CN116173096B - Pharmaceutical composition for treating cerebral apoplexy and preparation method and application thereof - Google Patents

Abstract

The invention provides a pharmaceutical composition for treating cerebral apoplexy, which is a preparation prepared from the following raw materials in parts by weight: 8-14 parts of Antrodia camphorata, 8-14 parts of earthworm, 8-14 parts of radix angelicae, and 4-8 parts of liquorice. The invention also provides a preparation method and application of the pharmaceutical composition. The medicine has the effects of regulating angiogenesis after cerebral ischemia to play a role in neuroprotection, improving limb movement dysfunction after cerebral apoplexy, and has precise and appropriate formula compatibility and obvious effect, and particularly has better formula effect under the same dosage as Antrodia camphorata.

Description

Pharmaceutical composition for treating cerebral apoplexy and preparation method and application thereof

Technical Field

The invention relates to a pharmaceutical composition for treating cerebral apoplexy, and belongs to the field of traditional Chinese medicines.

Background

Cerebral stroke is the second most fatal and third most disabling disease worldwide, and one of its main types is ischemic stroke. The occurrence of ischemic necrosis of brain tissue and the like due to occlusion or injury of cerebral blood vessels is a main cause of the occurrence of ischemic stroke. In recent years, cerebral apoplexy is not only easy to occur in middle-aged and elderly people, but also the incidence rate in young people is increased gradually, and the physical and mental health of people is seriously damaged by limb movement dysfunction caused by sequelae. Therefore, rehabilitation after cerebral apoplexy becomes one of the problems that needs to be overcome urgently.

After cerebral ischemia of a patient suffering from ischemic stroke for a certain period of time, cerebral blood flow can resume reperfusion, thereby causing serious damage or dysfunction of brain cells, which is called cerebral ischemia/reperfusion injury, and is one of important pathophysiological mechanisms of the ischemic stroke. Recent studies have found that one of the important factors in the progression of ischemic stroke is vascular injury following ischemia. If the new collateral circulation can be established as soon as possible, the angiogenesis in the ischemic area is promoted, the local blood flow supply is restored, not only the endangered nerve cells in the ischemic penumbra can be saved, but also a good microenvironment can be created for the plasticity of the nerve structure, the damaged nerve function can be helped to be restored, the prognosis of a patient is improved, and the angiogenesis is prompted to have important significance for the treatment of ischemic cerebral apoplexy. Angiogenesis is the formation of new vascular networks based on existing vascular networks through the steps of proliferation, migration, sprouting, etc. of vascular Endothelial Cells (ECs), thereby adapting to the biological processes of local functions. Although compensatory angiogenesis can occur in the body after cerebral ischemia, the process is slow and the effect is weak, and the defect of the nerve function after cerebral ischemia is not enough improved. Therefore, kumar D and other first-time therapeutic angiogenesis theory, also called molecular bypass, promotes angiogenesis and repairs damaged blood vessels through a therapeutic means, thereby increasing local blood supply and improving ischemic and anoxic damage, and is a potential important target point and a new strategy for clinically treating ischemic cerebral apoplexy.

Antrodia camphorata, also called Antrodia camphorata, belongs to the order of non-pleated fungus, polyporaceae, genus Hymenomycetes, perennial fungus, and Lawsonia inermis Ding Ming is Antrodia camphorata, which is a new species published by the biochemical world in 1990. Researches show that polysaccharide, terpenes, steroid compounds, maleic acid, succinic acid derivatives and the like are main active compounds of Antrodia camphorata, and have the effects of protecting liver, resisting cancer, regulating immunity, removing allergy, detoxifying, resisting inflammation and the like. It has been reported that for treating various cardiovascular and cerebrovascular diseases (Antrodia camphorata can reduce cholesterol and fat content in blood, adenosine can reduce coagulation function of blood platelet), thus can treat various cardiovascular and cerebrovascular diseases such as hypertension, hypotension, arteriosclerosis, thrombosis, myocardial infarction, cerebral apoplexy, and angina pectoris. In combination with the classification standard of Chinese medicinal materials in Chinese medicine, antrodia camphorata can be classified as a heat-clearing and toxin-removing medicine, and the nature and taste of Antrodia camphorata are pungent, bitter and slightly sweet, and are cold-nature medicines; the Chinese medicinal composition has the effects of clearing heat and detoxicating, eliminating carbuncles and resolving hard mass, relieving sore throat, clearing liver and purging fire, removing blood stasis and relieving pain. Therefore, the effect of Antrodia camphorata cannot be exerted by the single use, and the clinical use has certain limitation.

Disclosure of Invention

The technical scheme of the invention provides a novel pharmaceutical composition for treating cerebral apoplexy, and a preparation method and application of the pharmaceutical composition.

The invention provides a pharmaceutical composition for treating cerebral apoplexy, which is a preparation prepared from the following raw materials in parts by weight:

8-14 parts of Antrodia camphorata, 8-14 parts of earthworm, 8-14 parts of radix angelicae, and 4-8 parts of liquorice.

Further preferably, the preparation is prepared from the following raw materials in parts by weight:

12 parts of antrodia camphorata, 12 parts of earthworm, 12 parts of dahurian angelica root and 6 parts of liquorice.

The pharmaceutical composition is a preparation prepared by taking crude drug powder of Antrodia camphorata, earthworm, dahurian angelica root and liquorice, water or organic solvent extract as active ingredients and adding pharmaceutically acceptable auxiliary materials or auxiliary components.

Wherein the preparation is tablet, capsule, granule, pill, oral liquid, powder.

