Traditional Chinese medicine composition for nourishing heart, restoring consciousness and activating collaterals and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition for nourishing heart, restoring consciousness and activating collaterals and application thereof. The traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 50 parts of ginseng, 90-110 parts of astragalus membranaceus, 20-40 parts of safflower carthamus, 20-40 parts of cassia twig, 10-30 parts of ginger processed pinellia and 10-30 parts of gastrodia elata, can be used for treating the recovery period and sequelae period of cerebral arterial thrombosis, and can also be used for treating thrombolysis 24 hours after acute period.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a traditional Chinese medicine composition for nourishing the heart, refreshing the brain and activating collaterals and an application thereof. Background Art

[0002] Ischemic stroke (IS) is a series of neurological deficit syndromes caused by cerebral blood circulation disorders, characterized by high morbidity, high disability rate, and high mortality rate. Antiplatelet and anticoagulant therapy are mainly limited to secondary prevention and the prevention and treatment of deep vein thrombosis. The success rate of intravenous thrombolysis and mechanical thrombectomy with recombinant tissue plasminogen activator (rt-PA) is closely related to the treatment time window. While thrombolysis and other methods can recanalize cerebral blood vessels, they often further aggravate the inflammatory damage of cerebral ischemic tissue and even make it irreversible, that is, cerebral ischemia / reperfusion injury (CIRI). An article published in Stroke magazine pointed out that CIRI is a process of two different but interrelated stages: ischemia (primary energy failure) and reperfusion (secondary energy failure). The neuritis injury mediated by it is an important pathological process leading to neurological deficits after IS occurs, such as Figure 1 shown.

[0003] The neurovascular unit is the overall therapeutic target for brain cell protection after ischemic stroke. Most previous studies on cerebral ischemic injury have focused on neurons, or different cell populations and structures separated from the brain. The neurovascular unit (NVU) is mainly composed of neurons (NS), astrocytes (AS), and brain microvascular endothelial cells (BMEC). The introduction of its concept has transformed the single neuron protection into the protection of each component of the NVU, providing a new perspective for the comprehensive study of CIRI neurological function repair. The normal operation of brain nerve function depends on the interaction between the various components of the NVU, including blood-brain barrier (BBB) ​​regulation, cell preservation, inflammatory immune response regulation, and neurovascular repair, such as Figure 2As shown. CIRI includes neural structural damage including astrocytes, brain microvascular endothelial cells and matrix components, not just neurons, which makes the efficacy of drugs that simply target neurons or a certain pathogenesis link poor. As a holistic treatment target after ischemic stroke, NVU focuses on the overall protection of multi-target brain cells to repair the brain's neural function to the greatest extent. NVU as a holistic treatment target for CIRI neural function repair is consistent with the "holistic concept" of traditional Chinese medicine. Traditional Chinese medicine and its active ingredients have a regulatory effect on the components of NVU, including improving neuronal damage, inhibiting neuroinflammation, promoting the regeneration of brain microvascular endothelial cells, repairing BBB damage, and improving the activation state of astrocytes. It is feasible to use multi-target traditional Chinese medicine with NVU as the overall treatment target for brain protection strategy.

[0004] Therefore, exploring effective and safe traditional Chinese medicine solutions to combat inflammatory injury caused by cerebral ischemia / reperfusion is a clinical problem that needs to be solved urgently, which is of great significance for promoting neurological function recovery after IS.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The first purpose of the present invention is to provide a Chinese medicine composition for nourishing the heart, refreshing the brain and activating the collaterals in view of the deficiencies in the prior art.

[0007] The second object of the present invention is the use of the Chinese medicine composition.

[0008] To achieve the above first purpose, the technical solution adopted by the present invention is as follows:

[0009] A traditional Chinese medicine composition for nourishing the heart, refreshing the brain and activating collaterals is prepared from the following raw materials in parts by weight: 50 parts of ginseng, 90-110 parts of astragalus, 20-40 parts of safflower, 20-40 parts of cassia twig, 10-30 parts of ginger and pinellia, and 10-30 parts of gastrodia.

[0010] Furthermore, the Chinese medicine composition is made of the following raw materials in parts by weight: 50 parts of ginseng, 98-102 parts of astragalus, 28-32 parts of safflower, 28-32 parts of cassia twig, 18-22 parts of pinellia tuber, and 18-22 parts of gastrodia elata.

