Traditional Chinese medicine composition and application thereof in preparation of medicine for treating cerebral arterial thrombosis

Abstract

The invention provides a traditional Chinese medicine composition and application thereof in preparation of a medicine for treating cerebral arterial thrombosis, and relates to the technical field of biological medicine. The traditional Chinese medicine composition is prepared from the following traditional Chinese medicinal materials in parts by weight: 5-30 parts of Yulangsan, 5-30 parts of panax japonicus, 5-30 parts of ruta graveolens, 5-30 parts of camphorwood, 5-30 parts of millettia japonica, 5-30 parts of costus root, 5-30 parts of rhizoma curculiginis, 5-30 parts of clinopodium herb, 5-30 parts of white hyacinth bean and 5-30 parts of radix puerariae. The whole formula treats both qi and blood, regulates phlegm and blood stasis, is reasonable in compatibility of the medicines, and has the effects of tonifying qi and activating blood, dispersing blood stasis and dredging collaterals, clearing heat and dissipating blood stasis, restoring consciousness and dredging collaterals, and tonifying spleen and warming kidney. The traditional Chinese medicine composition can relieve cerebral infarction and encephaledema, improve neurological dysfunction, inhibit neuronal apoptosis and regulate inflammatory response to relieve inflammatory damage of brain tissue, and a good treatment effect on cerebral arterial thrombosis model rats is achieved. The traditional Chinese medicine composition provides a new choice for development of medicines for treating cerebral arterial thrombosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a traditional Chinese medicine composition and its application in the preparation of drugs for ischemic stroke. Background Technology

[0002] Ischemic stroke (IS), also known as "apoplexy," is an acute cardiovascular and cerebrovascular disease characterized by symptoms such as hemiplegia, numbness on one side of the body, and speech impairment. It is characterized by high incidence, high recurrence rate, high disability rate, and high mortality rate. It often occurs in middle-aged and elderly people, accounting for 69.6% to 70.8% of stroke patients in China, and in recent years there has been a trend of younger onset, seriously threatening the health and lives of middle-aged and elderly people.

[0003] Currently, the treatment of stroke (IS) mainly includes two categories: neuroprotective therapy and neurorehabilitation. Neuroprotective therapy includes intravenous thrombolysis and neuroprotective agents. For patients with very early and acute IS, the optimal treatment plan is intravenous administration of tissue plasminogen activator and endovascular thrombectomy to relieve large vessel occlusion and salvage the ischemic penumbra through rapid reperfusion. Tissue plasminogen activator is the only thrombolytic drug approved by the US Food and Drug Administration and is widely used worldwide. However, its therapeutic window is narrow; patients must undergo thrombolysis within 4.5 hours after stroke. Many patients miss the treatment window or cannot receive timely treatment due to their own reasons. Even patients who have received acute thrombolysis and thrombectomy, although reperfusion can restore blood oxygenation, the free radicals generated can trigger a series of inflammatory cascade reactions, further aggravating tissue damage and causing varying degrees of neurological dysfunction and behavioral impairment, seriously affecting the patient's quality of life. Therefore, developing safer and more effective therapies is crucial. Traditional Chinese medicine, with its advantages of multiple components and multiple targets, can regulate various pathological processes in IS, and recent studies have provided scientific evidence for its clinical application. Based on this, the present invention aims to provide a traditional Chinese medicine that can be used to treat ischemic stroke, in order to provide new strategies and methods for the clinical treatment of ischemic stroke. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a traditional Chinese medicine composition and its application in the preparation of drugs for treating ischemic stroke.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a traditional Chinese medicine composition, which is composed of the following traditional Chinese medicinal materials in parts by weight: 5-30 parts of *Gynostemma pentaphyllum*, 5-30 parts of *Panax japonicus*, 5-30 parts of *Rubus idaeus*, 5-30 parts of camphor wood, 5-30 parts of *Everlasting Heart*, 5-30 parts of *Vine of Fragrant Vine*, 5-30 parts of *Curculigo orchioides*, 5-30 parts of *Smilax china*, 5-30 parts of white hyacinth bean, and 5-30 parts of kudzu root.

