Traditional Chinese medicine component compound for treating ischemic encephaledema and application thereof

Abstract

The invention relates to a traditional Chinese medicine component compound. The traditional Chinese medicine component compound comprises baicalin, salidroside and catalpol. The traditional Chinese medicine component compound disclosed by the invention is a traditional Chinese medicine compound, pays attention to enhancement of an autoimmune system, and is free of toxic and side effects and reasonable in formula; the traditional Chinese medicine composition effectively aims at main pathogenesis of ischemic encephaledema and cerebral ischemia, such as reduction of cerebral moisture content, reduction of cerebral ischemia area, alleviation of neurological dysfunction, reduction of neuronal apoptosis and the like, and has a remarkable curative effect; the raw materials are wide in source and easy to obtain, can be extracted and biosynthesized by adopting crude drugs, can also be directly used as chemical synthesis products, and are convenient for preparation and large-scale production.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a compound of traditional Chinese medicine components for treating ischemic cerebral edema and its application. Background Technology

[0002] Stroke, also known as apoplexy or cerebrovascular accident, is generally classified into two categories based on its cause: ischemic stroke and hemorrhagic stroke. Ischemic stroke accounts for more than 85% of stroke cases. Cerebral edema is a common pathological phenomenon following ischemic stroke, with a complex pathophysiological mechanism. It is closely related to the disability rate, mortality rate, and recovery of neurological function after cerebral ischemia, and is a major cause of worsening, deterioration, and even death in stroke patients. Cerebral edema usually appears rapidly after a stroke, leading to swelling of brain tissue, increased intracranial pressure, and limiting the recovery of normal brain function. In the early stages of cerebral edema formation, the patient's symptoms are relatively mild, and compensatory regulation can occur within the brain, such as reducing cerebrospinal fluid and blood volume to balance the increase in cerebral water. However, due to the presence of the skull, if the water content increases rapidly and the rate of cerebral edema progression exceeds the maximum compensatory capacity, it will lead to increased intracranial pressure, and the patient will typically experience various functional impairments in motor, sensory, cognitive, and speech functions. Meanwhile, severe cerebral edema increases the difficulty and risk of surgical treatment. Without timely treatment, it can even lead to brain herniation and respiratory failure, resulting in patient death. Therefore, alleviating and eliminating cerebral edema is a crucial research area in the treatment of ischemic cerebral diseases, and in-depth research and development of effective treatment methods are essential for improving ischemic cerebral edema.

[0003] Traditional Chinese medicine (TCM) ingredient combination, through rational drug combinations and dosage adjustments, aims to fully leverage the efficacy of each drug against specific etiologies and pathogenesis, achieving synergistic therapeutic effects, thereby improving efficacy and reducing side effects. For complex pathological mechanisms, the needs of using only a single effective component of TCM are often insufficient, highlighting the necessity of TCM ingredient combination therapy. The pathogenesis of ischemic cerebral edema involves multiple pathological mechanisms, such as inflammatory responses, oxidative stress, and blood-brain barrier disruption. TCM ingredient combination can comprehensively regulate multiple mechanisms, providing more effective treatment. This holistic treatment concept aligns closely with traditional Chinese medicine theory and the needs of comprehensive rehabilitation after stroke. The Chinese herbal medicine database contains a large number of natural components with potential therapeutic effects. Through scientific research and rational combination, based on this theory, there is the potential to develop more drugs with clinical application value, enriching the treatment methods for clinical diseases.

[0004] The combination of traditional Chinese medicine (TCM) ingredients to treat specific diseases demonstrates significant advancements in modern TCM research and development. Firstly, TCM ingredient combinations, through the synergistic action of multiple components on multiple targets, can comprehensively regulate pathological processes and enhance therapeutic efficacy. Secondly, while high doses of single drugs often trigger side effects, TCM ingredient combinations, through the rational combination of components, can exert synergistic effects at low doses, reducing side effects. Finally, the clear composition and defined content of TCM ingredients are its most significant advantages, combining the strengths of both TCM and Western medicine. By combining effective components according to the fundamental principles of TCM compatibility and optimal dose-effect, it retains some of the medicinal properties and indications of traditional Chinese medicine decoctions while fully utilizing modern science and technology to elucidate the rationality of the combinations, clarify pharmacological mechanisms, and achieve quality control. This overcomes the shortcomings of traditional TCM compound formulas, such as unclear components, complex and ambiguous mechanisms, and difficulty in quality control, providing new ideas and methods for the modernization of traditional Chinese medicine. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a solution.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a complex of traditional Chinese medicine components, the complex comprising baicalin, rhodioloside, and catalpol.

[0007] Ischemic cerebral edema is primarily attributed to qi stagnation and blood stasis. Traditional Chinese medicine (TCM) theory holds that qi is the commander of blood; qi deficiency weakens the body's ability to propel blood circulation, leading to circulatory disorders and blood stasis. TCM's holistic approach emphasizes the harmony of qi and blood, considering qi deficiency and blood stasis not only as pathological manifestations but also as the foundation for disease development. Therefore, the key to treating ischemic stroke lies in regulating qi and blood, promoting blood circulation, and removing blood stasis to restore normal blood circulation and bodily function. Rhodiola rosea, a traditional qi-tonifying and blood-activating herb, is often used to treat diseases caused by qi deficiency and blood stasis. Rhodioloside (CAS No. 10338-51-9) is the main active ingredient in Rhodiola rosea. In treating cerebrovascular diseases, rhodioloside has a protective effect against neuronal damage caused by cerebral ischemia, both in vivo and in vitro. Studies have found that rhodioloside can exert a neuroprotective effect after cerebral ischemia, mainly through mechanisms such as protecting vascular endothelial cells, reducing oxidative stress, and inhibiting neuronal apoptosis. The effects of rhodioloside are highly consistent with the concept of "tonifying qi and promoting blood circulation" in traditional Chinese medicine. It can effectively improve the state of qi deficiency and blood stasis, promote blood circulation, and reduce cerebral ischemia damage.

[0008] Modern Traditional Chinese Medicine (TCM) has also proposed the theory of "toxic damage to brain collaterals," believing that the pathogenesis of cerebral ischemia is "internal generation of toxic pathogens, originating in the diseased collaterals, damaging the brain collaterals, leading to insufficient nourishment of the brain and dysfunction of the brain." This theory emphasizes the role of toxic pathogens in the process of cerebral ischemia, believing that toxic pathogens not only damage the brain collaterals but also further lead to brain dysfunction. Therefore, TCM therapies of clearing heat and detoxifying are also of great significance in the treatment of cerebral ischemia. Baicalin (CAS No. 21967-41-9) is the main effective component of Scutellaria baicalensis, a representative TCM herb for clearing heat and detoxifying. It belongs to a class of flavonoid glycosides, with low chemical polarity, and can cross the blood-brain barrier to exert its effects. In the treatment of cerebral ischemia, baicalin has the effects of protecting the permeability of the blood-brain barrier, promoting nerve cell regeneration, and inhibiting the release of various inflammatory factors. Its mechanism of action mainly includes anti-oxidative stress, anti-inflammation, anti-apoptosis, and anti-excitatory amino acid toxicity. These effects are consistent with the concepts of clearing heat and detoxifying, and protecting the brain network in the theory of "damage to brain network by toxins". They can effectively reduce the damage of toxins to the brain network and improve the state of cerebral ischemia.

