3) adding 95% ethanol which is 5 times the weight of the crude drugs to carry out reflux extraction on the filter residues, filtering, and mixing the obtained filtrate with the filtrate obtained in the step 2) to obtain mixed filtrate;

(4) carrying out reduced pressure concentration on the mixed filtrate obtained in the step (3) by using a rotary evaporator until no alcohol smell exists, and drying to obtain an extract;

(5) dissolving the extract obtained in the step (4) in distilled water of which the amount is 10 times that of the extract, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol of equal volume to obtain a petroleum ether extract, an ethyl acetate extract, an n-butanol extract and a residual water layer;

(6) and (4) carrying out reduced pressure concentration and drying on the ethyl acetate extract obtained in the step (5) to obtain an ethyl acetate part in the stem of cabbage, namely the traditional Chinese medicine extract for treating ischemic stroke.

3. The use of the extract of claim 1 for treating ischemic stroke.

4. Use according to claim 3, characterized in that: the application of ethyl acetate part in stem of leaf of said cabbage in the preparation of the medicine for reducing the cerebral infarction volume.

5. Use according to claim 3, characterized in that: the application of ethyl acetate part of stem of leaf of cabbage in preparing medicine for improving cognitive dysfunction and improving spatial learning and memory ability.

6. Use according to claim 3, characterized in that: the application of ethyl acetate part of stem of leaf of said stem of cabbage in the preparation of medicine for protecting neuron cell.

7. Use according to claim 3, characterized in that: the application of ethyl acetate part in stem of leaf of said stem of cabbage in the preparation of medicine for promoting angiogenesis and improving collateral circulation.

8. Use according to claim 3, characterized in that: the application of ethyl acetate part in stem of leaf of said stem of cabbage in the preparation of medicine for protecting blood brain barrier.

9. Use according to any one of claims 3 to 8, characterized in that: the ethyl acetate part in the stem of leaf of cabbage is a single-component or compound preparation.

Technical Field

The invention relates to a traditional Chinese medicine extract for treating cerebral arterial thrombosis and a preparation method and application thereof, belonging to the technical field of traditional Chinese medicines.

Background

Ischemic Stroke (CIS) accounts for about 80% of Stroke, has the outstanding characteristics of high incidence, high mortality and high disability rate, and is a major public health problem concerned by medicine. At present, the purpose of recovering blood flow by thrombolysis or intravascular thrombus extraction is the most main and effective method for treating acute ischemic stroke, but the benefit range is extremely narrow due to the time window or technical limitation, and even if a patient successfully receives thrombolysis or thrombus extraction treatment, the patient is influenced by multiple factors such as recanalization rate, blood vessel recanalization but ineffective blood flow, reperfusion injury and the like, and the prognosis of the patient after stroke is still unsatisfactory. Therefore, angiogenesis and neuroprotection remain the main therapeutic principles and directions for prevention and treatment and rehabilitation of most patients with ischemic stroke, and research on angiogenesis and neuroprotective agents is urgently needed.

The stem of leaf of Rubiaceae, including JIAOTENJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOJIAOQIANG, JIAOJIANDONGTU and SHANGSHUJIAOJIAOJIAOHUANG, are produced in southeast part of Zhejiang, eastern and southern part of Fujian, Taiwan, Guangdong, hong Kong, Hainan, Guangxi and southeaven part of Yunnan. The medicinal part is dry stem branch with leaves, has effects of relaxing muscles and tendons, activating collateral flow, dispelling pathogenic wind, relieving pain, cooling blood, and relieving swelling, and can be used for treating rheumatalgia, ischialgia, carbuncle, sore, toxic swelling, and sore throat. At present, research reports on chemical components and pharmacological activity of stem of cabbage are rare, and particularly few studies on neuropharmacology are available, so that the application of the stem of cabbage in preparation of a medicament for treating cerebral arterial thrombosis is not researched and reported.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine extract for treating ischemic stroke and a preparation method and application thereof aiming at the defects of the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme: a traditional Chinese medicine extract for treating ischemic stroke is characterized in that: the Chinese medicinal extract is the stem of cabbage and the stem of cabbage is the ethyl acetate part of stem of cabbage.