The invention also provides a method for preparing the pharmaceutical composition, which comprises the following steps:

a. taking the raw materials in each weight proportion;

b. pulverizing Antrodia camphorate into fine powder, and sieving; decocting Lumbricus, radix Angelicae Dahuricae and Glycyrrhrizae radix in water, filtering, mixing filtrates, concentrating the filtrate to obtain fluid extract, adding Antrodia Camphorata fine powder and pharmaceutically acceptable adjuvants or auxiliary components;

or (b)

b. Pulverizing Antrodia Camphorata, lumbricus, and radix Angelicae Dahuricae into fine powder, and sieving; decocting Glycyrrhrizae radix in water, concentrating the decoction, adding fine powder of Antrodia Camphorata, lumbricus and radix Angelicae Dahuricae, and adding pharmaceutically acceptable adjuvants or auxiliary components.

The invention provides application of the pharmaceutical composition in preparing medicines with the effects of activating blood, tonifying qi, dredging collaterals and relieving spasm.

The invention provides application of the pharmaceutical composition in preparing medicines for treating cerebral apoplexy.

Wherein the medicine is a medicine for treating ischemic cerebral apoplexy.

The invention provides application of the pharmaceutical composition in preparing medicines for promoting angiogenesis.

The invention also provides application of the pharmaceutical composition in preparing medicines for improving limb movement dysfunction after cerebral apoplexy.

The antrodia camphorate is used as a monarch drug in the raw material formula of the invention for activating blood circulation to dissipate blood stasis and replenishing qi to invigorate the spleen; lumbricus is good at relieving spasm, dredging meridian passage, and is ministerial drug; radix angelicae is used as an adjuvant drug for dispelling wind and relieving pain; so that the liquorice root is used for harmonizing various medicines. The whole formula is compatible, deficiency and excess are treated simultaneously, and the effects of activating blood, tonifying qi, dredging collaterals and relieving spasm are achieved. The medicine has the effects of regulating angiogenesis after cerebral ischemia to play a role in neuroprotection, improving limb movement dysfunction after cerebral apoplexy, and has the advantages of precise and appropriate formula compatibility and obvious effect, and particularly has better formula effect under the same dosage as Antrodia camphorata.

Drawings

FIG. 1 sets of HUVEC migration competence (x 50)

FIG. 2 comparison of the vascularity of the HUVEC groups (. Times.50)

FIG. 3 comparison of cerebral infarct volumes in groups of rats

FIG. 4 shows pathological changes of cerebral cortex tissue on the ischemic side (. Times.200) of rats in each group (note: the black arrow indicates that no obvious pathological changes are seen in the Sham group of rat cerebral cortex nerve cells, the yellow arrow indicates that nerve cell destruction occurs in the cerebral cortex on the ischemic side of the MCAO group of rat, and the nucleus is contracted and stained dark blue.)

FIG. 5 comparison of the number of Nissl positive cells of cerebral cortex on ischemia side of each group of rats (. Times.400) (note: a: nissl staining pattern of each group of rats; b: histogram of Nissl staining statistics of each group of rats: note: P <0.05vs the Sham group; #P <0.05, #P <0.01vs the MCAO group.)

FIG. 6 comparison of Tunel positive cell rates of rat cerebral cortex groups (. Times.400) (note: a: tunel staining pattern of rat groups; b: column chart of Tunel staining statistics of rat groups: note: P <0.01vs the Sham group; #P <0.05, #P <0.01vs the MCAO group.)

FIG. 7 comparison of cerebral cortex vascularity on ischemic side of rats in each group (. Times.200)

FIG. 8 comparison of the density of the new micro-blood vessels of the cerebral cortex on the ischemic side of each group of rats (. Times.200)

Detailed Description

EXAMPLE 1 preparation of the pharmaceutical granules of the invention

The formula comprises the following components: antrodia camphorate 12g, earthworm 12g, dahurian angelica root 12g, licorice root 6g

The preparation method comprises the following steps: pulverizing Antrodia camphorate into fine powder, and sieving; decocting Lumbricus, radix Angelicae Dahuricae and Glycyrrhrizae radix with 10 times of water for 2 times each for 1.5 hr, filtering, mixing filtrates, concentrating the filtrate to obtain fluid extract with relative density of 1.30 (30deg.C), adding Antrodia Camphorata fine powder and dextrin, mixing, granulating, drying at 50-60deg.C, and making into granule.

EXAMPLE 2 preparation of the medicament of the invention

The formula comprises the following components: antrodia camphorate 12g, earthworm 12g, dahurian angelica root 12g, licorice root 6g

Pulverizing Antrodia Camphorata, lumbricus and radix Angelicae Dahuricae into fine powder, and sieving; decocting Glycyrrhrizae radix with water for 2 times, adding fine powder of Antrodia Camphorata, lumbricus and radix Angelicae Dahuricae, and stirring.

EXAMPLE 3 preparation of the pharmaceutical capsules of the invention

The formula comprises the following components: 14g of Antrodia camphorate, 14g of earthworm, 14g of radix angelicae and 8g of liquorice are prepared into particles according to the method of the example 1 and are filled into capsules to prepare capsules.

EXAMPLE 4 preparation of the pharmaceutical powder of the present invention

Taking the raw materials: 14g of Antrodia camphorate, 14g of earthworm, 8g of radix angelicae and 8g of liquorice, and drying to obtain powder.

The beneficial effects of the invention are demonstrated by specific pharmacodynamic tests.

Experimental example 1 Effect of the drug of the present invention on HUVEC angiogenesis after OGD/R

1 Experimental materials

1.1 laboratory animals and cells

30 SPF-class healthy male SD rats with the body mass of (260+/-10) g are purchased from Shanghai Laike laboratory animal liability Limited (production license: SCXK (Shanghai) 2017-0005) and are fed to the Fujian university laboratory animal center (qualification: SYXK (Min) 2019-0007); SV40T transformed human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVEC) purchased from the GmbH (cat# CL-0675).