[0009] Traditional Chinese medicine (TCM) theory holds that the location of cerebral ischemia is in the brain, emphasizing that "the kidneys are connected to the brain," meaning the brain is the sea of ​​marrow, and the kidneys govern bones and produce marrow. Therefore, TCM believes that the pathogenesis of cerebral ischemia is also related to kidney qi deficiency leading to blood stasis, with qi and yin deficiency being the root cause and blood stasis and toxin accumulation as the manifestation. Treatment for cerebral ischemia should focus on nourishing yin and tonifying the kidneys, emphasizing improving the ischemic state by tonifying the kidneys and nourishing blood. Ziziphus jujuba var. spinosa (CAS No. 2415-24-9) is found in high concentrations in Rehmannia glutinosa, a TCM herb for tonifying the kidneys and nourishing blood. TCM believes that Rehmannia glutinosa nourishes yin and blood by entering the kidneys. In treating cerebral ischemia, Ziziphus jujuba var. spinosa works by inhibiting ischemic neuronal apoptosis, inhibiting astrocyte proliferation, and exhibiting anti-inflammatory effects. These effects of Ziziphus jujuba var. spinosa align closely with the TCM concept of "nourishing yin and tonifying the kidneys," effectively improving kidney qi deficiency and qi and yin deficiency, promoting blood circulation, and reducing cerebral ischemia damage.

[0010] In the concept of traditional Chinese medicine (TCM) formulation, TCM emphasizes holistic regulation, achieving comprehensive therapeutic effects through the synergistic effects of multiple components. The TCM composition provided in this invention contains ingredients with effects such as promoting blood circulation and removing blood stasis, tonifying qi and kidneys, and clearing heat and detoxifying. Through rational drug combination and dosage adjustment, the synergistic effects of each drug are utilized. Rhodiola rosea glycosides can invigorate qi and promote blood circulation, baicalin can clear heat and detoxify, and catalpol can nourish yin and tonify kidneys. These components work synergistically to exert a joint therapeutic effect. This multi-component, multi-target treatment approach can comprehensively regulate the pathological process and improve therapeutic efficacy. Therefore, the TCM formulation for treating cerebral edema and cerebral ischemia, with its multi-component, multi-target, and holistic regulatory characteristics, demonstrates unique advantages and potential. The TCM concepts of qi deficiency and blood stasis, toxin damage to brain collaterals, and the kidneys' connection to the brain provide a theoretical basis for the formulation of TCM components.

[0011] In some embodiments, the herbal compound contains, by weight, 5-200 parts of baicalin, 5-150 parts of rhodioloside, and 5-550 parts of catalpol. For example, the herbal compound may contain 5, 25, 50, 75, 100, 125, 150, 175, or 200 parts of baicalin, 5, 25, 50, 75, 100, 125, or 150 parts of rhodioloside, and 5, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, or 550 parts of catalpol.

[0012] In some embodiments, the herbal compound contains, by weight, 50-200 parts of baicalin, 25-150 parts of rhodioloside, and 75-400 parts of catalpol. For example, the herbal compound contains 50, 75, 100, 125, 150, 175, or 200 parts of baicalin, 25, 50, 75, 100, 125, or 150 parts of rhodioloside, and 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 parts of catalpol.

[0013] In some embodiments, the herbal compound contains, by weight, 150-200 parts of baicalin, 75-125 parts of rhodioloside, and 250-350 parts of catalpol. For example, the weight parts of baicalin in the aforementioned herbal ingredient complex are 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 parts; the weight parts of rhodioloside are 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 parts; and the weight parts of catalpol are 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 parts.

[0014] In some embodiments, the mass ratio of baicalin, rhodioloside, and catalpol in the herbal compound is (5-20):(2.5-10):(7.5-30).

[0015] In some embodiments, the mass ratio of baicalin, rhodioloside, and catalpol in the herbal compound is 1:(0.25-0.75):(1-2).

[0016] In some embodiments, the mass ratio of baicalin, rhodioloside, and catalpol in the herbal compound is 1:0.5:1.5.

[0017] In some embodiments, the molar ratio of baicalin, rhodioloside, and catalpol in the herbal compound is (1-6):(1-50):(1-100). For example, the molar ratio of baicalin, rhodioloside, and catalpol in the herbal compound is 1:1:1, 1.5:25:100, 6:50:100, 1.5:12.5:25, 3:12.5:100, or 3:25:25.

[0018] In some embodiments, the herbal compound also contains one or more of tanshinone, gastrodin, and geniposide.

[0019] In some embodiments, the herbal compound also contains tanshinone.

[0020] In some embodiments, the herbal compound contains, by weight, 150-200 parts of baicalin, 75-125 parts of rhodioloside, 250-350 parts of catalpol, and 150-200 parts of tanshinone. For example, the herbal compound may contain 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 parts of baicalin, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 parts of catalpol. The number of servings is 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345 or 350, and the number of tanshinone servings by weight is 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200.

[0021] In some embodiments, the herbal compound also contains gastrodin.

[0022] In some embodiments, the herbal compound contains, by weight, 150-200 parts of baicalin, 75-125 parts of rhodioloside, 250-350 parts of catalpol, and 150-200 parts of gastrodin. For example, the herbal compound may contain 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 parts of baicalin, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 parts of catalpol. The number of servings is 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345 or 350, and the number of gastrodin weight servings is 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200.

[0023] In some embodiments, the herbal compound also contains tanshinone and geniposide.

[0024] In some embodiments, the herbal compound contains, by weight, 150-200 parts of baicalin, 75-125 parts of rhodioloside, 250-350 parts of catalpol, 150-200 parts of tanshinone, and 250-350 parts of geniposide. For example, the weight percentages of baicalin in the aforementioned herbal ingredient complex are 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 parts; the weight percentages of rhodioloside are 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 parts; and the weight percentages of catalpol are 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 parts. 305, 310, 315, 320, 325, 330, 335, 340, 345 or 350 parts by weight; tanshinone in weight parts of 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200 parts by weight; geniposide in weight parts of 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345 or 350 parts by weight.

[0025] In some embodiments, the herbal compound also contains gastrodin and geniposide.

[0026] In some embodiments, the herbal compound contains, by weight, 150-200 parts of baicalin, 75-125 parts of rhodioloside, 250-350 parts of catalpol, 150-200 parts of gastrodin, and 250-350 parts of geniposide. For example, the weight percentages of baicalin in the aforementioned herbal ingredient complex are 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 parts; the weight percentages of rhodioloside are 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 parts; and the weight percentages of catalpol are 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 parts. 305, 310, 315, 320, 325, 330, 335, 340, 345 or 350 parts by weight; gastrodin in parts by weight of 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200 parts by weight; geniposide in parts by weight of 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345 or 350 parts by weight.

[0027] In some embodiments, the herbal compound also contains gastrodin, tanshinone, and geniposide.

[0028] In some embodiments, the herbal compound contains, by weight, 150-200 parts of baicalin, 75-125 parts of rhodioloside, 250-350 parts of catalpol, 150-200 parts of gastrodin, 150-200 parts of tanshinone, and 250-350 parts of geniposide. For example, the weight percentages of baicalin in the aforementioned herbal ingredient complex are 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 parts; the weight percentages of rhodioloside are 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 parts; and the weight percentages of catalpol are 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, or 335 parts. The weight percentages of gastrodin are 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 parts; the weight percentages of tanshinone are 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 parts; and the weight percentages of geniposide are 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, and 350 parts.

[0029] In some embodiments, the herbal compound is composed of baicalin, rhodioloside, and catalpol.

[0030] A second aspect of the present invention provides a pharmaceutical composition comprising the traditional Chinese medicine component complex described in the first aspect.