[0009] Furthermore, the traditional Chinese medicine composition comprises the following traditional Chinese medicinal materials in parts by weight: 10-30 parts of *Gynostemma pentaphyllum*, 10-30 parts of *Panax japonicus*, 10-30 parts of *Rubus idaeus*, 10-30 parts of camphor wood, 10-30 parts of *Everlasting Heart*, 10-30 parts of *Vinegaria fraseri*, 5-25 parts of *Curculigo orchioides*, 5-25 parts of *Hedyotis diffusa*, 5-25 parts of white hyacinth bean, and 5-25 parts of kudzu root.

[0010] Furthermore, the traditional Chinese medicine composition comprises the following traditional Chinese medicinal materials in parts by weight: 24 parts of *Gynostemma pentaphyllum*, 26 parts of *Codonopsis pilosula*, 20 parts of *Rubus idaeus*, 20 parts of *Camphora sinensis*, 18 parts of *Everlasting Heart*, 22 parts of *Vitis vinifera*, 12 parts of *Curculigo orchioides*, 14 parts of *Smilax china*, 16 parts of *Hymenopus pubescens*, and 16 parts of *Pueraria lobata*.

[0011] Secondly, the present invention provides the application of the above-mentioned traditional Chinese medicine composition in the preparation of a drug for treating ischemic stroke. The drug is prepared from the above-mentioned traditional Chinese medicine composition as a raw material, supplemented with pharmaceutically acceptable excipients, and formulated into any oral dosage form selected from oral liquid, tablets, pills, granules, powders, capsules, and decoctions.

[0012] Yulangsan: Scientifically known as Millettia pulchra, it is the dried root tuber of Millettia pulchra, a plant in the genus Millettia of the legume family. It is neutral in nature and sweet in taste, and has the effects of dispersing blood stasis and reducing swelling, tonifying deficiency and calming the mind, and clearing heat and detoxifying. It is often used to treat rheumatic pain, traumatic injuries, sore throat and other symptoms.

[0013] Panax japonicus: The dried rhizome of Panax japonicus, a plant of the Araliaceae family. It is sweet and slightly bitter in taste, warm in nature, and enters the liver and spleen meridians. It has the effects of tonifying deficiency and strengthening the body, promoting blood circulation and relieving pain, relieving cough and resolving phlegm. It is often used for symptoms such as physical weakness and fatigue, cough due to overwork, and traumatic injuries.

[0014] Rue: The whole herb of Rutagraveolens L., a plant belonging to the Rutaceae family. It has a pungent, slightly bitter taste and is cool in nature; it possesses the effects of clearing heat and detoxifying, dispersing blood stasis and relieving pain, relieving spasms and opening the orifices, promoting blood circulation and activating collaterals, and killing parasites. It is used for colds with fever, toothache, irregular menstruation, infantile eczema, and externally for boils, carbuncles, and traumatic injuries.

[0015] Camphor wood: Derived from the dried wood of the camphor tree (Cinnamomum camphora), a plant in the Lauraceae family. It has a pungent and warm nature, and enters the liver and spleen meridians. It has the effects of dispelling wind and cold, warming the middle and regulating qi, and promoting blood circulation and unblocking collaterals. It is mainly used to treat colds due to wind and cold, stomach cold and bloating, and rheumatic pain.

[0016] *Solanum cathayanum* CY Wu et SCHuang [S. dulcamara L. var. Chinense Dunal], a plant in the Solanaceae family, is sweet and bitter in taste and cold in nature. It has the effects of clearing heat and detoxifying, calming wind and relieving convulsions. It is commonly used for infantile fever and convulsions, jaundice, cough due to lung heat, toothache due to wind-heat, scrofula, and in women, metrorrhagia, leukorrhea, and pelvic inflammatory disease.

[0017] Guangxiangteng: The root of Fissistigma oldhamii (Hemsl.) Merr., a plant of the Annonaceae family; slightly pungent in taste, neutral in nature, and enters the liver and stomach meridians; it has the effects of dispelling wind and dampness, promoting blood circulation and relieving pain. It is used for rheumatic pain, back pain, stomach pain, and traumatic injuries.