[0011] Furthermore, the Chinese medicine composition is made of the following raw materials in parts by weight: 50 parts of ginseng, 100 parts of astragalus, 30 parts of safflower, 30 parts of cassia twig, 20 parts of pinellia tuber, and 20 parts of gastrodia elata.

[0012] Furthermore, the Chinese medicine composition also includes excipients, which include at least one of a filler, a binder, a disintegrant, a lubricant, an absorbent and a diluent.

[0013] Furthermore, the dosage form of the Chinese medicine composition is selected from any one of granules, pastes, pills, injections and oral liquids.

[0014] To achieve the above second purpose, the technical solution adopted by the present invention is as follows: application of the Chinese medicine composition in the preparation of a medicine for nourishing the heart, refreshing the brain and activating collaterals.

[0015] Furthermore, the heart-nourishing, brain-awakening and collateral-activating drug is used for the treatment of ischemic stroke in the recovery phase, sequelae phase and 24 hours after thrombolysis in the acute phase.

[0016] Furthermore, the heart-nourishing, brain-awakening and collateral-activating medicine can achieve at least one of the following efficacy indicators:

[0017] (1) Reduce cerebral infarction volume;

[0018] (2) Reduced pyknotic neuronal nuclei and neatly arranged pyramidal cells;

[0019] (3) Improve the loss of Nissl bodies and enhance neuronal activity;

[0020] (4) Improve cerebral blood flow, cerebral vascular complexity index and vascular skeleton density index, and reduce vascular diameter index;

[0021] (5) Down-regulate the increased IL-1β inflammatory level caused by cerebral ischemia-reperfusion.

[0022] The Chinese medicine composition of the present invention is a prescription for nourishing the heart, awakening the brain and activating the collaterals (YXXN), which is an empirical prescription of famous traditional Chinese medicine practitioners formed under the guidance of the theory of "five internal organs in one, holistic concept" of traditional Chinese medicine, and has a definite and safe clinical efficacy. The applicant previously confirmed through 2,302 high-quality evidence-based studies that the traditional Chinese medicine Qilong Capsule (astragalus, earthworm, angelica, red peony root, chuanxiong, safflower, peach kernel, salvia miltiorrhiza) for invigorating qi and activating blood circulation has an efficacy advantage in improving the neurological deficits of ischemic stroke with qi deficiency and blood stasis syndrome; system biology studies have shown that its mechanism is related to inhibiting the NF-κB inflammatory pathway and promoting neurological function repair.

[0023] Huang Yuanyu, a doctor in the Qing Dynasty, said in "Four Sacred Heart Sources": The symptoms of stroke are caused by damp soil, and the reason for damp soil is cold water. Cold water restrains soil, and soil failure cannot circulate qi in the limbs. Once it is injured by the seven emotions and attacked by wind from all directions, it will cause a stroke. Under the guidance of this theory, the applicant optimized the prescription of Qilong Capsule based on the clinical experience of famous old Chinese medicine practitioners, retaining its effects of invigorating qi and activating blood circulation (astragalus, safflower), adding drugs with related effects of greatly replenishing vital energy (ginseng), warming meridians and unblocking collaterals (cinnamon twigs), lowering turbidity and removing dampness (pinellia ternata), and dispelling wind and unblocking collaterals (Gastrodia elata), forming a prescription for nourishing the heart, refreshing the brain and activating collaterals composed of ginseng, astragalus, safflower, cinnamon twigs, pinellia ternata, and Gastrodia elata, which has the effects of invigorating qi and activating blood circulation, warming meridians and unblocking collaterals, strengthening the spleen and removing dampness, and dispelling wind and unblocking collaterals. It is used for the treatment of ischemic stroke recovery and sequelae, and also for the treatment of 24 hours after thrombolysis in the acute phase. The main ingredients of the Yangxin Xingnao Huoluo formula are ginseng and astragalus (greatly replenishing vital energy, strengthening the spleen and raising the clear, involving the heart, spleen, and kidney), the assistant ingredients are safflower and cinnamon twig (activating blood circulation and unblocking meridians, warming the meridians and assisting yang and transforming qi, involving the heart and liver), and the adjuvant ingredients are ginger, pinellia, and gastrodia (removing dampness and lowering turbidity, calming wind and stopping spasms and unblocking meridians, involving the spleen and liver). Therefore, the Yangxin Xingnao Huoluo formula is an optimized prescription based on the clinical experience of famous old Chinese medicine practitioners, under the guidance of the theory of "five internal organs in one", with the heart-liver-spleen-kidney-brain axis as the overall treatment strategy, focusing on the close connection between the internal organs, taking the neurovascular unit as the overall treatment target, and promoting the repair of nerve function around the brain cell protection strategy.