1.2 Experimental drugs

Antrodia camphorate (university of Fujian traditional Chinese medicine college, 210228); radix Angelicae Dahuricae (decoction pieces of Chinese medicinal materials, inc., 210515); earthworm (Anhuifeng Huifeng national medicine Co., ltd., 201233); licorice (Anhuifeng Huifeng national medicine Co., ltd., 20210201).

1.3 laboratory apparatus

Table 1 laboratory apparatus

1.4 Experimental reagent

Table 2 experimental reagents

2 Experimental methods

2.1 preparation of the serum containing the drugs in the formulation

30 rats fed adaptively for 7d were randomly divided into 3 groups of 10 rats each, and each group was filled with drinking water, formula (prepared in example 1) (8.4 g/kg), antrodia camphorata (8.4 g/kg), and administered continuously for 7d 1 time/d. 2h after the last administration, the rats were anesthetized with sodium pentobarbital (60 mg/kg), and the abdominal aorta was sampled. After the blood sample was allowed to stand on ice for 3 hours, it was centrifuged at 3500rpm for 15 minutes, and the supernatant was collected and filtered at 0.22. Mu.m. Inactivating in water at 60deg.C for 15min to obtain composition-H group drug-containing serum with concentration of 10%, composition-M group drug-containing serum with concentration of 5%, composition-L group drug-containing serum with concentration of 2.5% and Antrodia camphorate group drug-containing serum with concentration of 10%, and supplementing with blank serum with serum concentration less than 20%.

2.2 cell resuscitation and culture

HUVEC were removed from the liquid nitrogen tank and thawed in a 37℃water bath. The thawed HUVEC was transferred to a 15mL centrifuge tube, appropriate amount of medium was added, and centrifuged at 1000rpm for 5min. The upper liquid is discarded, DMEM high-sugar complete medium containing 2mL of 10% FBS is added, the medium is transferred to 2 cell culture flasks of 25cm < 2 >, 5mL of complete medium is added to each culture flask, and the culture flask is placed in a 5% CO2 incubator at 37 ℃ for culture, and the medium is changed for 1 time every day. HUVEC morphology: the cells are flat, polygonal or fusiform, and the edges of the cells are serrated and are mutually embedded.

2.3 passage of cells

When the HUVEC monolayer fusion reached 80%, the medium was discarded, washed 3 times with PBS, and digested with 2mL pancreatin; after digestion for 3min, observation under a microscope: after cytoplasmatic retraction and cell gap increase, the culture flask is shaken to make cells fall off. After the cells were detached from the flask wall, digestion was terminated by adding 4mL of complete medium, centrifuging at 1000rpm for 5min, discarding the supernatant, adding 2mL of new complete medium, and blowing uniformly, 1:2, subculturing in bottles, culturing in a 5% CO2 incubator at 37 ℃ for two days, and changing the liquid once. The cell monolayer was continued to passaged when it reached 80%.

2.4 preparation of an oxygen sugar deprivation/reoxygenation (oxygen glucose deprivation/reoxygenation, OGD/R) model

HUVEC are inoculated into a 6, 24 or 96-well plate, placed in an incubator for culturing until cells are in a 80% single-layer fusion state, the culture solution is discarded, washed for 3 times by PBS, a DMEM sugar-free culture medium is added, then the HUVEC is placed in a three-gas incubator for culturing for 4 hours at 37 ℃ in an incubator with 1% O2, 94% N2 and 5% CO2 environment, and OGD treatment is carried out to simulate ischemic injury. After 4 hours, the DMEM sugarless medium was aspirated, the medium was replaced with complete medium after PBS washing, the culture was continued in a CO2 incubator at 37℃and the proliferation of HUVEC was examined by CCK-8 to reflect the degree of OGD damage.

2.5CCK-8 method for detecting HUVEC proliferation

HUVECs were inoculated at 1X 105/well into 96-well plates, sugar-free medium was changed, and OGD/R treatment was performed according to the 2.4 experimental procedure. Removing sugar-free culture medium after molding, and culturing in CO2 incubator for 24 hr with high sugar culture medium containing different concentration of medicinal serum (the medicinal serum concentration of the formula is 2.5%, 5%, 10%, and the serum concentration is less than 20% with blank serum); a group of normal control groups (20% of blank serum is added) and a group of blank culture medium groups (without cells) are used as blank groups, and neither group participates in modeling, and the culture is carried out in a CO2 incubator for 24 hours. After the completion of the culture, 10. Mu.L of CCK-8 solution was added to each group of wells in the absence of light, incubated in a CO2 incubator in the absence of light for 2 hours, and after 2 hours, the OD value at 450nm was measured by using an ELISA reader to calculate the cell proliferation activity. And selecting the optimal intervention formula drug-containing serum concentration for subsequent experiments according to the detection result of proliferation activity.

Cell proliferation rate (%) = [ (dosing well OD value-blank medium well OD value)/(normal control well OD value-blank medium well OD value) ] ×100%

2.7 Experimental grouping

The HUVEC was divided into blank (Control), model (OGD/R), formula-L, formula-M, formula-H and Antrodia camphorata groups.

2.8 scratch test

HUVECs were inoculated into 24 well plates at 1X 105/well, OGD/R treatment was performed for 4h, a scratch was made at the centerline position of each well, PBS was added and gently washed 3 times to remove floating HUVECs, and the HUVECs were prevented from falling off and continued to grow at the scratch. After the scratch was scratched, the scratch width was photographed under an optical microscope, recorded, and measured as a baseline value. And after photographing, adding a complete culture medium containing the medicine-containing serum of the formula, culturing in a CO2 incubator for 8 hours, photographing again after 8 hours, and recording the width of the scratch area, wherein the migration distance of the HUVEC is obtained by subtracting the two times.