[0018] Curculigo orchioides: The dried rhizome of Curculigo orchioides, a plant in the Amaryllidaceae family. It is hot in nature and pungent in taste. It enters the kidney and liver meridians and has the effects of warming the kidney and strengthening yang, dispelling cold and dampness, and strengthening tendons and bones. It is often used for symptoms such as kidney yang deficiency, cold pain in the waist and knees, and rheumatic pain.

[0019] Clinopodium gracile (Benth.) Matsum. and its neighboring species Clinopodium confine (Hance) O. Ktze., belonging to the Lamiaceae family, are used as whole herbs. They possess properties that dispel wind and clear heat, promote qi circulation and blood flow, and detoxify and reduce swelling. They are commonly used for colds with fever, abdominal pain due to food stagnation, vomiting, diarrhea, dysentery, diphtheria, sore throat, carbuncles and erysipelas, urticaria, insect bites, traumatic injuries, and external bleeding.

[0020] White Hyacinth Bean: The dried, mature seed of the hyacinth bean plant (Lycium chinense). It is neutral in nature and sweet in taste, and enters the spleen and stomach meridians. It has the effects of strengthening the spleen and resolving dampness, relieving summer heat and detoxifying. It is used to treat summer heat and dampness causing vomiting and diarrhea, spleen deficiency causing nausea and vomiting, poor appetite and chronic diarrhea, water retention causing thirst, leukorrhea, and infantile malnutrition.

[0021] Kudzu root: The dried root of *Pueraria montana* (Lour.) Merr., a plant belonging to the genus *Pueraria* of the legume family; sweet, pungent, and cool in nature, it has the functions of raising yang and stopping diarrhea, promoting blood circulation and relieving alcohol poisoning; as a pungent and cool exterior-releasing herb, kudzu root has the effects of clearing heat, promoting body fluid production, and quenching thirst. It is commonly used for exogenous fever and headache, stiff neck and back pain, thirst, diabetes, measles that fail to erupt, dysentery, diarrhea, dizziness and headache, hemiplegia due to stroke, chest pain, and damage to the middle jiao from alcohol poisoning.