[0031] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0032] In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0033] In some embodiments, the excipients further include at least one of fillers, diluents, disintegrants, binders, lubricants, flow aids, surfactants, solvents, flavoring agents, and preservatives.

[0034] In some embodiments, the filler or diluent includes sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, α-starch, and dextrin; celluloses such as microcrystalline cellulose, gum arabic, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, pharmaceutical-grade calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0035] In some embodiments, the lubricant, flow aid, or anti-sticking agent includes stearic acid; metal stearate salts such as calcium stearate or magnesium stearate; talc; colloidal silica; micronized silica gel; hydrogenated vegetable oil; polyethylene glycol; lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicates such as silicic anhydride or silicate hydrates, etc.

[0036] In some embodiments, the adhesive includes distilled water, ethanol of varying concentrations, starch paste, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, ethyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, polyethylene glycol, and compounds similar to the excipients described above.

[0037] In some embodiments, the disintegrant includes cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, or croscarmellose sodium; croscarmellose; and chemically modified starch / cellulose, such as carboxymethyl starch or sodium carboxymethyl starch.

[0038] In some embodiments, the surfactant includes Tween, sodium dodecyl sulfate, sodium stearate sulfonate, etc.

[0039] In some embodiments, the antioxidant includes sodium bisulfite, sodium metabisulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine, thiourea, phosphoric acid, citric acid, etc.

[0040] In some embodiments, the preservative or antibacterial agent includes benzoic acid and sodium benzoate, sorbic acid, ethanol, parabens, benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenylethanol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil, and peppermint oil.

[0041] In some embodiments, the flavoring agent includes sweeteners such as sodium saccharin, aspartame, syrup, steviol, mannitol, sorbitol, mannose, galactose, maltose, fructose, glucose, sucrose, etc.; acid flavoring agents such as citric acid, malic acid or tartaric acid; and aroma agents such as fennel oil, peppermint oil, menthol, peppermint water, cinnamon oil, lemon essence, lemon oil and various flavorings.

[0042] In some embodiments, the pharmaceutical composition is in the form of a gastrointestinal or non-gastrointestinal dosage form.

[0043] In some embodiments, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0044] In some embodiments, the non-gastrointestinal dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0045] In some embodiments, the dosage form of the pharmaceutical composition is any one of powder, granules, tablets, capsules, gels, suspensions, nasal drops, pills, injections, suppositories, aerosols, oral liquids, ointments, emulsions, and irrigants.

[0046] The third aspect of this invention provides the application of the traditional Chinese medicine component complex described in the first aspect or the pharmaceutical composition described in the second aspect in any of the following aspects: preparing a drug for treating cerebral edema; preparing a drug for treating cerebral ischemia; preparing a drug for reducing the infarct area in patients with cerebral ischemia; preparing a drug for reducing the water content of brain tissue in patients with cerebral ischemia; preparing a drug for improving the neurological deficits in patients with cerebral ischemia.

[0047] In some embodiments, the cerebral edema is ischemic cerebral edema.

[0048] The beneficial effects of this invention are: The herbal compound of the present invention is a traditional Chinese medicine compound that focuses on enhancing the body's own immune system, has no toxic side effects, and has a reasonable formulation. It effectively targets the main pathogenesis of ischemic cerebral edema and cerebral ischemia, such as reducing brain water content, reducing the area of ​​cerebral ischemia, alleviating neurological dysfunction, and reducing neuronal apoptosis, with significant therapeutic effects. The raw materials are widely available and easy to obtain. They can be extracted from crude drugs, biosynthesized, or directly used as chemically synthesized products, which facilitates preparation and large-scale production. Attached Figure Description

[0049] Figure 1 The effects of the herbal compound complexes from Examples 6 and 7 on the viability of HCMEC cells induced by OGD / R damage were demonstrated. Compared with the normal group, Compared with the model group, # P <0.05, ## P <0.01.

[0050] Figure 2The effects of the herbal compound complexes from Examples 6 and 7 on the viability of SVGp12 cells induced by OGD / R damage were demonstrated. Compared with the normal group, Compared with the model group, # P <0.05, ## P <0.01.

[0051] Figure 3 The effects of the herbal compound complexes from Examples 6 and 7 on the viability of SH-SY5Y cells induced by OGD / R damage were demonstrated. Compared with the normal group, Compared with the model group, # P <0.05, ## P <0.01.

[0052] Figure 4 The study demonstrated the effect of the drug on the area of ​​cerebral infarction in rats after cerebral ischemia-reperfusion.

[0053] Figure 5 The study demonstrated the effects of the drug on brain tissue damage in rats following cerebral ischemia-reperfusion.

[0054] Figure 6 This study demonstrates the effects of drugs on DWI and T2WI sequences in rats after cerebral ischemia-reperfusion injury. A shows DWI images in MRI; B shows DWI ADC value analysis; and C shows quantitative analysis of cerebral infarction area on T2WI sequences, compared with the sham-operated group. Compared with the model group, # P <0.05, ## P <0.01.

[0055] Figure 7 This study demonstrated the effect of the drug on blood-brain barrier permeability in rats after cerebral ischemia-reperfusion.

[0056] Figure 8 This study demonstrated the effect of the drug on cell apoptosis in rat brain tissue after cerebral ischemia-reperfusion.

[0057] Figure 9 The effects of the drug on the ultrastructure of the rat brain after cerebral ischemia-reperfusion were demonstrated.

[0058] Figure 10 The results of the Western blot (WB) experiment on brain edema-related proteins were presented.

[0059] Figure 11 The relative expression levels of proteins associated with cerebral edema are shown. Compared with the sham-operated group: Compared with the model group, # P <0.05,## P <0.01. Detailed Implementation

[0060] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0061] The baicalin in this embodiment of the invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd., batch number: B20570, specification: 1g. The rhodioloside in this embodiment of the invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd., batch number: B20504, specification: 1g. The catalpol in this embodiment of the invention was purchased from Chengdu Mansite Biotechnology Co., Ltd., batch number: A0215, specification: 1g.

[0062] Example 1: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine components, consisting of 1.5 μmol baicalin, 25 μmol rhodioloside, and 100 μmol catalpol.

[0063] Example 2: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine components, consisting of 6 μmol baicalin, 50 μmol rhodioloside, and 100 μmol catalpol.

[0064] Example 3: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine components, consisting of 1.5 μmol baicalin, 12.5 μmol rhodioloside, and 25 μmol catalpol.

[0065] Example 4: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine components, consisting of 3 μmol baicalin, 12.5 μmol rhodioloside, and 100 μmol catalpol.

[0066] Example 5: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine components, consisting of 3 μmol baicalin, 25 μmol rhodioloside, and 25 μmol catalpol.

[0067] Example 6: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine components, consisting of 12.5 μmol baicalin, 12.5 μmol rhodioloside, and 12.5 μmol catalpol.

[0068] Example 7: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine components, consisting of 6.25 μmol baicalin, 6.25 μmol rhodioloside, and 6.25 μmol catalpol.

[0069] Example 8: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine ingredients, consisting of 5g baicalin, 2.5g rhodioloside, and 7.5g catalpol.

[0070] Example 9: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine ingredients, consisting of 5g baicalin, 5g rhodioloside, and 30g catalpol.

[0071] Example 10: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine ingredients, consisting of 10g baicalin, 10g rhodioloside, and 7.5g catalpol.

[0072] Example 11: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine ingredients, consisting of 20g baicalin, 5g rhodioloside, and 15g catalpol.