2.9 angiogenesis experiments

Matrigel was frozen and thawed overnight on ice the day prior to the experiment. Other items required for the experiment were also pre-cooled and all operations were performed on ice. 200 μl of the carefully aspirated matrigel was added to a 24-well plate and the plate was gently shaken to allow the glue to lay flat, taking care to avoid air bubbles. Placing the culture plate paved with the matrigel into a CO2 incubator horizontally, and standing for 30min at 37 ℃ until the matrigel solution is solidified. HUVECs and HUVECs transfected with inhibitors were inoculated at 1X 105 cells/well into matrigel-plated well plates, OGD/R treated for 4h, complete medium with drug-containing serum was added to the formulation, incubated in a CO2 incubator for 8h, and the vessel formation of HUVECs was observed and photographed under an optical microscope, and analyzed using Image J Image software.

2.13Elisa method for detecting eNOS Activity in HUVEC

After 24h of formula intervention, cells were collected from the well plate, disrupted using an ultrasonic disrupter, centrifuged at 1500rpm for 10min at 4℃and assayed for eNOS activity according to the kit instructions.

2.14Elisa method for detecting NO content in HUVEC

After the formula is interfered for 24 hours, cells are collected from a pore plate, centrifuged at 1000rpm for 10 minutes, the supernatant is discarded, PBS is used for washing twice, the cells are subjected to ultrasonic disruption in ice water bath, and after the cells are disrupted, the NO content is detected according to the instruction of the kit.

2.15 statistical analysis

By mean ± standard deviationExperimental results are shown and statistically analyzed using SPSS 26.0. The experimental results were analyzed by non-parametric tests (Kruskal-Wallis test) if the normal distribution was not met, by single-factor analysis of variance (LSD test) if the normal distribution was met and the variance was not met, and by single-factor analysis of variance (gas-Howell test) if the normal distribution was met and the variance was not met. The statistical result has statistical significance by taking P <0.05 as a difference.

3 results of experiments

3.1 Effect of the formulation on HUVEC proliferation Activity after OGD/R4 h

The proliferation activity of HUVEC was examined in this experiment using CCK-8. As shown in table 3: after OGD/R modeling, the cell proliferation rate of the OGD/R group is obviously reduced (P is less than 0.01) compared with that of the Control group; compared with the OGD/R group, the cell proliferation rate of HUVEC after the prescription drug-containing serum stem prognosis is obviously increased (P is less than 0.05 or 0.01), and is obviously higher than that of the Antrodia camphorata group singly used.

TABLE 3 Effect of the formulations on HUVEC proliferation Activity

And (3) injection: ^** P<0.01vs the Control group； ^# P<0.05vs the OGD/R group； ^## P<0.01vs the OGD/R group；

3.2 Effect of the formulation on HUVEC migration ability after OGD/R4 h

The migration ability of HUVEC was tested in this experiment using the scratch method. See table 4 and fig. 1: after 8h culture, the HUVEC scratches in the Control group are overgrown and covered again, which shows that the growth state of the HUVEC is good; after OGD/R molding, the scratch distance of the OGD/R group is not obviously reduced; after the intervention of the formula, compared with the OGD/R group, the migration distance of scratches is obviously increased (P is less than 0.05), which shows that the formula has the capability of obviously promoting the migration of HUVEC, and the migration distance is obviously higher than that of the Antrodia camphorata group singly used.

Table 4 comparison of HUVEC migration Capacity for groups

And (3) injection: ^** P<0.01vs the Control group； ^## P<0.01vs the OGD/R group

3.3 Effect of the formulation on HUVEC vascularization ability after OGD/R4 h

The angiogenic capacity of HUVECs was tested in this experiment using matrigel. As shown in table 5, fig. 2: HUVEC in the Control group has remarkable vascularization capability, and the formed blood vessels are netlike, have obvious nodes and can see the lumen structure; HUVEC in OGD/R group had significantly reduced vascularization (P < 0.01), discontinuous vascularization, invisible nodes compared to Control group; after the prescription is dried, the HUVEC vascularization capacity in the prescription is obviously improved (P is less than 0.01), blood vessels gradually appear in a net shape, a small number of nodes are visible, and the prescription is suggested to have the function of promoting vascularization. And the angiogenesis promoting effect of the formula is obviously stronger than that of the Antrodia camphorata singly used.

Table 5 comparison of HUVEC angiogenic potential of groups

And (3) injection: ^** P<0.01vs the Control group； ^# P<0.05vs the OGD/R group； ^## P<0.01vs the OGD/R group；

3.4 Effect of the formulation on eNOS Activity in HUVEC after OGD/R4 h

The eNOS activity was detected in this experiment by the Elisa method. As shown in table 6: the eNOS activity in the OGD/R group was significantly increased (P < 0.01) compared to the Control group; after the intervention of the formula, compared with the OGD/R group, the eNOS activity of the formula is obviously increased (P is less than 0.01), which indicates that the eNOS activity of the formula can be improved, and the formula is obviously higher than that of an antrodia camphorata group singly used.

Table 6 comparison of eNOS Activity in HUVECs of groups

And (3) injection: ^** P<0.01vs the Control group； ^## P<0.01vs the OGD/R group；

3.6 Effect of the formulation on NO content in HUVEC after OGD/R4 h

The experiment adopts an Elisa method to detect the NO content. As shown in table 7: the NO content in the HUVEC of the Control group is extremely low; compared with the Control group, the NO content in the OGD/R group is obviously increased (P < 0.01); after the intervention of the formula, compared with the OGD/R group, the NO content in the formula group is obviously increased (P is less than 0.01), which indicates that the formula can promote ECs to release NO, and the formula is obviously higher than that of an Antrodia camphorata group singly used.

TABLE 7 comparison of NO content in HUVECs of groups

And (3) injection: ^** P<0.01vs the Control group； ^# P<0.05, ^## P<0.01vs the OGD/R group；

test example 2 neuroprotective effect of the inventive drug on cerebral ischemia/reperfusion injury rats

1 Experimental materials

1.1 laboratory animals

105 healthy male SD rats (260+ -10 g) with no specific pathogen (specific pathogen free, SPF) grade were selected, purchased from Shanghai Laek laboratory animal liability Co., ltd (production license: SCXK (Shanghai) 2017-0005), and fed to Fujian university laboratory animal center (certificate: SYXK (Min) 2019-0007). The study is approved by the ethical committee of animals of Fujian university, ethical examination number: FJTCM IACUC 2021087.