[0022] Ischemic stroke, commonly known as "apoplexy," is mostly due to qi deficiency and blood stasis, or phlegm and blood stasis obstructing the collaterals. Based on the theory of traditional Chinese medicine, the herbal composition of this invention uses *Gynostemma pentaphyllum* and *Codonopsis pilosula* as the principal herbs. *Gynostemma pentaphyllum* is good at promoting blood circulation, removing blood stasis, reducing swelling and relieving pain, and nourishing deficiency and calming the mind. *Codonopsis pilosula* has the effects of invigorating qi and supporting the body's resistance, promoting blood circulation and unblocking the collaterals, and nourishing deficiency and strengthening the body. The two herbs work together to invigorate qi and nourish deficiency, remove blood stasis and unblock the collaterals, and support the body's resistance while eliminating pathogenic factors, directly addressing the root pathogenesis of qi deficiency and blood stasis. The formula also includes four auxiliary herbs: *Heliotropium indicum*, *Cinnamomum camphora*, *Rubus idaeus*, and camphor wood. *Heliotropium indicum* clears heat and detoxifies, dispels wind and dampness; *Cinnamomum camphora* dispels wind and dampness, invigorates blood and unblocks the meridians. The two herbs work together to enhance the overall formula's effects of resolving blood stasis, unblocking the meridians, and clearing away stagnant heat. *Rubus idaeus* helps the formula to unblock the brain meridians and also has the functions of relieving spasms, opening the orifices, clearing heat and detoxifying, primarily targeting symptoms of stagnant heat obstructing the orifices. Camphor wood warms and unblocks the meridians, dispels cold stagnation and stasis in the meridians, and also promotes qi circulation to enhance the overall formula's effects of invigorating blood and removing blood stasis. Its strong aroma, combined with *Rubus idaeus*, achieves the effect of opening the orifices and refreshing the mind. The four herbs combined clear heat without causing cold stagnation and unblock the meridians without causing dryness or heat, helping the principal herb to achieve the effects of clearing heat and resolving phlegm, promoting qi and blood circulation, relieving spasms and unblocking the meridians, and opening the orifices and refreshing the mind. Then, the herbs *Scissors Grass*, *Curculigo orchioides*, *White Hyacinth Bean*, and *Pueraria lobata* are used as adjuvant herbs. *Scissors Grass* clears heat and detoxifies, disperses blood stasis and reduces swelling, assisting the principal and assistant herbs in dispersing blood stasis and clearing away stagnant heat. *Curculigo orchioides* warms and tonifies kidney yang to aid the circulation of qi and blood, strengthens tendons and bones to facilitate limb recovery. *White Hyacinth Bean* invigorates the spleen and replenishes qi, nourishing the root of qi deficiency, and harmonizes the middle jiao and eliminates dampness to prevent the generation of phlegm and turbidity. Its sweet and warm nature can counteract the dryness of the blood-activating and warming herbs in the formula, ensuring that the entire formula eliminates pathogens without harming the body's vital energy. *Pueraria lobata* raises clear yang, unblocks meridians and collaterals, and its light and upward-moving nature can guide the herbs directly to the head, face, and brain orifices, serving as the guiding herb in the formula. At the same time, it raises clear yang, guiding qi and blood to nourish the brain marrow, improving dizziness, drowsiness, and mental dullness, and harmonizes the herbs, ensuring that the entire formula has a proper balance of ascending and descending properties and a mild medicinal nature. This formula treats both qi and blood, regulates phlegm and blood stasis, and the combination of various herbs works synergistically to invigorate qi and blood, remove blood stasis and unblock collaterals, clear heat and disperse blood stasis, awaken the mind and unblock the meridians, and strengthen the spleen and warm the kidneys.

[0023] This invention uses a rat model of ischemic stroke as the research subject to systematically evaluate the efficacy of the traditional Chinese medicine combination of this invention in the treatment of ischemic stroke. Specifically, the traditional Chinese medicine combination of this invention can reduce cerebral infarction and cerebral edema, improve neurological dysfunction, inhibit neuronal apoptosis, regulate inflammatory response and reduce inflammatory damage to brain tissue, thus achieving a good therapeutic effect on rat model of ischemic stroke. Attached Figure Description

[0024] Figure 1 The results of neurological deficit scores in each group of rats; compared with the sham-operated group, **P<0.01; compared with the model group, ## P<0.01; compared with the positive control group, & P<0.05.

[0025] Figure 2 The results show the ratio of cerebral infarction volume in each group of rats; compared with the sham-operated group, **P<0.01; compared with the model group,## P<0.01; compared with the positive control group, & P<0.05.

[0026] Figure 3 The results show the brain tissue water content of rats in each group; compared with the sham-operated group, **P<0.01; compared with the model group, ## P<0.01; compared with the positive control group, & P<0.05.

[0027] Figure 4 The results show the number of Nissl bodies (a) and the apoptosis rate of nerve cells in the brain tissue of rats in each group (b); compared with the sham-operated group, **P<0.01; compared with the model group, ## P<0.01; compared with the positive control group, & P<0.05.

[0028] Figure 5 The levels of TNF-α(a) and IL-10(a) in the brain tissue of rats in each group were compared; compared with the sham-operated group, **P<0.01; compared with the model group, ## P<0.01; compared with the positive control group, & P<0.05, && P<0.05. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1

[0031] A drug for preparing ischemic stroke, wherein the drug is prepared by using the following traditional Chinese medicine composition as raw material, supplemented with pharmaceutically acceptable excipients, into any oral dosage form selected from oral liquid, tablets, pills, granules, powders, capsules, and decoctions; wherein the traditional Chinese medicine composition consists of the following traditional Chinese medicinal materials in parts by weight: 9 parts of *Gynostemma pentaphyllum*, 9 parts of *Codonopsis pilosula*, 6 parts of *Rubus idaeus*, 6 parts of *Camphora japonica*, 5 parts of *Cinnamomum camphora*, 5 parts of *Cephalotaxus fortunei*, 5 parts of *Curculigo orchioides*, 50 parts of *Hedyotis diffusa*, 5 parts of *Hymenopus pubescens*, and 5 parts of *Pueraria lobata*.