[0073] Example 12: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine ingredients, consisting of 5g baicalin, 10g rhodioloside, and 15g catalpol.

[0074] Example 13: Complex of Traditional Chinese Medicine Components A complex of traditional Chinese medicine ingredients, consisting of 10g baicalin, 2.5g rhodioloside, and 15g catalpol.

[0075] Example 14: Solution of Traditional Chinese Medicine Component Complex Complex composition: baicalin 5g, rhodioloside 2.5g, catalpol 7.5g.

[0076] Preparation method: Dissolve the precisely weighed Chinese herbal ingredients in physiological saline (pH=6.5~8.0), stir until evenly dissolved, adjust to a suitable pH (pH=6.5~8.0), and filter aseptically to obtain the physiological saline solution of the Chinese herbal ingredient complex.

[0077] Example 15: Solution of Traditional Chinese Medicine Component Complex Complex composition: 50g baicalin, 25g rhodioloside, 75g catalpol, 50g tanshinone.

[0078] Preparation method: Dissolve the precisely weighed Chinese herbal ingredients in physiological saline (pH=6.5~8.0), vortex until evenly dissolved, adjust to a suitable pH (pH=6.5~8.0), and filter aseptically to obtain the physiological saline solution of the Chinese herbal ingredient complex.

[0079] Example 16: Solution of Traditional Chinese Medicine Component Complex Complex composition: baicalin 5g, rhodioloside 2.5g, catalpol 7.5g, gastrodin 5g.

[0080] Preparation method: Dissolve the precisely weighed Chinese herbal ingredients in physiological saline (pH=6.5~8.0), vortex until evenly dissolved, adjust to a suitable pH (pH=6.5~8.0), and filter aseptically to obtain the physiological saline solution of the Chinese herbal ingredient complex.

[0081] Example 17: Solution of Traditional Chinese Medicine Component Complex Complex composition: baicalin 50g, rhodioloside 25g, catalpol 75g, tanshinone 50g, geniposide 75g.

[0082] Preparation method: Dissolve the precisely weighed Chinese herbal ingredients in physiological saline (pH=6.5~8.0), vortex until evenly dissolved, adjust to a suitable pH (pH=6.5~8.0), and filter aseptically to obtain the physiological saline solution of the Chinese herbal ingredient complex.

[0083] Example 18: Solution of Traditional Chinese Medicine Component Complex Complex composition: 100g baicalin, 50g rhodioloside, 150g catalpol, 50g gastrodin, 50g tanshinone, 75g geniposide.

[0084] Preparation method: Dissolve the precisely weighed Chinese herbal ingredients in physiological saline, vortex until evenly dissolved, adjust to a suitable pH (pH=6.5~8.0), and filter aseptically to obtain the physiological saline solution of the Chinese herbal ingredient complex.

[0085] Example 19: Solution of Traditional Chinese Medicine Component Complex Complex composition: baicalin 5g, rhodioloside 2.5g, catalpol 7.5g, gastrodin 5g, geniposide 7.5g.

[0086] Preparation method: Dissolve the precisely weighed Chinese herbal ingredients in physiological saline, vortex until evenly dissolved, adjust to a suitable pH (pH=6.5~8.0), and filter aseptically to obtain the physiological saline solution of the Chinese herbal ingredient complex.

[0087] Example 20: Injection of Traditional Chinese Medicine Component Complex Complex composition: baicalin 5g, rhodioloside 2.5g, catalpol 7.5g.

[0088] Preparation method: Dissolve the precisely weighed Chinese herbal ingredients in physiological saline, stir until uniformly dissolved, adjust to a suitable pH (pH=6.5~8.0), filter aseptically, and adjust to a suitable osmotic pressure (280~290mmol / L) to obtain the Chinese herbal ingredient complex injection.

[0089] Example 21: Dry powder of traditional Chinese medicine component complex Complex composition: 0.5 kg baicalin, 0.25 kg rhodioloside, and 0.75 kg catalpol.

[0090] Preparation method: Dissolve the precisely weighed Chinese herbal ingredients in physiological saline, vortex until evenly dissolved, adjust to a suitable pH (pH=6.5~8.0), filter under sterile conditions, and spray dry to obtain the dry powder of the Chinese herbal ingredient complex.

[0091] Example 22: Compound Particles of Traditional Chinese Medicine Components Complex composition: baicalin 50g, rhodioloside 25g, catalpol 75g.

[0092] Preparation: Accurately weighed Chinese herbal ingredients are dissolved in physiological saline, vortexed until uniformly dissolved, and adjusted to a suitable pH (6.5-8.0). The solution is then aseptically filtered and freeze-dried. 100g of the dry extract powder is taken and mixed with excipients in a 1:1 mass ratio (1:1:1 mass ratio of maltodextrin, microcrystalline cellulose, and lactose). The mixture is pressed into thin sheets using a dry extrusion granulator, and then pulverized into granules. The granules are then sized, re-granulated, and pulverized to obtain granule formulations.

[0093] Example 23: Traditional Chinese Medicine Ingredient Complex Capsules Complex composition: baicalin 50g, rhodioloside 25g, catalpol 75g.

[0094] Preparation: Dissolve the precisely weighed Chinese herbal ingredients in physiological saline, vortex until uniformly dissolved, adjust to a suitable pH (6.5-8.0), aseptically filter and freeze-dry. Take 100g of the dried powder and mix it with excipients at a mass ratio of 1:2. The excipients include soluble starch, microcrystalline cellulose, and mannitol in a mass ratio of 1:1:1. Press the mixture into thin sheets using a dry extrusion granulator, then pulverize it into granules. Fill the fine granules into #0 empty capsules to prepare capsules.

[0095] Example 24: Nasal Drops Containing Traditional Chinese Medicine Ingredients Complex composition: 100g baicalin, 50g rhodioloside, 150g catalpol.

[0096] Preparation: Dissolve the precisely weighed Chinese herbal ingredients in physiological saline, vortex until evenly dissolved, adjust to a suitable pH (pH=6.5~8.0), filter aseptically, adjust to a suitable osmotic pressure (280~290mmol / L), add appropriate amounts of mannitol and glycerin to obtain the Chinese herbal ingredient complex nasal drops.

[0097] Comparative Example 1 A complex of traditional Chinese medicine ingredients, consisting of 1.5 μmol of baicalin.

[0098] Comparative Example 2 A complex of traditional Chinese medicine components, consisting of 3 μmol baicalin and 50 μmol catalpol.

[0099] Comparative Example 3 A complex of traditional Chinese medicine components, consisting of 100 μmol catalpol.

[0100] Comparative Example 4 A complex of traditional Chinese medicine components, consisting of 25 μmol of rhodioloside.

[0101] Comparative Example 5 A complex of traditional Chinese medicine components, consisting of 3 μmol baicalin and 50 μmol rhodioloside.

[0102] Comparative Example 6 A complex of traditional Chinese medicine components, consisting of 6 μmol baicalin and 12.5 μmol rhodioloside.

[0103] Comparative Example 7 A complex of traditional Chinese medicine components, consisting of 6 μmol baicalin and 25 μmol catalpol.

[0104] Comparative Example 8 A complex of traditional Chinese medicine components, consisting of 12.5 μmol rhodioloside and 50 μmol catalpol.

[0105] Comparative Example 9 A complex of traditional Chinese medicine ingredients, consisting of 20g baicalin and 2.5g rhodioloside.