1.2 Experimental drugs

Antrodia camphorate (university of Fujian traditional Chinese medicine college, 210228); radix Angelicae Dahuricae (decoction pieces of Chinese medicinal materials, inc., 210515); earthworm (Anhuifeng Huifeng national medicine Co., ltd., 201233); licorice (Anhuifeng Huifeng national medicine Co., ltd., 20210201).

1.3 laboratory apparatus

Table 8 laboratory apparatus

1.4 Experimental reagent

Table 9 Experimental reagent

2 Experimental methods

2.1 preparation of MCAO model rats

Rat middle cerebral artery occlusion (middle cerebral artery occlusion, MCAO) model was prepared using the wire-plug method: after the rats were fasted and not water-inhibited for 12 hours, sodium pentobarbital (60 mg/kg) was anesthetized and fixed in the supine position. The common carotid artery (common carotid artery, CCA), internal carotid artery (internal carotid artery, ICA) and external carotid artery (external carotid artery, ECA) were isolated blunt, with a incision along the cervical midline of the rat. Ligating the CCA and ECA, and clamping the ICA with an arterial clamp. A small opening is cut at the proximal end of the CCA, a wire plug is inserted until the black point on the wire plug completely enters the ICA to plug the middle cerebral artery, and the wire plug is partially pulled out after 2 hours to realize reperfusion. After the rats were awake, the rats were subjected to neurological scoring and myotonic ratings by referring to the modified neurological scoring method (modified neurological severity score, mNSS) and Ashworth ratings method [56-57], and those with neurological scores of 8 to 16 points and myotonic ratings of no less than 1 were considered to be successful in molding (the scoring was completed independently by two persons who did not participate in MCAO model preparation but had scoring experience at the same time).

2.2 neurological deficit scoring

The experiments were scored for neurological deficit in groups of rats at 1, 3, 5, and 7d, respectively, using mNSS scoring. The scoring is composed of 0 to 18 points, the higher the score is, the higher the nerve function defect degree is, and the scoring method comprises a sensory experiment, a motor experiment, a balance beam experiment, a reflection loss and an abnormal motor experiment, and the rat nerve function defect condition is comprehensively evaluated.

2.3 muscle tension rating

The experiment uses a modified Ashworth rating method to rate the degree of hemiparalysis side muscle cramp in each group of rats at 1, 3, 5, and 7d, respectively.

2.4 grouping and administration

Rats successfully modeled were randomly divided into 4 groups: model group (MCAO group), low, medium and high dose group (group-L, group-M, group-H) (prepared in example 1), antrodia camphorata group; a set of Sham surgical groups (Sham operation group, sham; the rest of the procedure was identical to the modeling group except that the middle cerebral artery was not occluded), 12 per group. All groups of rats are administrated by drenching according to the mass of 10mL/kg after grading on the day of operation, the administration doses of the formula-L, M, H groups are respectively 2.1g/kg, 4.2g/kg and 8.4g/kg, the administration doses of the Antrodia camphorata group are respectively 2.4g/kg, and drinking water is administrated by both the sham operation group and the model group for 1 d/time and 7d continuously.

2.5MRI detection of cerebral infarct volume

After the last dose, each group of rats was anesthetized with isoflurane and the brain of the rat was scanned with T2-weighted imaging using MRI. Parameter setting: the sequence of use: TSE; scanning thickness: 1mm; scanning the number of layers: 21 layers; the field of view: 32mm by 32mm; average signal number: 2; scanning time: 4min 28s800ms. Rat cerebral infarction volumes were determined using Image J analysis.

2.6 preparation of Paraffin sections of tissue

After the MRI scan, the rats were anesthetized and the hearts were perfused with physiological saline and 4% paraformaldehyde. And rapidly cutting the head and taking the brain after the limb of the rat is stiff, and fixing the brain tissue for 24 hours under the condition of 4% paraformaldehyde and 4 ℃. After the brain tissue was fixed, it was rinsed with ultrapure water until no irritating odor was obtained, and was cut into 2mm coronal sections with a rat brain model, followed by gradient ethanol dehydration using an automatic biological tissue dehydrator. The dehydration and permeabilization sequence is as follows: 75% ethanol (24 h) →80% ethanol (1 h) →90% ethanol (1 h) →95% ethanol (1 h) →absolute ethanol I (40 min) →absolute ethanol II (40 min) →absolute ethanol III (40 min) →xylene I (30 min) →xylene II (20 min) →paraffin I (40 min) →paraffin II (40 min) →paraffin III (50 min). In the xylene permeabilization process, the permeabilization condition is observed at fixed time, and the permeabilization is stopped immediately after the whole tissue is transparent cutin. Paraffin embedding and slicing are carried out after gradient dehydration is completed. Cutting the tissue wax block into slices with the thickness of 4 mu m and 20 mu m, spreading the slices at the water temperature of 40 ℃, and fishing out the slices after the tissue is completely unfolded. And then baking the slices at 55 ℃, transferring to 65 ℃ for 8 hours after the baking of the slices is completed, and finally storing in a refrigerator at 4 ℃ for standby.

2.7 dewaxing and rehydration of Paraffin sections

The paraffin section is baked for 1h at 60 ℃, and then dewaxed and rehydrated in a biosafety cabinet, and the specific steps are as follows: xylene I (15 min), xylene II (15 min), absolute ethanol I (5 min), absolute ethanol II (5 min), 95% ethanol (5 min), 90% ethanol (5 min), 80% ethanol (5 min), 75% ethanol (5 min), ultrapure water (10 min), PBS buffer (10 min).