[0032] Example 2

[0033] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine composition consists of the following traditional Chinese medicine materials in parts by weight: 30 parts of *Gynostemma pentaphyllum*, 30 parts of *Panax japonicus*, 26 parts of *Rubus idaeus*, 28 parts of *Camphora sinensis*, 24 parts of *Everlasting Heart*, 26 parts of *Vinegarica sinensis*, 22 parts of *Curculigo orchioides*, 22 parts of *Smilax china*, 26 parts of *Hymenopus pubescens*, and 26 parts of *Pueraria lobata*.

[0034] Example 3

[0035] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine composition consists of the following traditional Chinese medicine materials in parts by weight: 10 parts of *Gynostemma pentaphyllum*, 10 parts of *Codonopsis pilosula*, 10 parts of *Rubus idaeus*, 10 parts of *Camphora sinensis*, 10 parts of *Everlasting Heart*, 10 parts of *Vinegaria fraseri*, 5 parts of *Curculigo orchioides*, 5 parts of *Smilax china*, 5 parts of *Hymenopus pubescens*, and 5 parts of *Pueraria lobata*.

[0036] Example 4

[0037] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine composition consists of the following traditional Chinese medicine materials in parts by weight: 30 parts of *Gynostemma pentaphyllum*, 30 parts of *Panax japonicus*, 30 parts of *Rubus idaeus*, 30 parts of *Camphora sinensis*, 30 parts of *Everlasting Heart*, 30 parts of *Vinegarica sinensis*, 25 parts of *Curculigo orchioides*, 5-25 parts of *Smilax china*, 25 parts of *Hymenopus pubescens*, and 25 parts of *Pueraria lobata*.

[0038] Example 5

[0039] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine composition consists of the following traditional Chinese medicine materials in parts by weight: 15 parts of *Gynostemma pentaphyllum*, 15 parts of *Panax japonicus*, 12 parts of *Rubus idaeus*, 12 parts of *Camphora sinensis*, 12 parts of *Everlasting Heart*, 12 parts of *Vitis thunbergii*, 9 parts of *Curculigo orchioides*, 9 parts of *Smilax china*, 12 parts of *Hymenopus pubescens*, and 9 parts of *Pueraria lobata*.

[0040] Example 6

[0041] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine composition consists of the following traditional Chinese medicine materials in parts by weight: 18 parts of *Gynostemma pentaphyllum*, 20 parts of *Panax japonicus*, 14 parts of *Rubus idaeus*, 16 parts of *Camphora sinensis*, 16 parts of *Everlasting Heart*, 14 parts of *Vinegarica sinensis*, 10 parts of *Curculigo orchioides*, 10 parts of *Smilax china*, 10 parts of *Hymenopus pubescens*, and 12 parts of *Pueraria lobata*.

[0042] Example 7

[0043] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine composition is composed of the following traditional Chinese medicine materials in parts by weight: 24 parts of *Gynostemma pentaphyllum*, 26 parts of *Codonopsis pilosula*, 20 parts of *Rubus idaeus*, 20 parts of *Camphora sinensis*, 18 parts of *Everlasting Heart*, 22 parts of *Vitis thunbergii*, 12 parts of *Curculigo orchioides*, 14 parts of *Smilax china*, 16 parts of *Hymenopus pubescens*, and 16 parts of *Pueraria lobata*.

[0044] Example 8

[0045] The therapeutic effect of the traditional Chinese medicine composition of the present invention on rats with ischemic stroke.

[0046] 1. Materials and Methods

[0047] 1.1 Experimental Materials

[0048] 1.1.1 Laboratory Animals

[0049] Eighty 8-9 week old SPF-grade female SD rats with a body weight of (230±20) g were selected. All animals were housed in a standard experimental animal room with controlled temperature and humidity and a 12-hour light-dark cycle. They were acclimatized for one week before the experiment.