[0106] Comparative Example 10 A compound of traditional Chinese medicine ingredients, consisting of 2.5g rhodioloside and 30g catalpol.

[0107] Comparative Example 11 A compound of traditional Chinese medicine ingredients, consisting of 10g baicalin and 30g catalpol.

[0108] Comparative Example 12 A complex of traditional Chinese medicine ingredients, consisting of 5g rhodioloside and 7.5g catalpol.

[0109] Example 25: Verification of the efficacy of traditional Chinese medicine component complex using brain endothelial cells In this embodiment, human brain microvascular endothelial cells (HCMEC) were used as the basis to analyze and verify the effects of the herbal compound components in Examples 1-5 and Comparative Examples 1-8 using cell viability (OD) values.

[0110] Cells: Human brain microvascular endothelial cells (HCMEC).

[0111] Test methods 1. Drug preparation and experimental arrangement: The compound of Chinese medicine components was prepared into stock solutions using DMSO for later use; 10% fetal bovine serum and 1% penicillin-streptomycin bispecific antibody were added to the special culture medium for human brain microvascular endothelial cells to obtain the complete cell culture medium.

[0112] 2. Cell culture and model establishment: Room temperature cells were purchased for this experiment. The cell growth status was observed and the cells were placed in an incubator (37 ℃, 5% CO2) for 24 hours. The medium was changed afterward. When the cell status was stable and the growth density reached 80%, the cells were passaged at a ratio of 1:3. After passage 2 times, the cells were retained for subsequent experiments, and the remaining cells were cryopreserved.

[0113] Before cell seeding, discard the culture medium in the culture flask, wash the cells three times with 1 mL PBS, add a small amount of trypsin to cover the cells, and let them stand at room temperature for 10 minutes to allow the cells to digest and detach. Add culture medium to stop the digestion. Collect the cell suspension in the flask, count the cells, and adjust the HCMEC concentration to 1×10⁻⁶. 5 The inoculation volume was 100 μL / well in a 96-well culture plate. After the plate was laid, it was placed in an incubator and subsequent experiments could be carried out after 24 hours.

[0114] The culture medium for cells seeded in 96-well plates for 24 hours was discarded, and the cells were washed three times with PBS. 100 μL of complete culture medium was added to each well of the control group, and 100 μL of sugar-free DMEM culture medium was added to each well of the model group. 100 μL of pre-dissolved sugar-free DMEM culture medium was added to each of the remaining wells according to the experimental group. The concentrations of each herbal component in the sugar-free DMEM culture medium for each experimental group are shown in Table 1. Table 1: Concentrations of various traditional Chinese medicine components in sugar-free DMEM culture medium

[0115] 96-well plates were placed in a hypoxic chamber filled with 5% CO2 and 95% N2 for closed culture. After hypoxia, the 96-well plates were placed in an incubator for reoxygenation and reglucose culture; this process constitutes an OGD / R-induced damage model for cells. Finally, cell viability was measured to evaluate the effect of the drug on the cells. The OGD / R conditions for HCMEC cells were: HCMEC OGD / R 2 h / 2 h.

[0116] 3. Cell viability assay: After 24 hours of cell culture and OGD / R-induced damage, 10 μL of CCK8 solution was added to each well. The culture plate was gently shaken to mix the solution thoroughly and incubated in an incubator for 2 hours. After incubation, the absorbance was measured using a microplate reader at a wavelength of 450 nm, and the cell OD value was read.

[0117] 4. Statistical methods: GraphPad Prism software was used for data analysis. Data are expressed as mean ± standard deviation. One-way ANOVA was used to compare multiple groups of data. P <0.05 indicates a statistically significant difference. P <0.01 indicates a statistically significant difference.

[0118] Test results The cell viability results for each group are shown in Table 2: Table 2: Results of cell viability assay

[0119] Based on the above results, it can be seen that the herbal compound in Examples 1-5 has a better effect on improving cell viability of HCMEC cells with OGD / R induced damage than the herbal compound in Comparative Examples 1-8, indicating that the combination of baicalin, rhodioloside and catalpol can achieve better results.

[0120] Example 26: Verification of the efficacy of the traditional Chinese medicine component complex using cerebral endothelial cells, astrocytes, and neurons. In this embodiment, human brain microvascular endothelial cells (HCMEC), human brain astrocytes (SVGp12), and human neuroblastoma cells (SH-SY5Y) were used as the basis to analyze and verify the effects of the traditional Chinese medicine component complex in Examples 6-7 using cell viability values ​​(OD values).

[0121] Cells: Human brain microvascular endothelial cells (HCMEC), human brain astrocytes (SVGp12), and human neuroblastoma cells (SH-SY5Y).

[0122] Test methods 1. Drug Preparation and Experimental Arrangement: The herbal compound components were prepared as stock solutions using DMSO. 10% fetal bovine serum and 1% penicillin-streptomycin were added to specific culture media for human brain microvascular endothelial cells, human brain astrocytes, and human neuroblastoma cells, respectively, to obtain the corresponding complete cell culture media. In cell experiments, drug concentrations were serially diluted starting from the highest concentration.

[0123] 2. Cell culture and model establishment: Room temperature cells were purchased for this experiment. The cell growth status was observed and the cells were placed in an incubator (37 ℃, 5% CO2) for 24 hours. The medium was changed afterward. When the cell status was stable and the growth density reached 80%, the cells were passaged at a ratio of 1:3. After passage 2 times, the cells were retained for subsequent experiments, and the remaining cells were cryopreserved.

[0124] Before cell seeding, discard the culture medium in the culture flask, wash the cells three times with 1 mL PBS, add a small amount of trypsin to cover the cells, and let them stand at room temperature for 10 minutes to allow the cells to digest and detach. Add culture medium to stop the digestion. Collect the cell suspension in the flask, count the cells, and adjust the concentration of HCMEC and SVGp12 cells to 1×10⁻⁶. 5 The concentration of SH-SY5Y cells was 3 × 10⁶ cells / mL. 5 The inoculation volume was 100 μL / well in a 96-well culture plate. After the plate was laid, it was placed in an incubator and subsequent experiments could be carried out after 24 hours.

[0125] The culture medium for cells seeded in 96-well plates for 24 hours was discarded. Cells were washed three times with PBS. 100 μL of the corresponding complete culture medium was added to each well of the Control group (normal group), and 100 μL of sugar-free DMEM medium was added to each well of the Model group. 100 μL of pre-dissolved sugar-free DMEM medium was added to each of the remaining wells according to the experimental group. The concentrations of each herbal component in the sugar-free DMEM medium for each experimental group are shown in Table 3. Table 3: Concentrations of various traditional Chinese medicine components in sugar-free DMEM culture medium

[0126] 96-well plates were placed in a hypoxic chamber filled with 5% CO2 and 95% N2 for closed culture. After hypoxia, the 96-well plates were placed in an incubator for reoxygenation and reglucose culture; this process constitutes an OGD / R-induced damage model for cells. Cell viability was then measured to evaluate the effects of the drug on the cells. Based on previous laboratory experience and experimental results, the final OGD / R conditions for the three cell types were determined as follows: HCMEC OGD / R 2h / 2h; SVGp12 OGD / R 4h / 2h; SH-SY5Y OGD / R 6h / 2h.

[0127] 3. Cell viability assay: After 24 hours of cell culture and OGD / R-induced damage, 10 μL of CCK8 solution was added to each well. The culture plate was gently shaken to mix the solution thoroughly and incubated in an incubator for 2 hours. After incubation, the absorbance was measured using a microplate reader at a wavelength of 450 nm, and the cell OD value was read.