2.8HE staining method for observing pathological changes of cerebral cortex tissue on ischemic side

Dewaxing and rehydrating the paraffin sections with the size of 4 mu m, staining with hematoxylin dye solution at room temperature for 2min, and differentiating the differentiation solution for 2min after washing with ultrapure water. After differentiation, eosin dye solution was added dropwise for 1min, followed by rinsing with ultrapure water. And (5) after the flushing is finished, placing the mixture in a baking oven at 37 ℃ for baking, and finally sealing the sheet by using neutral resin. The pathological changes of the cerebral cortex tissue on the ischemic side of each group of rats were observed using an optical microscope, and images were taken under a 200-fold microscope.

2.9Nissl staining observations of the number of Nissl positive cells of the cerebral cortex on the ischemic side

Dewaxing and rehydrating the paraffin sections with the size of 4 mu m, and dripping Nib dye liquor at 57 ℃ for dyeing for 40min. After dyeing, washing with ultrapure water, differentiating for 10s with 95% ethanol, drying at 37 ℃, and sealing with neutral resin. The morphology and number of cerebral cortex nerve cells on the ischemic side of each group of rats were observed using an optical microscope, and images were taken under a 400-fold microscope and analyzed using Image J Image software.

2.10Tunel method for detecting apoptosis rate of cerebral cortex nerve cells on ischemia side

Dewaxing and rehydrating the paraffin sections with the size of 4 mu m, incubating with proteinase K and a balancing buffer solution at room temperature for 10min and 5min respectively, then dripping a reaction buffer solution, incubating for 2h at 37 ℃ in a dark place, dying nuclei by DAPI, and sealing the tablet by anti-fluorescence quenching. The ischemic side cortical neural cell apoptosis was observed and photographed under a 400-fold microscope using an inverted fluorescence microscope and analyzed using Image J Image software.

2.11 statistical analysis

By mean ± standard deviationExperimental results are shown and statistically analyzed using SPSS 26.0. The mNSS score and Ashworth rating results were analyzed using a non-parametric test (Kruskal-Wallis test); the rest experimental results are analyzed by adopting a non-parametric test (Kruskal-Wallis test) if the normal distribution is not met, a single-factor analysis of variance (LSD test) if the normal distribution is met and the variances are uniform, and a single-factor analysis of variance (gas-Howell test) if the normal distribution is met and the variances are not uniform. The statistical result has statistical significance by taking P <0.05 as a difference.

3 results of experiments

3.1 Effect of the formulation on neurological function and muscular tension in rats with cerebral ischemia/reperfusion injury

The experiments adopt a mNSS scoring method and an Ashworth scoring method to score the neurological deficit degree of the rats and rank the spasticity of limbs in the 1 st, 3 rd, 5 th and 7 th days after molding. As shown in tables 10 and 11, the mNSS score and Ashworth rating of the MCAO rats after molding are obviously increased (P is less than 0.01) compared with that of the Sham rats, which indicates that the preparation of the MCAO limb spasm rat model is successful; after drug intervention for 3, 5 and 7d, both the mNSS score and Ashworth rating of the rats in the formula-H group were significantly lower than those of the MCAO group (P <0.05 or 0.01), and the formula-L and formula-M groups were reduced but not significantly different (P > 0.05); the prescription is suggested to be capable of remarkably improving the nerve function injury and limb spasticity of the MCAO model rat. And is significantly stronger than the Antrodia camphorata group used alone.

Table 10 comparison of mNSS scores for groups of rats at different time points

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And (3) injection: ^** P<0.01vs the Sham group； ^## P<0.01vs the MCAO group；

table 11 comparison of the rats' muscle tension ratings for each group

And (3) injection: ^** P<0.01vs the Sham group； ^# P<0.05vs the MCAO group； ^## P<0.01vs the MCAO group；

3.2 Effect of the formulation on cerebral ischemia/reperfusion injury rat cerebral infarction volume

Experiments brain scans were performed on each group of rats using MRI after the last dose. The results are shown in Table 12 and FIG. 3, and the color and brightness of the brain tissue of the Sham group rats are uniform, and no infarct area is formed; compared with the Sham group, the brain tissue of the MCAO group rat has a large white area, which indicates that the cerebral infarction volume is obviously increased (P is less than 0.01); after 7d of formula intervention, compared with a model group, the cerebral infarction volume of rats in each dose group is obviously reduced (P is less than 0.01), which indicates that the formula can obviously reduce the cerebral infarction volume of MCAO rats. And the infarct size is significantly smaller than that of Antrodia camphorata alone.

Table 12 comparison of cerebral infarct volumes in groups of rats

And (3) injection: ^** P<0.01vs the Sham group； ^## P<0.01vs the MCAO group；

3.3 Effect of the formulation on pathological changes in cerebral cortex tissue on the ischemic side of cerebral ischemia/reperfusion injury rats

The experiment adopts HE staining method to observe the pathological changes of rat tissues. As shown in fig. 4, the rat brain cortex nerve cells of the Sham group have complete structure, clear outline, regular arrangement and no obvious pathological change; compared with the Sham group, the rat ischemia side cerebral cortex nerve cells of the MCAO group are damaged in a large amount and the cell nuclei are contracted and stained in dark blue; after 7d of formula intervention, the pathological changes of cerebral cortex nerve cells of the ischemic side of rats in each dose group are recovered to a certain extent. And the improvement effect is obviously stronger than that of the antrodia camphorate group singly used.