[0050] 1.1.2 Experimental Drugs

[0051] Preparation of Chinese medicine: Weigh the following Chinese medicinal materials according to the following weights: 24g of *Gynostemma pentaphyllum*, 26g of *Panax japonicus*, 20g of *Rubus idaeus*, 20g of *Camphora sinensis*, 18g of *Everlasting Heart*, 22g of *Caulis Fragrantissima*, 12g of *Curculigo orchioides*, 14g of *Herba Scissorsicae*, 16g of *Radix Hyacinthi*, and 16g of *Radix Puerariae*. After washing, soak them in 8 times their weight of water for 30 minutes, then decoct for 40 minutes. Take the decoction. Add 6 times their weight of water to the dregs and decoct for 30 minutes. Take the decoction. Combine the two decoctions and concentrate under reduced pressure to a drug concentration of 1g / mL, which is equivalent to about 1g of raw drug per milliliter. Refrigerate for later use. When using, prepare the appropriate concentration with physiological saline.

[0052] Positive control drug: Butylphthalide soft capsules, 0.1g / capsule.

[0053] 1.2 Experimental Methods

[0054] 1.2.1 Model Construction

[0055] Rats were randomly divided into a sham-operated group (n=12) and a model group (n=68). A rat model of ischemic stroke was established using the middle cerebral artery occlusion (MCAO) method. After anesthetizing the rats, the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed. The CCA was briefly ligated, and a small incision was made distal to the ECA. A nylon filament was inserted into the ICA through the incision on the ECA, and the filament was advanced into the middle cerebral artery. After 2 hours of ischemia, the nylon filament was removed, and the wound was sutured. In the sham-operated group, the carotid artery was exposed and immediately sutured. Twenty-four hours post-operation, the Zea Longa neurological function score was performed on the rats in the model group. A score of 1 or higher was considered a successful model. Sixty rats that successfully established the model and survived were selected.

[0056] 1.2.2 Grouping and Drug Administration

[0057] Sixty rats that successfully underwent sham surgery were randomly divided into five groups: model group, positive control group, low-dose traditional Chinese medicine (TCM) group, medium-dose TCM group, and high-dose TCM group, with 12 rats in each group. The positive control group was administered butylphthalide soft capsules by gavage at a dose of 54 mg / kg; the low, medium, and high doses of TCM were administered by gavage at doses of 4.3 g / kg, 8.5 g / kg, and 16.9 g / kg, respectively; the model group and sham surgery group were administered the same dose of physiological saline by gavage. All rats in each group were given the medication once daily for 14 consecutive days.

[0058] 1.2.3 Neurological deficit score

[0059] After drug administration, neurobehavioral changes in neurological deficits in each group of rats were assessed using the Zea-Longa scoring method. The scoring criteria are shown in Table 1.

[0060]

[0061] 1.2.4 Detection of cerebral infarction area

[0062] Twenty-four hours after drug administration, rats were euthanized by decapitation under anesthesia. Brain tissue was rapidly separated and frozen at -20°C for 20 min. The cerebellum, brainstem, and anterior olfactory bulb were removed. The brain tissue was cut into five 3 mm sections, which were immediately incubated in 2% TTC solution for 20 min. The brain sections were placed in the order of coronal sections and photographed. The infarct volume was measured using ImageJ.

[0063] Infarct volume ratio % = Infarct volume / Total brain slice volume × 100%

[0064] 1.2.5 Measurement of brain tissue water content

[0065] Brain tissue was obtained according to method 1.2.4. The injured cerebral hemisphere was cut and placed in a pre-weighed dry beaker. The injured hemisphere was weighed and then dried to constant weight. The water content of the brain tissue was calculated.

[0066] Brain tissue water content % = [(wet weight - dry weight)] / wet weight × 100%

[0067] 1.2.6 Nissl staining

[0068] Brain tissue was obtained according to method 1.2.4. The hippocampus of the ischemic rat brain was taken for analysis. Paraffin sections were prepared according to conventional methods, then Nissl staining was performed, followed by dehydration and drying. Subsequently, the pathological morphological characteristics of the brain tissue were observed using a panoramic scanner of a digital slide system. After taking pictures, the number of Nissl bodies was calculated using ImageJ software.