[0024] The applicant previously conducted a series of experimental studies using the cerebral ischemia / reperfusion (MCAO / R) rat model as a carrier, and found that the Yangxin Xingnao Huoluo prescription can significantly reduce the volume of rat cerebral infarction after 28 days of treatment; the nuclei of pyknotic neurons are significantly reduced, the pyramidal cells are neatly arranged, and the layers are relatively complete; the loss of Nissl bodies is significantly improved, the neuronal activity is significantly improved, and no obvious vacuolar structure is observed; the rat cerebral blood flow, rat cerebral vascular complexity index, vascular skeleton density index, and vascular diameter index are significantly improved; the increased IL-1β inflammatory level caused by cerebral ischemia reperfusion is significantly downregulated. And the Yangxin Xingnao Huoluo prescription is equivalent to the positive control drug edaravone in the above efficacy indicators; it has a greater efficacy advantage than Qilong capsule in reducing the volume of cerebral infarction. The above results all indicate that the Yangxin Xingnao Huoluo prescription can significantly promote the angiogenesis of neurovascular units in the cerebral ischemic injury site of rats, improve the cerebral microcirculation after cerebral ischemia reperfusion in rats, and thus improve the cerebral ischemia reperfusion injury in rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 It is a diagram of the important pathological process of inflammatory injury mediated by cerebral ischemia / reperfusion (i.e., neurological deficit after IS occurs);

[0027] Figure 2 This is a diagram of the role of NVU in cranial nerve function;

[0028] Figure 3 The TTC staining images (a) and ischemic area volume ratio data (b) of rats in each group, where the six images from top to bottom in each column of (a) are the detection results of different brain slices of the same rat in the group after 28 days of treatment, and the ordinate of (b) is the ischemic area volume ratio (Infarct Volume (%));

[0029] Figure 4 HE staining images of rats in each group;

[0030] Figure 5 The Nissl body staining images of rats in each group;

[0031] Figure 6 The laser Doppler blood flow distribution diagram (a) and cerebral blood flow result diagram (b) of each group of rats, where the ordinate of (b) is cerebral blood flow (ContralateralcerebralFluxmeanratio(%));

[0032] Figure 7 Figure 1 shows the optical coherence tomography blood flow imaging of rats in each group (a), average area data (b), average vascular complexity index (c), average vascular diameter index (d), and average vascular skeleton density index (e). The ordinate of (b) is the average area (AverageArea (mm 2 )), the ordinate of (c) is the average vascular complexity index (AverageComplexity), the ordinate of (d) is the average vascular diameter index (AverageDiameter(μm)), and the ordinate of (e) is the average vascular skeleton density index (AverageSkeletonDensity);

[0033] Figure 8 is the graph of IL-1β concentration of rats in each group;

[0034] Note: In the above figures, compared with the Sham group (sham operation group), # P<0.05, ## P<0.01, ### P<0.001; compared with MCAO / R (model group), *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. The process parameters of the following embodiments that do not specify specific conditions are usually based on conventional conditions.

[0036] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in the present invention.

[0037] All Chinese medicinal materials used in the present invention comply with the description of the corresponding medicinal materials in the 2020 edition of the Chinese Pharmacopoeia, especially the medicinal parts and the content of the effective ingredients. For example, the content of astragaloside I (C 41 H 68 O 14 ) shall not be less than 0.080%. The raw materials used in the following examples are their corresponding medicinal material slices, which are all obtained by the processing methods in the 2020 edition of the "Chinese Pharmacopoeia".

[0038] The Chinese medicine composition of the present invention can be prepared into the following dosage forms: granules, pastes, pills, injections, oral liquids, etc.

[0039] When preparing different dosage forms, corresponding auxiliary materials are added, such as fillers, binders, disintegrants, lubricants, absorbents and diluents, etc. The preparation of different dosage forms can be carried out by conventional methods in the art, which will not be described in detail here.