A preparation method of a traditional Chinese medicine extract for treating ischemic stroke is characterized by comprising the following steps: the method comprises the following steps:

(1) pulverizing dry stem of cabbage with a traditional Chinese medicine pulverizer, and grinding into coarse powder;

(2) extracting the stem of cabbage with 95% ethanol in an amount of 8 times the weight of the medicinal materials at 85 deg.C under reflux for 3 hr, and filtering the obtained extractive solution to obtain filtrate and residue;

(3) adding 95% ethanol which is 5 times the weight of the crude drugs to carry out reflux extraction on the filter residues, filtering, and mixing the obtained filtrate with the filtrate obtained in the step 2) to obtain mixed filtrate;

(4) carrying out reduced pressure concentration on the mixed filtrate obtained in the step (3) by using a rotary evaporator until no alcohol smell exists, and drying to obtain an extract;

(5) dissolving the extract obtained in the step (4) in distilled water of which the amount is 10 times that of the extract, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol of equal volume to obtain a petroleum ether extract, an ethyl acetate extract, an n-butanol extract and a residual water layer;

(6) and (4) carrying out reduced pressure concentration and drying on the ethyl acetate extract obtained in the step (5) to obtain an ethyl acetate part in the stem of cabbage, namely the traditional Chinese medicine extract for treating ischemic stroke.

The application of a traditional Chinese medicine extract for treating cerebral arterial thrombosis is utilized.

The application of ethyl acetate part in stem of leaf of said cabbage in the preparation of the medicine for reducing the cerebral infarction volume.

The application of ethyl acetate part of stem of leaf of cabbage in preparing medicine for improving cognitive dysfunction and improving spatial learning and memory ability.

The application of ethyl acetate part of stem of leaf of said stem of cabbage in the preparation of medicine for protecting neuron cell.

The application of ethyl acetate part in stem of leaf of said stem of cabbage in the preparation of medicine for promoting angiogenesis and improving collateral circulation.

The application of ethyl acetate part in stem of leaf of said stem of cabbage in the preparation of medicine for protecting blood brain barrier.

The ethyl acetate part in the stem of leaf of cabbage is a single-component or compound preparation.

The invention observes the neuroprotective effect of ethyl acetate parts in stem of cabbage on mouse model of ischemic stroke.

According to the invention, the cerebral infarction volume of the mouse, the spatial learning and memory ability of the mouse and the neuronal death condition are observed by TTC staining, Morris water maze experiment and immunohistochemical staining methods. The experimental result shows that the stem of cabbage rattan extract can obviously reduce the cerebral infarction volume of mice, increase the cerebral blood flow, enhance the space learning and memory ability of mice with ischemic stroke and reduce the death of neurons in hippocampus of the mice.

The invention observes the promoting effect of the stem of cabbage on collateral circulation reconstruction and angiogenesis in the semi-dark zone after ischemic stroke.

The invention uses immunohistochemical staining and Western Blot method to observe the effect of stem of Bhattaceae on blood brain barrier of mouse. Immunohistochemical results show that the stem extract of stem of leaf of stem of leaf of stem of leaf of cabbage obviously increases the expression of adhesion molecule of platelet (CD31), vascular endothelial growth factor of growth (VEGFR2) of stem of leaf of stem of leaf of stem of leaf of mouse; western Blot results showed that the stem of cabbage leaf extract significantly increased the expression of the tight junction proteins ZO-1, Claudin-5 in the mouse semi-dark zone.

The invention has the beneficial effects that: the invention discovers that the stem of cabbage leaves has the effect of treating ischemic stroke for the first time, is a new application of the stem of cabbage leaves, enlarges the clinical application range of the stem of cabbage leaves, and is expected to provide a basis for the research and development of new drugs for treating ischemic stroke.