[0069] 1.2.7 TUNEL assay for neuronal apoptosis

[0070] Brain tissue was harvested according to method 1.2.4. Rats with ischemic brain tissue were used for hippocampal analysis. Paraffin sections were prepared using standard methods and treated sequentially with xylene and graded ethanol solutions. 100 μL of proteinase K working solution was added, and the sections were incubated at room temperature for 10 min. TUNEL reaction solution was then added for 1.0–1.5 h. The slides were then immersed in a humidified chamber containing 4,6-diamidinyl-2-phenylindole (DAPI) solution and incubated in the dark for 5 min. After washing with phosphate-buffered saline (PBS), the slides were mounted and observed under a fluorescence microscope. Five different fields of view were randomly selected, TUNEL-positive cells were counted, and the apoptosis index was calculated.

[0071] 1.2.8 ELISA method for detecting the level of inflammatory factors in brain tissue

[0072] Brain tissue was obtained according to method 1.2.4. Hippocampal tissue from the ischemic brain of rats was collected, homogenized, and centrifuged to obtain supernatant. The levels of TNF-α and IL-10 were detected using an ELISA kit.

[0073] 1.3 Statistical Analysis

[0074] SPSS 22.0 software was used for statistical analysis. Data were expressed as mean ± standard deviation. One-way ANOVA and SNK-q test were performed for comparisons between groups. P < 0.05 was considered statistically significant.

[0075] 2 Results

[0076] 2.1 Effects of the traditional Chinese medicine of this invention on the neurological function of rats with ischemic stroke

[0077] Compared with the sham-operated group, the neurological deficit scores of rats in the model group were significantly increased (P<0.01); compared with the model group, the neurological deficit scores of rats in the positive control group, and the low, medium, and high dose groups of traditional Chinese medicine were all significantly decreased (P<0.01). The neurological deficit score of rats in the high dose group of traditional Chinese medicine was significantly lower than that of the positive control group (P<0.05). The results are as follows. Figure 1 As shown.

[0078] 2.2 Effects of the traditional Chinese medicine of this invention on the infarct volume in rats with ischemic stroke

[0079] Compared with the sham-operated group, rats in the model group showed significant cerebral infarction, with a significantly increased infarct volume ratio (P<0.01). Compared with the model group, the infarct volume ratios in the positive control group, and the low, medium, and high dose groups of traditional Chinese medicine were all significantly decreased (P<0.01). The infarct volume ratio in the high dose group of traditional Chinese medicine was significantly lower than that in the positive control group (P<0.05). Results are as follows. Figure 2 As shown.

[0080] 2.3 Effects of the traditional Chinese medicine of this invention on the water content of brain tissue in rats with ischemic stroke

[0081] Compared with the sham-operated group, the water content of brain tissue in the model group was significantly increased (P<0.01); compared with the model group, the water content of brain tissue in the positive control group, and the low, medium, and high dose groups of traditional Chinese medicine was significantly decreased (P<0.01). The water content of brain tissue in the high dose group of traditional Chinese medicine was significantly lower than that in the positive control group (P<0.05). The results are as follows. Figure 3 As shown.

[0082] 2.4 Effects of the traditional Chinese medicine of this invention on neurons in the hippocampus of rats with ischemic stroke

[0083] Compared with the sham-operated group, the number of Nissl bodies in the hippocampus of rats in the model group was significantly decreased (P<0.01), and the apoptosis rate of nerve cells was significantly increased (P<0.01). Compared with the model group, the number of Nissl bodies in the positive control group and the low, medium, and high dose groups of traditional Chinese medicine were all significantly increased (P<0.01), and the apoptosis rate of nerve cells was significantly decreased (P<0.01). The apoptosis rate of nerve cells in the high dose group of traditional Chinese medicine was significantly lower than that in the positive control group (P<0.05). The results are as follows. Figure 4 As shown.