[0040] The present invention is further described in detail below in conjunction with specific experiments and results.

[0041] Animal experiment process and results of drug efficacy verification

[0042] 1. Experimental Materials

[0043] 1.1 Drugs and Reagents

[0044] Qilong Capsule (National Medicine Standard No. Z20000097), Yangxin Xingnao Huoluo Recipe (extract), Edaravone (MCE, batch number: HY-B0099), TTC (Sigma, batch number: T8877-10G), 4-0 non-absorbable surgical suture (Yangzhou Liyuan Medical Equipment Co., Ltd., batch number: 20210102), MCAO suture (Beijing Xinong Technology Co., Ltd., batch number: A5-304050), 4% paraformaldehyde (Solebao, batch number: 20201225), RIPA tissue / cell lysis buffer (Solebao, batch number: 20211211), normal saline (Shijiazhuang Siyao Xi'an Company, batch number: 2401172105), RatIL-1βELISAkit (Shanghai ELISA Biotechnology Co., Ltd., Jun2024).

[0045] 1.2 Instruments and Equipment

[0046] Optical coherence tomography blood flow imager (OLYMPUS, Japan), laser imaging system (Moor Instruments, UK), skull drill (Shanghai Yuyan Scientific Instrument Co., Ltd.), -80℃ ultra-low temperature refrigerator (Qingdao Haier Special Electrical Appliance Co., Ltd.), TGL-16M low-temperature ultracentrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), AL104 electronic balance (METTLERTOLEDO Instrument (Shanghai) Co., Ltd.).

[0047] 1.3 Animals

[0048] 120 SPF SD rats, male, weighing 260±20g, were provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with the laboratory animal production license number: SCXK(Beijing)2021-0006. Before the experiment, the animals were acclimatized to the laboratory environment for 5 days, with a room temperature of 20-25℃, fed with standard pellet feed, and had free access to water.

[0049] 2. Experimental Methods

[0050] 2.1 Therapeutic effect of Yangxinxingnaohuoluo prescription on cerebral ischemia-reperfusion injury in rats

[0051] 2.1.1 Establishment of the animal model of cerebral ischemia-reperfusion injury in MCAO / R rats

[0052] The rat model of focal cerebral ischemia / reperfusion injury was prepared by middle cerebral artery embolization. Before the experiment, the nylon suture (MCAO suture) was cleaned with 75% alcohol, marked at 18.5 mm from the ball end, and placed in 0.9% sterile saline for later use. The rats were weighed and treated with ketamine (80 mg kg -1 ) and xylazine (10 mg kg -1) anesthesia, lying supine on the operating table. Make a midline incision in the neck, separate the left common carotid artery (CCA), external carotid artery (ECA), and branch arteries of the internal carotid artery (ICA) in turn, and ligate the distal end of the ECA and the centripetal end of the common artery (CCA). Use an artery clamp to clamp the CCA and ICA, make an incision in the ECA equivalent to 2 / 3 of the diameter of the ECA, insert the prepared suture into the ICA to block the blood flow of the ICA. And use surgical thread to gently ligate. Loosen the artery clamp of the ICA, insert the nylon suture into the ICA, insert 18.5±0.5mm to the beginning of the anterior cerebral artery (ACA), and block the blood supply of the middle cerebral artery MCA. After 2 hours of MCA occlusion, pull out the nylon suture from the ACA, withdraw it to the ECA for recovery, and reperfuse for 24 hours.

[0053] Rats in the sham operation group were only treated with carotid artery isolation without ligation or suture insertion to induce cerebral ischemia.

[0054] All rats were scored for neurological function and behavior according to the Longa 5-grade neurological function scoring standard. Rats with a neurological function score of 1 to 3 indicated a successful modeling. Laser Doppler blood flowmetry was used to detect cerebral blood flow. A decrease in cerebral blood flow of approximately 70% of the basal value after embolization was considered a successful modeling of focal cerebral ischemia due to middle cerebral artery occlusion.