[0128] 4. Statistical methods: GraphPad Prism software was used for data analysis. Data are expressed as mean ± standard deviation. One-way ANOVA was used to compare multiple groups of data. P <0.05 indicates a statistically significant difference. P <0.01 indicates a statistically significant difference.

[0129] Test results 1. Effects of the herbal compound components in Examples 6 and 7 on the viability of HCMEC cells induced by OGD / R damage. Compared with the normal group, the HCMEC cell viability in the model group was significantly reduced. P <0.01); Compared with the model group, the HCMEC cell viability was significantly increased after administration of the traditional Chinese medicine component complex in Examples 6 and 7 ( P <0.05), see Figure 1.

[0130] 2. Effects of the herbal compound components in Examples 6 and 7 on the viability of SVGp12 cells induced by OGD / R damage. Compared with the normal group, the viability of SVGp12 cells in the model group was significantly reduced. P <0.01); Compared with the model group, the viability of SVGp12 cells after administration of the traditional Chinese medicine component complex in Examples 6 and 7 was significantly increased ( P <0.05), see Figure 2.

[0131] 3. Effects of the herbal compound components in Examples 6 and 7 on the viability of SH-SY5Y cells induced by OGD / R damage. Compared with the normal group, the viability of SH-SY5Y cells in the model group was significantly reduced. P <0.01); Compared with the model group, the SH-SY5Y cell viability was significantly increased after administration of the traditional Chinese medicine component complex in Examples 6 and 7 ( P <0.05), see Figure 3.

[0132] Example 27: Verification of the efficacy of traditional Chinese medicine component complex in a rat model of cerebral ischemia-reperfusion. This embodiment uses a rat model of cerebral ischemia-reperfusion injury and combines pharmacodynamic indicators such as cerebral infarction area, brain water content, brain index and neurobehavioral scores to verify the effects of the traditional Chinese medicine compound in Examples 8-13.

[0133] Test materials 1. Drug Preparation: The herbal compound components in Examples 8-13 were dissolved in 0.9% physiological saline. Baicalin has low polarity and poor water solubility; therefore, the pH needs to be adjusted to 7 using NaOH. At this point, the solution is yellow-green and transparent, and baicalin can be completely dissolved. Rhodioloside and ziziphus jujuba var. spinosa are more polar and have better water solubility, and can be directly dissolved in physiological saline.

[0134] 2. Animals: Male SD rats, SPF grade, 200-220 g, provided by Spiford (Beijing) Biotechnology Co., Ltd.

[0135] Test methods 1. Rat MCAO / R Model Establishment: Rats underwent MCAO / R modeling. Anesthesia was administered via intraperitoneal injection of 2% sodium pentobarbital. The neck was shaved and disinfected. A midline incision was made, and the right common carotid artery, external carotid artery, and internal carotid artery were isolated. The internal carotid artery was clamped with a microartery clip. The common carotid artery and external carotid artery were ligated proximally. An incision was made 2 mm from the bifurcation of the common carotid artery, and a suture was inserted into the internal carotid artery. The suture was lightly tied at the distal end of the common carotid artery. The suture was gently pushed forward with ophthalmic forceps. The suture diameter was 0.24 mm, and the head diameter was (0.32±0.02) mm. The insertion depth was calculated from the bifurcation and reached 2 cm, securing the cells at the distal end of the common carotid artery. Drug administration was administered immediately after suture insertion. Reperfusion was performed 90 minutes later, allowing blood to re-enter the middle cerebral artery through the Circle of Willis, achieving cerebral vascular reperfusion. Finally, the animal wound was sutured, and the animals were returned to their cages, provided with normal water and feed, and their condition was observed.

[0136] 2. Animal grouping and administration: Rats were divided into a sham-operated group, a model group (administered with physiological saline), and an experimental group (administered with the compound solution of the traditional Chinese medicine components in Examples 8-13). Administration was via tail vein injection, with a volume of 2 mL / kg. All groups had normal food and water intake. Dosage for each group is shown in Table 4. Table 4: Dosage

[0137] Each group consisted of 12 rats. After 24 hours of cerebral ischemia, rats that failed to establish the model or died were removed. Eight rats were randomly selected from each group to measure the cerebral infarction area, brain water content, brain index, and neurobehavioral score.

[0138] 3. Statistical Methods: The experimental data are expressed as mean ± standard deviation (Mean ± SD). IBM SPSS and GraphPad Prism software were used for data analysis and graphing. The statistical methods used in this section include range analysis and variance analysis.

[0139] Test results The experimental results are shown in Table 5: Table 5: Experimental Results

[0140] The same method was used to detect the effects of the herbal compound components in Comparative Examples 9-12. The dosages are shown in Table 6. Table 6: Dosage

[0141] The results showed that after administration of the herbal compound in Comparative Example 9, the brain water content (%) of rats was 82.344±2.551; after administration of the herbal compound in Comparative Example 10, the brain water content (%) of rats was 82.381±1.142; after administration of the herbal compound in Comparative Example 11, the brain water content (%) of rats was 82.605±1.419; and after administration of the herbal compound in Comparative Example 12, the brain water content (%) of rats was 83.086±1.391.

[0142] The above results indicate that, based on the cerebral ischemia-reperfusion model rats, the herbal compound complexes in Examples 8-12 are superior to those in Comparative Examples 9-12 in improving brain water content. Particularly noteworthy is the presence of 2 parts by weight of baicalin, 1 part by weight of rhodioloside, and 3 parts by weight of catalpol in the herbal compound complex, achieving optimal results across multiple key indicators. This combination significantly reduced the infarct area, decreased brain tissue water content, and improved neurological deficit scores, demonstrating a good neuroprotective effect on MCAO / R model rats. Simultaneously, this herbal compound complex minimizes dosage while maximizing efficacy, achieving truly high efficiency and low toxicity.

[0143] Example 28: Verification of the efficacy of a traditional Chinese medicine compound in a rat model of focal cerebral ischemia. This embodiment further verifies the effect of the herbal compound in Example 20 using a focal cerebral ischemia model rat.

[0144] Test materials 1. Drug preparation: Sodium aescinate (positive control drug), purchased from Shandong Luye Pharmaceutical Co., Ltd., batch number: 231102106, specification: 5 mg, dosage: 2.5 mg / kg body weight.

[0145] 2. Animals: Male SD rats, SPF grade, 200-220 g, provided by Spiford (Beijing) Biotechnology Co., Ltd.

[0146] Test methods 1. Model Establishment: A rat model of focal cerebral ischemia was established by inserting a nylon suture embolism through the common carotid artery, following the method of Longa et al. Animals were anesthetized and fixed in a supine position on a warming blanket. The carotid artery (CCA), intracerebral artery (ICA), and extracerebral artery (ECA) were separated. The ECA was ligated and cut. The CCA was temporarily blocked with a slipknot. After clamping the ICA with an arterial clamp, an incision was made in the ECA, and a 0.24 mm diameter nylon suture was inserted until significant resistance was felt, to a depth of 20 ± 2 mm (measured from the bifurcation of the common carotid artery), causing occlusion and ischemia of the middle cerebral artery, thus establishing a rat model of focal cerebral ischemia. In the sham-operated group, only the CCA was separated and the ECA ligated, without inserting the nylon suture. After 1.5 hours, the nylon suture was removed, and the CCA suture was released to achieve reperfusion. During the experiment, the temperature of the warming blanket was set at 37 ℃ to 37.5 ℃ to maintain the rat's body temperature.