[0084] 2.5 Effects of the traditional Chinese medicine of this invention on the levels of inflammatory factors in the brain tissue of rats with ischemic stroke

[0085] Compared with the sham-operated group, the level of TNF-α in the brain tissue of rats in the model group was significantly increased, and the level of IL-10 was significantly decreased (P<0.01). Compared with the model group, the level of TNF-α in the brain tissue of rats in the positive control group and the low, medium, and high dose groups of traditional Chinese medicine was significantly decreased, and the level of IL-10 was significantly increased (P<0.01). The level of TNF-α in the brain tissue of rats in the high dose group of traditional Chinese medicine was significantly lower than that in the positive control group (P<0.01), and the level of IL-10 was significantly higher than that in the positive control group (P<0.05).

[0086] 3. Conclusion

[0087] This experiment successfully established a rat model of ischemic stroke using the MCAO method. The rats in the model group exhibited significant neurological dysfunction, marked cerebral infarction and edema, significant neuronal apoptosis, a significant increase in the level of the pro-inflammatory factor TNF-α, and a significant decrease in the level of the anti-inflammatory factor IL-10. After administration of the herbal medicine of this invention to the ischemic stroke model rats, neurological dysfunction was significantly improved, the infarct volume and brain water content were significantly reduced, the number of Nissl bodies in the brain tissue was significantly increased, the neuronal apoptosis rate in the brain tissue was significantly reduced, and the levels of the pro-inflammatory factor TNF-α and the anti-inflammatory factor IL-10 in the brain tissue were significantly decreased. These results indicate that the herbal medicine of this invention exerts a good therapeutic effect on the ischemic stroke model rats by alleviating cerebral infarction and edema, improving neurological dysfunction, inhibiting neuronal apoptosis, and regulating the inflammatory response to reduce inflammatory damage to brain tissue.

[0088] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A traditional Chinese medicine composition, characterized in that, The traditional Chinese medicine composition consists of the following medicinal materials in parts by weight: 5-30 parts of *Gynostemma pentaphyllum*, 5-30 parts of *Panax japonicus*, 5-30 parts of *Rubus idaeus*, 5-30 parts of camphor wood, 5-30 parts of *Everlasting Heart*, 5-30 parts of *Vine of Fragrant Vine*, 5-30 parts of *Curculigo orchioides*, 5-30 parts of *Scissors Grass*, 5-30 parts of white hyacinth bean, and 5-30 parts of kudzu root.

2. The traditional Chinese medicine composition as described in claim 1, characterized in that, The traditional Chinese medicine composition consists of the following medicinal materials in parts by weight: 10-30 parts of *Gynostemma pentaphyllum*, 10-30 parts of *Panax japonicus*, 10-30 parts of *Rubus idaeus*, 10-30 parts of *Camphora sinensis*, 10-30 parts of *Euphorbia lathyris*, 10-30 parts of *Cephalotaxus fortunei*, 5-25 parts of *Curculigo orchioides*, 5-25 parts of *Hedyotis diffusa*, 5-25 parts of *Hymenopus pubescens*, and 5-25 parts of *Pueraria lobata*.

3. The traditional Chinese medicine composition as described in claim 1, characterized in that, The traditional Chinese medicine composition consists of the following parts by weight of medicinal materials: 24 parts of *Gynostemma pentaphyllum*, 26 parts of *Panax japonicus*, 20 parts of *Rubus idaeus*, 20 parts of *Camphora sinensis*, 18 parts of *Everlasting Heart*, 22 parts of *Vitis thunbergii*, 12 parts of *Curculigo orchioides*, 14 parts of *Smilax china*, 16 parts of *Hymenopus pubescens*, and 16 parts of *Pueraria lobata*.

4. The use of the traditional Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a drug for treating ischemic stroke, characterized in that, The drug is made from the traditional Chinese medicine composition as raw material, supplemented with pharmaceutically acceptable excipients, and prepared into any oral dosage form among oral liquid, tablets, pills, granules, powders, capsules, and decoctions.

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