[0055] 2.1.2 Dosage and grouping

[0056] 2.1.2.1 Preparation of drug solution

[0057] Preparation of Yangxinxingnaohuoluo Recipe (YXXN): Composition includes 50g ginseng, 100g astragalus, 30g safflower, 30g cinnamon twig, 20g ginger pinellia, 20g gastrodia, the above medicinal flavors are decocted twice, the first time add 8 times water for 1.5h, the second time add 6 times water for 1h, combine the decoction, concentrate, each 1ml extract is equivalent to 3g of the slice. The decoction conditions are 130℃, 800w; the concentration conditions are 270℃, 2100w at the beginning, and will be adjusted according to the actual situation in the middle, and the final concentration temperature is 130℃, 800w.

[0058] Preparation of Edaravone (EDA): Weigh a certain amount of Edaravone solid powder, prepare it with normal saline (concentration of 0.6 mg / mL), and mix it thoroughly by ultrasonication;

[0059] Preparation of Qilong Capsules (QL): Weigh a certain amount of solid powder in Qilong Capsules, prepare it with normal saline, the three concentrations of low, medium and high doses are 12 mg / mL, 24 mg / mL and 48 mg / mL respectively, and mix it by ultrasound until it is fully mixed.

[0060] 2.1.2.2 Grouping

[0061] After feeding for a period of time, healthy and qualified SPF-grade SD rats weighing 280-340 g were selected and randomly divided into 7 groups, 6 rats in each group: sham operation group (Sham), model (MCAO / R) group, Yangxinxingnaohuoluo (YXXN) group (480 mg·kg -1 )+MCAO / R, edaravone injection (EDA) control group (6 mg·kg -1 )+MCAO / R, QL low-dose group (120 mg·kg -1 )+MCAO / R, QL medium dose group (240 mg·kg -1 )+MCAO / R, QL high-dose group (480 mg·kg -1 )+MCAO / R. Each group was given oral administration for 28 days according to the dose, once a day. The sham group (Sham) and the model group were given an equal volume of sodium carboxymethylcellulose (CMC) solution (concentration 0.5%) by oral administration every day. The dose of Yangxinxingnaohuoluo was calculated according to the clinical prescription experience and the equivalent dose of 1:7 for adults 70kg and rats 260g. The final calculated dose was 480mg·kg -1 , that is, 480 mg of the decoction tablets are administered per kilogram of rat. The oral dose of Qilong Capsule (QL) is calculated according to the drug instructions based on the 1:7 equivalent dose of 70 kg adults and 260 g rats, and based on the previous research basis of Qilong Capsule, the high dose is calculated to be 480 mg kg -1 The medium dose is 1 / 2 of the high dose ((240 mg kg -1 ), low dose is 1 / 2 of the medium dose (120 mg kg -1 ).

[0062] 2.1.3TTC staining

[0063] The rats to be tested were anesthetized for 28 days and blood was taken from the abdominal aorta within 20 minutes after treatment, and the brain was quickly removed to maintain the integrity of the brain. The brain was frozen in a -20℃ refrigerator for about 20 minutes to make it easy to slice. The brain tissue was then placed in the brain trough (accurate calculation of the infarct area), and a slice was cut every 2mm, for a total of 5-6 slices. The slices were placed in a 2% TTC solution preheated to 37℃, placed in a 37℃ incubator and incubated in the dark for 10 minutes, and the brain slices were turned over from time to time to make them evenly contacted with the staining solution. After staining, normal tissue was rose red, and infarcted tissue was not stained and appeared white. The brain slices were taken out and placed in 4% paraformaldehyde and fixed at room temperature in the dark for 24 hours. The fixed brain slices were placed on a light-colored operating table and photographed with a digital camera. The ischemic area volume ratio was calculated according to the formula = (the sum of the white ischemic area of ​​each slice) / (the sum of the brain slice area of ​​each slice) × 100%.