[0147] 2. Experimental grouping and administration method: The experiment was divided into 4 groups, with 8 rats randomly selected in each group: (1) sham operation group; (2) model group, given an equal volume of physiological saline, administration volume 2 mL / kg; (3) positive drug group (sodium aescinate 2.5 mg / kg body weight), administration volume 2 mL / kg; (4) the compound of traditional Chinese medicine components in Example 20 (administration dosage: baicalin 5 mg / kg body weight + rhodioloside 2.5 mg / kg body weight + catalpol 7.5 mg / kg body weight), administration volume 2 mL / kg.

[0148] Administration method: Immediately after cerebral ischemia, administer the drug and dosage according to the group, via the tail vein.

[0149] 3. Inspection indicators and standards 3.1 Neurobehavioral studies: Neurobehavioral studies were observed using a single-blind method 24 hours after ischemia (using the Longa 5-point scoring system). The criteria were: 0 points: no neurological damage symptoms; 1 point: inability to fully extend the left forepaw; 2 points: circling towards the non-ischemic side; 3 points: falling towards the non-ischemic side while walking; 4 points: inability to walk spontaneously, with coma. 3.2 Brain water content: Blood and cerebrospinal fluid were absorbed from the surface of the rat brain using filter paper. The brain was placed in a pre-weighed culture dish and weighed wet (accurate to 0.001 g) using an electronic balance. The brain was then baked in a 60℃ oven for 72 hours until equilibrium was reached, and the dry weight was measured. The brain water content was calculated. Brain water content (%) = (wet weight - dry weight) / wet weight × 100%.

[0150] 3.3 Infarct Extent: The rat brain was divided into 6 slices on average, stained with TTC, and the total brain area and infarct area were statistically analyzed using a multimedia color pathological image analysis system. The infarct extent was expressed as infarct area / total brain area. Statistical analysis data are expressed as mean ± SD. Differences between groups in infarct extent were analyzed using a t-test; neurobehavioral disorders were statistically analyzed using ordinal values.

[0151] 3.4 Brain Pathological Morphology: Complete brain tissue was sectioned coronally into three sections. The middle section was fixed in 4% paraformaldehyde for 72 hours. The fixed brain tissue was then dehydrated, cleared, and embedded in paraffin. The paraffin sections were then dewaxed to water. The sections were sequentially immersed in xylene I for 20 minutes, xylene II for 120 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and 75% ethanol for 5 minutes, followed by washing with water. The sections were then stained with hematoxylin for 3–5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, and then blued with a blueing solution, followed by rinsing with running water. The sections were then sequentially dehydrated in a gradient of 85% and 95% ethanol for 5 minutes each, followed by eosin staining for 5 minutes. The sections were then dehydrated with anhydrous ethanol, cleared with xylene for 5 minutes, and mounted with neutral resin. Finally, images were taken under a microscope for image acquisition and analysis.

[0152] 3.5 Brain Tissue MRI Results: Twenty-four hours after cerebral ischemia, rats were placed in an animal-specific anesthesia box, and inhalation anesthesia was administered using isoflurane at an adjusted concentration. The rats were placed in a prone position with their teeth clenched into a restraint device, maintaining unobstructed breathing, and their heads were positioned midline within the MRI coil. T2WI images were first scanned using a spin-echo sequence: FOV=35mm, TE=30 ms, MTX=256×256, TR=3.0 s, ST=0.65 mm, 30 slices in total, FOV=35×35 mm. DWI imaging sequences were then measured in the coronal plane using a single-shot spin-echo planar sequence to measure ADC. ADC value is one of the more sensitive parameters for measuring the integrity of water diffusion. TR=20 ms, TE=2000 ms, MTX=64×64, ST=0.56 mm, FOV=35×35 mm, diffusion coding direction is 6, 2b=200 s / mm 2 (b is the diffusion weighting coefficient). Finally, functional magnetic resonance imaging (fMRI) image processing was performed. The T2WI and DWI images were imported into Paravision version 5.1 software configured in Bruker 8.0 TMRI, and then the T2WI and DWI images were processed.

[0153] 3.6 Blood-brain barrier permeability test: Two hours before sacrifice, 4 mL / kg of 2% Evans blue solution was injected via the tail vein. Successful injection was indicated by the rat's blood vessels and entire body turning blue within 5 seconds. Two hours later, the animals were sacrificed, perfused, fixed, and the brain was harvested. Frozen sections of the brain were prepared, with 1 section taken every 10 sections, each 40 μm thick. After mounting, the sections were observed and photographed under green excitation light.

[0154] 3.7 Detection of Apoptosis in Brain Tissue Cells: Complete brain tissue was sectioned coronally into three sections. The middle section was used, and after being frozen, it was air-dried at room temperature for 20 minutes and fixed with paraformaldehyde for 30 minutes. The sections were washed three times with PBS for 5 minutes each time. The sections were perforated with 0.3% Triton-100 for 15 minutes. After washing three times with PBS for 5 minutes each time, 100 μL of pre-diluted 1×Equilibration Buffer was added to each brain slice, and the sections were equilibrated at room temperature for 20 minutes. During equilibration, TdT labeling solution was prepared under light-protected conditions. After equilibration, 50 μL of TdT labeling solution was added to each brain slice and incubated at 37°C for 60 minutes under light-protected conditions. The sections were washed three times with PBS for 5 minutes each time (under light-protected conditions). The sections were mounted with DAPI-containing mounting medium and observed and photographed using an inverted fluorescence microscope.

[0155] 3.8 Ultrastructure of brain tissue: A sample of approximately 1 mm in size was taken from the lateral border of the ischemic side of the rat brain cortex. 3 The tissue was fixed in electron microscopy fixative and stored at 4°C. The fixed brain tissue was fixed with 2.5% glutaraldehyde and then with 1% osmium tetroxide. After a series of dehydration operations, it was embedded in situ, ultrathin (60 nm thick) sections were prepared, and the sections were stained with lead double lead and observed and photographed under an electron microscope.

[0156] 3.9 Detection of Brain Tissue and Serum Factors: Arterial blood was collected from the abdominal aorta of rats, allowed to stand for approximately 1 hour, and then centrifuged (3000 rpm, 4 ℃, 15 minutes). The supernatant was collected and stored for later use. Brain tissue and serum samples were processed and tested according to the kit instructions to detect the levels of factors such as ET-1, NO, IL-1β, TNF-α, TXB2, and PGI2 in the samples.

[0157] 3.10 Results of Brain Edema-Related Proteins: Brain tissue from the affected side of rats was collected, and total protein was extracted. The total protein concentration was determined using the BCA method, and the concentrations were adjusted to be consistent. 30–50 μg of total protein was loaded and separated by 10% (w / w) gel electrophoresis. The membrane was transferred at a constant current (220 mA) for 1 hour in an ice-water bath, blocked with 5% (v / v) bovine serum albumin for 1 hour, and then incubated overnight at 4 °C with primary antibody. After washing with TBST, the membrane was incubated with secondary antibody for 1 hour. Solution A and solution B from the ECL chemiluminescence buffer were mixed at a 1:1 ratio for color development, and the protein expression level was quantitatively analyzed using ImageJ software, with the gray value of β-actin used as a standard. Three samples were used in each group, and each sample was repeated three times.

[0158] 4. Statistical methods: GraphPad Prism software was used for data analysis. Data are expressed as mean ± standard deviation. One-way ANOVA was used to compare multiple groups of data. P <0.05 indicates a statistically significant difference. P<0.01 indicates a statistically significant difference.