Drawings

FIG. 1 shows the effect of stem extract of stem of cabbage leaves on the volume of cerebral infarction and spatial learning and memory ability of mice (. word p < 0.05 statistically significant compared to Sham group; # p < 0.05 statistically significant compared to MCAO group). Note: (A) TTC dyeing; (B) histogram of infarct volume percentage; (C) longa score histogram; (D) the water maze represents a track diagram after 24h of molding; (E) escape latency histogram.

FIG. 2 shows immunohistochemical staining of mouse hippocampal CA1 region Neun, CD31, VEGFR2 after ischemic stroke (. p < 0.05 statistically significant compared to Sham group; p < 0.05 statistically significant compared to MCAO group). (A) Neun immunohistochemical staining of hippocampal CA1 area; (B) immunohistochemical staining of CD31 in the CA1 region of the hippocampus; (C) immunohistochemical staining of VEGFR2 in hippocampal CA1 zone; (D) histogram of the number percentage of Neun cells; (E) histogram of the percentage of CD31 cell numbers; (F) percentage of cell numbers for VEGFR2 histogram.

FIG. 3 shows the expression of the proteins involved in the tight junctions in the hemizonal regions of mice after ischemic stroke (. p < 0.05, statistically significant compared to the Sham group; p < 0.05, statistically significant compared to the MCAO group). Note: (A) ZO-1, Claudin5 Western blot banding pattern; (B) ZO-1 protein gray value ratio results; (C) claudin5 protein gray value ratio results.

Detailed Description

The present invention is further illustrated by the following specific examples.

Example 1

The stem leaf of Yangzhou leaf of Yangzhou. Taking 15kg of dry stem leaf of stem of cabbage, crushing and grinding into coarse powder, performing hot reflux extraction with 95% ethanol at 85 ℃, concentrating the extracting solution under reduced pressure until no alcohol smell exists to obtain thick extract, dissolving the extract in water, and extracting by using ethyl acetate to obtain the ethyl acetate part of the stem of cabbage.

Example 2: preparation of animal models

The invention adopts male ICR mice with 8-week-old males recognized by the international academia, the weight of the male ICR mice is 18-22 g, the male ICR mice are 2-3 months old, the male ICR mice are purchased from the comparative medicine center of Yangzhou university, the appropriate temperature (23 ℃) and humidity (60%) are kept, the environment of 12-hour illumination/12-hour darkness is kept, and the male ICR mice are cultured for 1 week adaptively.

Experimental reagent: isoflurane (reweld technologies ltd);

an experimental instrument: MCAO line bolt (Beijing Western science and technology Co., Ltd.), and small animal anesthesia machine (Midmark, USA)

1) Grouping: normal control group (Sham group), normal administration group (Sham + SJT group), model group (MCAO group), model administration group (MCAO + SJT group) (SJT,100mg/kg), and n is 30.

2) Molding (MCAO): healthy male ICR mice are selected, are fasted for 12 hours before operation without water supply, and are firstly prepared from 33 percent of O₂、67%N₂O and 2.5% isoflurane mixed gas continuous anesthesia, after conventional skin preparation and iodophor sterilization, incision is made along the median line of the neck first, the neck fascia, glands and muscles are exposed, the left Common carotid artery (Common carotid artery), the external carotid artery (external carotid artery ECA), the internal carotid artery (internal carotid artery ICA) and the vagus nerve are isolated, the ECA distal end is ligated, an articulation is made by threading a thin wire, the CCA proximal end and the ECA distal end are temporarily clamped by an artery clamp, a V-shaped incision is made at the clamp close to the ECA, the ECA is pulled down to be in line with the ICA, a plug wire is inserted into the ECA, the artery clamp of the ICA is released when entering the ICA, the plug wire is slowly entered into the blood vessel along the direction, and detection is performed to prevent insertion of the temporal artery, a part of the plug wire is ligated after reaching the expected position and is cut off, the artery clamp is detected, the artery is sterilized and the plug wire is extracted after 45min operation, the thin thread is ligated. In addition, the body temperature is maintained at about 37 ℃ by using a heat preservation blanket in the whole process of the operation until the animals are resuscitated.