[0064] 2.1.4 Immunohistochemical staining

[0065] 2.1.4.1 HE staining

[0066] Within 20 minutes after anesthesia, the rats to be tested were blooded from the abdominal aorta, the chest was cut open, and about 100 mL of normal saline was perfused into the heart until a clear and transparent liquid flowed out of the right atrium. After the perfusion, the brain tissue was quickly removed and fixed in 10% paraformaldehyde at room temperature for 24 hours. Paraffin-sealed tissue of the rat hippocampus was prepared and sliced. The paraffin sections were decolorized twice with xylene for 20 minutes each time, then dewaxed twice with anhydrous ethanol for 5 minutes each time, and then dewaxed with 75% ethanol for 5 minutes. Washed with PBS three times for 5 minutes each time. The dewaxed sections were placed in hematoxylin staining solution for 10 minutes and rinsed with distilled water three times for 2 minutes each time. After color separation with 1% hydrochloric acid ethanol for 20 seconds, rinsed with distilled water three times for 2 minutes each time, and then returned to blue with 37°C warm water for 3 minutes. Stained with aqueous eosin staining solution for 12 minutes and rinsed with distilled water three times for 2 minutes each time. The sections were placed in 50% ethanol, 75% ethanol, 95% ethanol, anhydrous ethanol and xylene for 5 min each for dehydration. The xylene around the tissue was quickly wiped off with filter paper, and then the sections were quickly sealed with glycerol gelatin.

[0067] 2.1.4.2 Nissl body staining

[0068] Within 20 minutes after anesthesia, the rats to be tested were blooded from the abdominal aorta, the chest cavity was cut open, and about 100 mL of normal saline was perfused into the heart until a clear and transparent liquid flowed out of the right atrium. After the perfusion, the brain tissue was quickly removed and fixed in 10% paraformaldehyde at room temperature for 24 hours. Paraffin-sealed tissues of the classical hippocampus of rats were prepared and sliced. The paraffin sections were decolorized twice with xylene for 20 minutes each time, then dewaxed twice with anhydrous ethanol, and then dewaxed with 75% ethanol for 5 minutes. Washed three times with PBS for 5 minutes each time. The dewaxed sections were placed in Nissl staining solution for 10 minutes and rinsed with distilled water three times for 2 minutes each time. The sections were placed in 50% ethanol, 75% ethanol, 95% ethanol, anhydrous ethanol and xylene for 5 minutes each for dehydration, and the xylene around the tissue was quickly wiped off with filter paper, and then the sealing was quickly completed with glycerol gelatin.

[0069] 2.1.5 Laser Doppler technology to detect blood flow distribution

[0070] Place the anesthetized rat to be tested flat on a tray, disinfect the hair and skin on the top of the rat's head with alcohol, cut the skin on the top of the rat's head with surgical scissors, clean the remaining hair at the cut position of the head with a cotton ball dipped in physiological saline, and face the top of the rat's head directly under the laser Doppler blood flowmeter camera. The evaluation criteria are: the rat's cerebral blood flow drops to less than 40-30% of the pre-ischemic level, which is considered a successful ischemia; the cerebral blood flow recovers to more than 60-70% of the pre-ischemic level, which is considered a successful reperfusion.

[0071] 2.1.6 Detection of cerebral microcirculation-related indices after cerebral ischemia-reperfusion in rats by optical coherence tomography blood flow imaging

[0072] OCTA uses the relative movement of red blood cells and surrounding tissues as an endogenous blood flow marker feature, replacing conventional exogenous fluorescent markers, and comprehensively utilizes the spatial scattering signal acquisition capability of optical coherence imaging technology and the motion recognition capability of dynamic optical scattering characteristics to distinguish dynamic blood flow areas and static surrounding tissues in three-dimensional space. In order to cooperate with the OCT imaging system to better capture and record the cerebral cortical blood vessels, a stable optical window suitable for long-term observation of rat cortical blood vessels is made on the rat head. The craniotomy window surgery is usually performed on the right parietal bone. First, rats are anesthetized by intraperitoneal injection of ketamine (80 mg / kg) and xylazine (10 mg / kg), and the body temperature is maintained between 36.8-37°C using a constant temperature heating pad. After anesthesia induction, the animal is placed on a stereotactic frame in a prone position to stabilize the head. Lubricant ointment is applied to the eyes to prevent dry eyes. A 4 mm × 4 mm craniotomy is performed using a dental drill at 1 mm posteriorly and laterally. A round piece of skull is carefully removed with forceps and replaced with a round transparent glass cover with a diameter of 5 mm. The glass cover was sealed to the skull using cyanoacrylate bone cement to form a 3mm×3mm flat tissue surface as an imaging window, through which the main meningeal vessels in the somatosensory cortex area can be seen under a microscope. Subsequently, the ischemic lateral brain area of ​​the rat was tomographically scanned using optical coherence tomography blood flow imaging technology, and the vascular signals in the target area were reconstructed through an algorithm to form a vascular network with a three-dimensional space, and the relevant parameters of the microvessels were detected.