3.4 Effect of the formulation on the number of Nissl-positive cells in the cerebral cortex on the ischemic side of a rat with cerebral ischemia/reperfusion injury

Experiments Nissl positive cell numbers were measured for each group of rats using Nissl staining. As shown in table 13 and fig. 5, the morphology of the rat brain cortex Nissl positive cells of Sham group is clear and complete and the number is high; compared with the Sham group, the number of Nissl positive cells of the cerebral cortex of the ischemia side of the MCAO group rat is reduced (P is less than 0.05) and the morphological structure is contracted; after 7d of formula intervention, the number of Nissl positive cells of cerebral cortex on ischemia side of the rats in the formula-M, H group is obviously increased (P is less than 0.05 or 0.01), and the cell morphology is also improved. And compared with the Antrodia camphorate group which is singly used, the formula-H group significantly increases the number of Nissl positive cells.

Table 13 comparison of the number of Nissl positive cells in the cerebral cortex on the ischemic side of rats in each group

And (3) injection: ^* P<0.05vs the Sham group； ^# P<0.05vs the MCAO group； ^## P<0.01vs the MCAO group；

3.5 Effect of the formulation on apoptosis of cerebral cortex nerve cells on ischemic side of cerebral ischemia/reperfusion injury rats

The experiment adopts a Tunel method to detect the apoptosis rate of the rat nerve cells in each group. As shown in table 14, fig. 6, sham group rats had few apoptotic nerve cells; compared with the Sham group, the MCAO group rat has obviously increased Tunel positive cell rate (P < 0.01) of cerebral cortex on ischemia side, which indicates that nerve cells undergo massive apoptosis; after 7d of formula intervention, the rate of Tunel positive cells of cerebral cortex on the ischemia side of the rats in the formula-M, H group is reduced to different degrees (P is less than 0.05 or 0.01). group-H rats significantly reduced neuronal apoptosis compared to the antrodia group alone.

Table 14 comparison of the Tunel positive cell Rate of rat cerebral cortex of each group

And (3) injection: ^** P<0.01vs the Sham group； ^# P<0.05vs the MCAO group； ^## P<0.01vs the MCAO group；

experimental example 3 effect of formulation on angiogenesis after cerebral ischemia/reperfusion injury in rats

1 Experimental materials

1.1 laboratory animals

As above.

1.2 Experimental drugs

As above.

1.3 laboratory apparatus

Table 15 laboratory apparatus

1.4 Experimental reagent

Table 16 Experimental reagent

2 Experimental methods

2.1 gelatin-ink infusion

The rats were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg). After anesthesia, the heart was perfused with physiological saline and 4% paraformaldehyde, followed by 200mL of gelatin-ink solution (preparation of 200mL gelatin-ink solution: 8g gelatin, 8mL ink and 184mL heparin-containing physiological saline) preheated to 60℃at 55℃until the gelatin was completely dissolved, and filtered to obtain the final product. After the end of the perfusion, the ventral position of the rat was kept in a refrigerator at 4 ℃ and refrigerated for at least 2 hours until the gelatin was completely coagulated. The head is broken, the brain tissue is placed in 4% paraformaldehyde solution, and the brain tissue is fixed in a refrigerator at 4 ℃ for 24 hours.

2.2 preparation of gelatin-ink frozen sections

After fixation of the gelatin-ink infused brain tissue was completed, it was rinsed with ultrapure water until no irritating odor was obtained, and it was cut into 2mm coronal sections with a rat brain mold, followed by transfer into a 30% sucrose solution, and dehydration for 24 hours. And (3) placing the dehydrated brain tissue coating embedding agent on a freezing table of a frozen slicing machine for freezing, immediately cutting into slices with the thickness of 100 mu m after the freezing is finished, fishing out the slices in antifreeze solution for collection, and airing at room temperature for 8 hours. The air-dried sections were stained with eosin dye solution for 40s and then rinsed with ultrapure water. And (5) after the flushing is finished, placing the mixture in a baking oven at 37 ℃ for baking, and finally sealing the sheet by using neutral resin. The microvascular distribution and trend in the rat cerebral cortex tissues of each group was observed and photographed under 200-fold mirror using an optical microscope, and analyzed using Image J Image software.

2.3 detection of ischemia-side cerebral cortex neogenesis microvascular Density by immunohistochemical method

Dewaxing and rehydrating the paraffin sections with the diameter of 20 mu m, immersing the paraffin sections in sodium citrate antigen retrieval liquid which is heated to a micro-boiling state in advance after rehydration, and heating the paraffin sections with low fire in a microwave oven for 15min. After antigen retrieval, sections were removed and washed with PBS after recovery to room temperature. And (3) dripping endogenous peroxidase blocking liquid to block for 30min after the washing is finished, and washing with PBS. 5% BSA was added dropwise to cover brain tissue completely and blocked at room temperature for 2h. After blocking, BSA was discarded, no washing was performed, and a pre-formulated CD34 primary antibody (antibody dilution ratio 1:200) was added dropwise, and incubated overnight in a refrigerator at 4 ℃. The next day the sections were removed and washed with PBS and then run with immunohistochemical kit, and secondary antibody (incubation at room temperature for 2 h) and SABC solution (incubation at room temperature for 1 h) were added dropwise, respectively. After the incubation, PBS is used for washing again, and the DAB chromogenic kit is used for chromogenic. After the color development is completed, the solution is washed by PBS, hematoxylin is dripped to counterstain the nucleus, differentiation liquid is dripped to carry out blue reflection, and finally sealing is carried out. The density of new-born microvessels in rat cerebral cortex tissue was observed and photographed under 200-fold mirror using an inverted fluorescence microscope, and analyzed using Image J Image software.

2.4 statistical analysis

By mean ± standard deviationExperimental results are shown and statistically analyzed using SPSS 26.0. The experimental results were analyzed by non-parametric tests (Kruskal-Wallis test) if the normal distribution was not met, by single-factor analysis of variance (LSD test) if the normal distribution was met and the variance was not met, and by single-factor analysis of variance (gas-Howell test) if the normal distribution was met and the variance was not met. The statistical result has statistical significance by taking P <0.05 as a difference.