3) Administration: after the molding is finished, selecting mice which are successfully molded, performing gavage administration on the stem of cabbage 6h, 12h and 18h after MCAO operation by a molding administration group, performing gavage administration on the normal administration group at the same time interval, and performing gavage administration on the stem of cabbage at the same time interval by a normal administration group at the same time interval.

Example 3: TTC staining and determination of cerebral infarct volume;

experimental reagent: 2,3, 5-Triphenyltetrazolium chloride (TTC) (Sigma Co., USA)

In this section, using the animal model described in example 2, mice were anesthetized 24h after cerebral ischemia reperfusion and sacrificed rapidly, and brain tissue was gently removed. The mouse brains were placed in brain troughs and excised every 2mm coronal section. These brain pieces were then stained in 2% TTC stain at 37 ℃ for 30 minutes, with care being taken to avoid light. After the dyeing is finished, the brain slices are fixed in 4% paraformaldehyde and kept in a refrigerator at 4 ℃ overnight. The red part of the brain slice represents the normal area and the white part represents the infarcted area. Measured analysis of the taken TTC stained photographs was performed with Image-Pro Plus 6.0 software, cerebral infarction volume = brain slice thickness (2 mm) × area.

The results are shown in figure 1A, B, in which mice in the model-created group (approximately 12% of the whole brain) had significantly reduced infarct volume compared to the model-created group (approximately 40% of the whole brain).

Example 4: a neurological score;

the part adopts the animal model described in the embodiment 2, adopts a Longa scoring method to score the nerve function of each group of mice, and the reference standard is divided into 5 grades: 1) 0 minute: no neurological deficit; 2) 1 minute: left forelimb flexion (paralyzed side) of the mouse; 3) and 2, dividing: when walking, the mouse turns to the paralyzed side; 4) and 3, dividing: when walking, the mice are hemiplegic; 5) and 4, dividing: mice can not walk spontaneously and lose consciousness. Random evaluation trials were performed over a specified time and a score greater than 1 indicated successful MCAO modeling.

As a result, as shown in fig. 1C, the mice in the model group scored high (3.5 points for nerve function), while the mice in the model group scored good (about 1 point for nerve function).

Example 5: morris water maze experiments;

behavioral testing was performed on each group of mice to assess learning and memory. The apparatus was filled with water (25. + -. 1 ℃ C.) and the animals were allowed to become familiar with the environment before the start of the experiment. On day 1, all mice were allowed to swim freely. From day 2 to day 7, the mouse needs to find only one underwater platform with several spatial distance cues, the platform is hidden 1.5 cm below the water surface, the mouse is trained (n =7) to find the platform within 60s, and if the mouse does not find the platform within the specified time, the mouse is placed on the platform to rest for 15 s. The distance travelled, latency and swimming speed were recorded. From day 8 to day 12, and 4 trials per mouse per day were performed for up to 60s per lane. The wait time to climb onto the hidden platform is recorded to assess learning ability. At day 13, animals received the probe trial. The hidden platform was removed and the number of raspberries crossing the previous location of the hidden platform was recorded to test memory.

The results are shown in fig. 1D, E, and compared with the modeling group, the modeling administration group has the advantages of obviously shortened latent escape time, increased platform crossing times, clearer track and definite purpose. After the model-making administration, the mouse nerve dysfunction is reduced, and the spatial learning and memory ability is enhanced.