[0073] 2.1.7 Interleukin 1β (IL-1β) detection

[0074] The IL-1β concentration in rat serum was detected using the RatIL-1β ELISA kit, and the absorbance of each well was detected using a microplate reader to calculate the IL-1β release percentage. IL-1β concentration (pg / mL) = standard curve slope × dilution factor × (sample well absorbance - control well absorbance).

[0075] 3. Experimental results

[0076] 3.1 Therapeutic effect of Yangxinxingnaohuoluo prescription (YXXN) on cerebral ischemia-reperfusion injury in rats

[0077] 3.1.1 TTC staining

[0078] The results of TTC staining (a) and ischemic area volume ratio (b) of rats in each group are shown in Figure 3Compared with the sham group, the infarct volume of the model group (MCAO / R) rats increased significantly (P<0.001), which again indicated that the rat cerebral ischemia-reperfusion model was successfully established. Compared with the model group, YXXN (480 mg·kg -1 ), QL (high, medium, and low doses), and EDA (6 mg kg -1 ) treatment for 28 days significantly reduced the infarction volume of rats (P<0.001), indicating that the Yangxin Xingnao Huoluo prescription can significantly improve cerebral ischemia-reperfusion injury in rats. Compared with the positive control drug edaravone group, which had a cerebral infarction volume of 5.776% after 28 days of administration, the Yangxin Xingnao Huoluo prescription group had a lower cerebral infarction volume of 3.098% after 28 days of administration. The cerebral infarction volume of the Yangxin Xingnao Huoluo prescription group was smaller than that of the QL high, medium, and low dose groups after 28 days, indicating better efficacy.

[0079] 3.1.2 Immunohistochemical staining

[0080] HE staining and Nissl staining are often used to observe pathological changes in the morphology of rat brain neurons.

[0081] 3.1.2.1 HE staining

[0082] HE staining results of rats in each group are shown in Figure 4 Compared with the sham group, the neuronal cells in the cortex, hippocampal CA1 and CA3 regions of the model group were irregular in morphology, with condensed nuclei and blurred outlines, unclear nucleoli, reduced nuclei, and disordered arrangement of pyramidal cells. -1 ), QL (high, medium, and low doses), and EDA (6 mg kg -1 ) After 28 days of treatment, the nuclei of pyknotic neurons were significantly reduced, the pyramidal cells were arranged neatly, and the layers were relatively complete. There was no significant difference in the efficacy of Yangxinxingnaohuoluo prescription compared with Qilong capsule and edaravone.

[0083] 3.1.2.2 Nissl body staining

[0084] The Nissl body staining results of rats in each group are shown in Figure 5 Compared with the sham group, the model group had a serious loss of Nissl bodies in the cortex, hippocampal CA1 and CA3 regions, decreased neuronal activity, sparse neuronal arrangement, and a large number of vacuolar structures. -1 ), QL (high, medium, and low doses), and EDA (6 mg kg -1 ) After 28 days of treatment, the loss of Nissl bodies was significantly improved, the neuronal activity was significantly improved, and no obvious vacuolar structure was observed. There was no significant difference in the efficacy of Yangxinxingnaohuoluo prescription compared with Qilong capsule and edaravone.

[0085] 3.1.3 Laser Doppler technology to detect blood flow distribution

[0086] Tissue blood flow detection can directly reflect the functional state of microcirculatory blood perfusion and is an important indicator of microcirculation research. Doppler technology is an important technical means to detect tissue blood flow.

[0087] The results of laser Doppler examination of blood flow distribution (a) and cerebral blood flow (b) in rats in each group are shown in Figure 2. Figure 6 Compared with the sham group, the cerebral blood flow of the model group rats was decreased. -1 ), QL (high, medium, and low doses), and EDA (6 mg kg -1 ) treatment increased the cerebral blood flow of rats after 28 days of administration. Compared with the positive control drug edaravone group, the cerebral blood flow of the Yangxin Xingnao Huoluo Fang group improved by 89.08% after 28 days of administration. Compared with the QL (high, medium, and low dose) groups, there was no significant difference in cerebral blood flow in the Yangxin Xingnao Huoluo Fang group after 28 days of administration.