3 results of experiments

3.1 Effect of the formulation on the distribution of cerebral cortex microvasculature on the ischemic side of cerebral ischemia/reperfusion injury rats

The experiment adopts a gelatin-ink perfusion method to detect the distribution of cerebral cortex microvessels on the ischemia side of each group of rats. The results are shown in Table 17, FIG. 7: the number of capillaries in the cerebral cortex of the Sham group rat is more, and the filling performance is better; compared with the Sham group, the micro-vascular distribution area in the cerebral cortex of the ischemia side of the MCAO group rat is obviously reduced (P is less than 0.01), which indicates that the micro-vascular of the cortex is greatly destroyed after molding; after 7d of formula intervention, the distribution area of the microvessels in the cerebral cortex of the ischemic side of the rats of each administration group is increased to different degrees (P <0.05 or 0.01). And the increase of the microvascular density is significantly stronger than that of Antrodia camphorata alone.

Table 17 comparison of cerebral cortex vascularity on ischemic side of rats of each group

And (3) injection: ^** P<0.01vs the Sham group； ^# P<0.05vs the MCAO group； ^## P<0.01vs the MCAO group；

3.2 Effect of the formulation on the Density of the neo-microvasculature of the cerebral cortex on the ischemic side of the ischemia/reperfusion injury rats

The experiment adopts an immunohistochemical method to detect the density of cerebral cortex microvessels on the ischemia side of each group of rats. The results are shown in Table 18, FIG. 8: the density of the new micro-blood vessels of the cerebral cortex on the ischemia side of the MCAO group rat is obviously increased (P is less than 0.01) compared with that of the Sham group; after 7d of formula intervention, the density of the cerebral cortex neogenesis microvessels of each administration group of rats is obviously increased (P is less than 0.05 or 0.01), which shows that the formula can obviously promote the angiogenesis after cerebral ischemia/reperfusion injury. And the increase of the microvascular density is significantly stronger than that of Antrodia camphorata alone.

Table 18 comparison of the densities of the cerebral cortex neo-capillaries on the ischemic side of rats in each group

And (3) injection: ^** P<0.01vs the Sham group； ^# P<0.05vs the MCAO group； ^## P<0.01vs the MCAO group。

3.3 influence of the formulation on related proteins such as vascular endothelial growth factor in cerebral ischemia/reperfusion injury rats

The experiment adopts a Western blot method to detect the relative expression quantity of VEGF and VEGFR2 proteins. As shown in table 19: compared with the Sham group, the expression of VEGF and VEGFR2 proteins in the MCAO group is obviously increased (P is less than 0.05), which indicates that the angiogenesis process starts to be activated after cerebral ischemia; after 7d of formula intervention, the expression level of VEGF and VEGFR2 proteins in the formula is obviously increased (P is less than 0.05 or 0.01) compared with that in the MCAO group, which shows that the formula can promote the expression of VEGF and VEGFR2 proteins after cerebral ischemia and promote angiogenesis.

Table 19 comparison of the expression levels of VEGF, VEGFR2 protein in the cerebral cortex on the ischemic side of rats of each group

And (3) injection: ^* P<0.05vs the Sham group； ^# P<0.05vs the MCAO group； ^## P<0.01vs the MCAO group；

3.4 Effect of the formulation on the NO content of vasodilator in brain ischemia/reperfusion injured rats

The experiment adopts an Elisa kit method to detect the content of NO. As shown in table 20: sham group rats had very low levels of NO in the ischemic side of the cerebral cortex; the NO content in the MCAO group was significantly increased (P < 0.05) compared to Sham group; after 7d of formula intervention, compared with the MCAO group, the NO content in the formula is obviously increased (P is less than 0.05), which indicates that the formula has the function of promoting vascular endothelial cells to generate NO.

Table 20 comparison of NO content in cerebral cortex on ischemic side of rats of each group/>

And (3) injection: ^* P<0.05vs the Sham group； ^# P<0.05vs the MCAO group。

Claims (6)

1. a pharmaceutical composition for treating ischemic stroke, which is characterized in that: the preparation is prepared from the following raw materials in parts by weight:

12 parts of antrodia camphorate, 12 parts of earthworm, 12 parts of radix angelicae, and 6 parts of liquorice;

the medicine has the effects of activating blood, tonifying qi, dredging collaterals and relieving spasm; the medicine is a medicine for promoting angiogenesis.

2. The pharmaceutical composition according to claim 1, wherein: the preparation is prepared by taking crude drug powder of Antrodia camphorata, earthworm, dahurian angelica root and liquorice, water or organic solvent extract as active ingredients and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

3. The pharmaceutical composition according to claim 2, wherein: the preparation is tablet, capsule, granule, pill, oral liquid, powder.

4. A process for preparing a pharmaceutical composition according to any one of claims 1 to 3, comprising the steps of:

a. taking the raw materials in each weight proportion;

b. pulverizing Antrodia camphorate into fine powder, and sieving; decocting Lumbricus, radix Angelicae Dahuricae and Glycyrrhrizae radix in water, filtering, mixing filtrates, concentrating the filtrate to obtain fluid extract, adding Antrodia Camphorata fine powder and pharmaceutically acceptable adjuvants or auxiliary components;

or (b)

b. Pulverizing Antrodia Camphorata, lumbricus, and radix Angelicae Dahuricae into fine powder, and sieving; decocting Glycyrrhrizae radix in water, concentrating the decoction, adding fine powder of Antrodia Camphorata, lumbricus and radix Angelicae Dahuricae, and adding pharmaceutically acceptable adjuvants or auxiliary components.

5. Use of a pharmaceutical composition according to any one of claims 1-3 for the preparation of a medicament for the treatment of ischemic stroke.

6. Use of a pharmaceutical composition according to any one of claims 1-3 for the preparation of a medicament for improving limb movement dysfunction after ischemic stroke.