Example 6: immunohistochemical staining;

reagent: rabbit anti-Neun (1:1000, abcam, USA), rabbit anti-CD 31(1:50, abcam, USA), rabbit anti-VEGFR 2(1:1000, abcam, USA), horseradish peroxidase-labeled secondary antibody: goat anti-rabbit-HRP (1:1000, Santa Cruz, USA), microscope with neutral gum (Beijing Soilebao science and technology Co., Ltd.);

this section was stained using the animal model described in example 2, using ABC-DAB immunohistochemistry. To eliminate endogenous peroxidase activity, brain tissue sections were first treated with 0.3% hydrogen peroxide (H)₂O₂) Incubate for 20min, followed by treatment in 5% BSA for 30 min. These brain pieces were then immersed in diluted rabbit anti-Neun, CD31, VEGFR2 overnight in a refrigerator at 4 ℃. Thereafter, they were incubated with biotinylated goat anti-rabbit IgG for 90 minutes, developed with DAB, and the tissues were sequentially adhered to microscope slides. Finally, after dehydration by gradient alcohol and xylene, sealing with neutral resin. A negative control experiment was performed with immune serum instead of primary antibody to determine the specificity of immunostaining, and all solutions involved in this experimental procedure were prepared by dilution with 0.01M PBS. Neun immunoreactivity was evaluated according to OD value, calculated as OD = log (256/mean gray). Adobe Photoshop version 8.0 and NIH image1.59 software were used to correct relative OD values and analysis results, respectively.

As shown in FIG. 2A, D, Neun-positive neurons were difficult to be observed in CA1 region of hippocampus of model-made mice (about 10%), whereas Neun-positive neurons were significantly reduced after administration of dry dose, and increased to about 52%. Fig. 2B, E shows that the expression of CD31 in the model-making group was doubled compared to the normal control group, and the expression of CD31 in the model-making administration group was more significantly increased, which was about 7.5 times that in the normal control group. Fig. 2C, F shows that VEGFR2 in the normal administration group was slightly increased to about 1.2 times that in the normal control group, VEGFR2 expression in the molding group was significantly increased to about 2.3 times that in the normal control group, and VEGFR2 expression in the molding group was significantly increased to about 4 times that in the normal control group, as compared with the normal control group. The dry administration prognosis can reduce the death of neurons and promote angiogenesis.

Example 7: performing a western blot experiment;

reagent: BCA protein concentration determination kit (Biyuntian Biotech Co., Ltd.), rabbit anti-ZO-1 (1:1000, abcam, USA), rabbit anti-Claudin 5(1:1000, abcam, USA), rabbit anti-beta-actin (1:1000, Santa Cruz Biotechnology, USA), horseradish peroxidase-labeled secondary antibody: goat anti-rabbit-HRP (1:3000, Santa Cruz, USA);

in this section, the animal model described in example 2 was used, and after 24h of cerebral ischemia reperfusion, mice were anesthetized and sacrificed, brains were removed, and hippocampal regions on the ischemic side were isolated and placed on ice. The semi-dark band tissue was fully lysed using the whole protein extraction kit. After centrifugation, the supernatant was taken up, and the protein concentration was determined according to the BCA protein concentration determination kit, so that each sample was adjusted to an equal amount of protein (40. mu.g). Then adding corresponding reducing Loading buffer into the sample, and boiling in a water bath kettle at 100 ℃ for 5min to denature the protein. The denatured proteins were separated by electrophoresis on a 10% SDS-PAGE gel and then transferred to a PVDF membrane. To prevent non-specific protein binding sites from binding to the antibody, the PVDF membrane was blocked in TBS containing 0.1% Tween 20 and 5% BSA for 90 min. And cutting the PVDF membrane according to the corresponding molecular weight, respectively incubating with the rabbit anti-ZO-1, Claudin5 and beta-actin, and performing overnight refrigeration at 4 ℃. Incubation with the corresponding secondary antibody was performed for 2h at room temperature, and then the PVDF membrane was exposed to the hypersensitive ECL chemiluminescent reagent and visually analyzed by the Bio-Rad automated gel imaging system. Finally, grey value analysis of the bands was performed using Image Pro Plus 6.0 to assess the relative expression levels of the proteins.

As shown in FIG. 3, the expression of Claudin5 and ZO-1 in the normal administration group was slightly increased compared with the normal control group, the expression of ZO-1 and Claudin5 in the modeling group was significantly reduced, and the expression of ZO-1 and Claudin5 in the modeling group was significantly increased compared with the modeling group. Indicating that the post-administration prognosis can preserve the integrity of the blood brain barrier.