[0088] 3.1.4 Detection of cerebral microcirculation-related indices after cerebral ischemia-reperfusion in rats by optical coherence tomography blood flow imaging

[0089] Blood perfusion is an important indicator for measuring the physiological function and pathological state of living organisms. Blood flow detection is extremely important for the evaluation and diagnosis of tissue physiological and pathological states. OCTA technology realizes in vivo, label-free, three-dimensional blood flow motion angiography, which can quickly obtain blood perfusion information in the horizontal direction of capillaries, and plays an important role in the dynamic visualization of microcirculation and the detection of multiple indicators.

[0090] The results of optical coherence tomography blood flow imaging and multi-index detection of rats in each group are as follows Figure 7 The results showed that compared with the sham group, the average cerebral vascular area of ​​the model group (MCAO / R) rats had no significant difference, the vascular complexity index and vascular skeleton density index decreased significantly (p < 0.05), and the vascular diameter index increased significantly; compared with the model group, YXXN (480 mg·kg -1 )、QL(120、240、480mg·kg -1 ) and EDA (6mg·kg -1 ) treatment for 28 days significantly increased the rat cerebral vascular complexity index and vascular skeleton density index, and significantly reduced the vascular diameter index. -1 ) and the positive control group EDA (6 mg kg -1 ) and QL (120, 240, 480 mg kg-1 ), there was no significant difference in vascular complexity index, vascular skeleton density index and vascular diameter after 28 days of treatment. The above results indicate that YXXN can significantly promote angiogenesis in the ischemic injury site of rat brain and improve the cerebral microcirculation after cerebral ischemia-reperfusion in rats.

[0091] 3.1.5ELISA detection of interleukin-1β

[0092] The IL-1β concentrations of rats in each group were as follows Figure 8 Compared with the Sham group, the IL-1β level in the model group (MCAO / R) was significantly increased, and YXXN (480 mg·kg -1 )、QL(120、240、480mg·kg -1 ) and EDA (6mg·kg -1 ) Treatment for 28 days could significantly reduce the elevated IL-1β level caused by the model group, and the Yangxinxingnaohuoluo recipe was as effective as Qilong capsules and the positive control drug edaravone.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Chinese medicine composition for nourishing the heart, refreshing the brain and activating collaterals, characterized in that: The Chinese medicine composition is prepared from the following raw materials in parts by weight: 50 parts of ginseng, 90-110 parts of astragalus, 20-40 parts of safflower, 20-40 parts of cassia twig, 10-30 parts of pinellia tuber, and 10-30 parts of gastrodia elata.

2. The Chinese medicine composition according to claim 1, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 50 parts of ginseng, 98-102 parts of astragalus, 28-32 parts of safflower, 28-32 parts of cassia twig, 18-22 parts of ginger and pinellia, and 18-22 parts of gastrodia.

3. The Chinese medicine composition according to claim 2, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 50 parts of ginseng, 100 parts of astragalus, 30 parts of safflower, 30 parts of cassia twig, 20 parts of ginger pinellia, and 20 parts of gastrodia.

4. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition also includes auxiliary materials, which include at least one of a filler, a binder, a disintegrant, a lubricant, an absorbent and a diluent.

5. The Chinese medicine composition according to claim 1, characterized in that: The dosage form of the Chinese medicine composition is selected from any one of granules, pastes, pills, injections and oral liquids.

6. Use of the Chinese medicine composition as described in any one of claims 1 to 5 in the preparation of a medicine for nourishing the heart, refreshing the brain and activating collaterals.

7. The use according to claim 6, characterized in that: The heart-nourishing, brain-awakening and collateral-activating medicine is used for the treatment of ischemic stroke in the recovery stage, sequelae stage and 24 hours after thrombolysis in the acute stage.

8. The use according to claim 7, characterized in that: The heart-nourishing, brain-awakening and collateral-activating medicine can achieve at least one of the following efficacy indicators: (1) Reduce cerebral infarction volume; (2) Reduced pyknotic neuronal nuclei and neatly arranged pyramidal cells; (3) Improve Nissl body loss and enhance neuronal activity; (4) Improve cerebral blood flow, cerebral vascular complexity index and vascular skeleton density index, and reduce vascular diameter index; (5) Down-regulate the increased IL-1β inflammatory level caused by cerebral ischemia-reperfusion.

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