[0159] Test results 1. Effects on neurological function 24 hours after administration, compared with the sham surgery group, the model group showed a significant increase in neurobehavioral scores. P <0.01); compared with the model group, the neurobehavioral score of the traditional Chinese medicine component complex group was significantly lower ( P The score was <0.05 in the positive control group, but the neurobehavioral score decreased, but the difference was not statistically significant. The results are shown in Table 7.

[0160] Table 7: Effects on neurological and behavioral scores in the cerebral ischemia-reperfusion model ( ±SD)

[0161] Note: Compared with the sham surgery group: Compared with the model group, # P <0.05, ## P <0.01 2. Effects on brain water content 24 hours after administration, compared with the sham-operated group, the brain water content in the model group was significantly increased. P <0.05); Compared with the model group, the brain water content of the positive drug group and the traditional Chinese medicine component complex group was significantly reduced ( P <0.05, P <0.01). The results are shown in Table 8.

[0162] Table 8: Effects on brain water content in the cerebral ischemia-reperfusion model ( ±SD)

[0163] Note: Compared with the sham surgery group: Compared with the model group, # P <0.05, ## P <0.01 3. Impact on the area of ​​cerebral infarction 24 hours after administration, compared with the sham surgery group, the infarct area in the model group was significantly increased. P <0.01); Compared with the model group, the cerebral infarction area in the traditional Chinese medicine component complex group was significantly reduced ( P <0.05), the neurobehavioral score decreased in the positive drug group, but the difference was not statistically significant. Results are shown in Table 9 and... Figure 4 .

[0164] Table 9: Effects on the infarct area in the cerebral ischemia-reperfusion model ( ±SD)

[0165] Note: Compared with the sham surgery group: Compared with the model group, # P <0.05, ## P <0.01 4. Observation of brain tissue structure In the sham-operated group, the cortical neurons of rats showed normal cell structure, neat cell arrangement, large nuclei, clear nucleoli, uniform chromatin distribution, intact structure, and abundant cytoplasm, without degeneration, necrosis, or inflammatory cell infiltration. In the model group, the cortical cells of rats showed deformation and necrosis, with large areas of reticular structure, large infarct areas, microglial proliferation, and neutrophil infiltration. The positive control group and the compound of traditional Chinese medicine components showed reduced lesions compared to the model group, with partial degeneration and edema of neurons, more neat cell arrangement, and occasional microglial proliferation, significantly improving the ultrastructural damage of neurons and synapses in the peripheral penumbra of the cortical ischemia area in rats with cerebral ischemia-reperfusion injury. Results are shown in the table below. Figure 5 .

[0166] 5. Brain tissue MRI results Infarct volume was analyzed by T2WI sequence signal intensity, and the infarct volume in the model group was significantly higher than that in the sham surgery group. P <0.01); compared with the model group, the traditional Chinese medicine component complex group was able to significantly reduce infarct volume ( P <0.01. Analysis of ADC values ​​using DWI sequences showed that, in the model group, the ADC values ​​in selected regions of the affected brain tissue were significantly lower than those of the unaffected side (DWI sequence analysis). P <0.01 indicates that the edema of the affected brain tissue is relatively severe; compared with the affected brain tissue in the model group, the ADC value of the affected brain tissue in the sham surgery group is significantly higher than that in the Model group. P <0.01, the ADC value of the affected brain tissue in the herbal compound group was significantly higher than that in the Model group ( P The value <0.05 indicates that the edema of the affected side of the brain tissue in the group treated with the traditional Chinese medicine compound was significantly improved. Results are shown below. Figure 6 .

[0167] 6. Observation of blood-brain barrier permeability No significant Evans blue leakage was observed in the brain tissue of the sham-operated group, and the vascular structure remained intact. In contrast, the vascular integrity of the model group was disrupted, resulting in a large area of ​​concentrated leakage and significant Evans blue leakage. P<0.01%. The Evans blue leakage in the herbal compound group was less than that in the model group; the vessel wall morphology remained continuous and intact, and no large-area exudation was observed. P <0.01). Results are shown in Table 10 and Figure 7 .

[0168] Table 10: Effects on blood-brain barrier permeability in a cerebral ischemia-reperfusion model ( ±SD)

[0169] Note: Compared with the sham surgery group: Compared with the model group, # P <0.05, ## P <0.01 7. Results of brain tissue cell apoptosis detection TUNEL staining results showed that, compared with the sham-operated group, the model group rats had an increased number of apoptotic neurons in the ischemic penumbra region and a significantly higher apoptosis rate. P <0.01. Compared with the model group, the number of apoptotic neurons in the ischemic penumbra region of rats in the positive control group and the group containing traditional Chinese medicine components was reduced, and the apoptosis rate was significantly decreased. P <0.01). Results are shown below. Figure 8 .

[0170] 8. Ultrastructural observation of brain tissue Transmission electron microscopy was used to observe the ultrastructure of neurons and astrocytes in the brain. Neuronal ultrastructure: In the sham-operated group, neurons showed normal morphology, intact cell membranes and nuclei, regular neuronal shapes, and clear membrane boundaries. In the model group, neurons exhibited more abnormal morphology, with irregular spindle-shaped structures and deeply stained nuclei. The ultrastructure of astrocytes in the cerebral cortex was also observed. Astrocyte ultrastructure: In the sham-operated group, astrocytes showed intact structure and morphology, without swelling, and were mostly connected to neurons. In the model group, swollen astrocytes were observed, mostly adhering to the damaged area, with abundant chromatin condensation on the top of the nuclear membrane and large nucleoli. The positive control drug and the traditional Chinese medicine compound group could alleviate astrocyte damage. Results are shown below. Figure 9 .

[0171] 9. Detection results of various factors in serum Compared with the sham-operated group, the serum levels of ET-1, NO, IL-1β, TNF-α, TXB2, and PGI2 in the model group rats were significantly altered. P <0.01); Compared with the model group, the contents of each factor in the positive drug group and the traditional Chinese medicine composition group 1 of this invention were significantly improved ( P <0.05; P <0.01). The results are shown in Table 11.

[0172] Table 11: Effects of various factors on serum levels in a cerebral ischemia-reperfusion model ( ±SD)

[0173] Note: Compared with the sham surgery group: Compared with the model group, # P <0.05, ## P <0.01 Twenty-four hours after ischemia-reperfusion, the expression of proteins AQP4, MMP-9, claudin-5, Occludin, and ZO-1 in brain tissue was detected. Results are shown below. Figure 10 and Figure 11 .

[0174] Experimental conclusions The compound of traditional Chinese medicine components in Example 20 can treat ischemic cerebral edema, improve the neurobehavior, brain tissue damage, and morphology of astrocytes and neurons in rats with cerebral ischemia, reduce brain water content, cerebral infarction area, and neuronal apoptosis in rats with cerebral ischemia, and regulate the expression of cerebral edema-related proteins.

[0175] Example 29: Verification of the therapeutic effects of different dosage forms of traditional Chinese medicine compound in a rat model of focal cerebral ischemia. Animals: Male SD rats, SPF grade, 200-220g, provided by Spiford (Beijing) Biotechnology Co., Ltd.

[0176] Test methods 1. Model building: The model building method is the same as that in Example 28.

[0177] 2. Experimental grouping and administration method: The experiment was divided into 4 groups, and 8 rats were randomly selected in each group: (1) sham operation group; (2) model group, given an equal volume of physiological saline, administration volume 2 mL / kg, tail vein administration; (3) the injection in Example 20 (administration dose: baicalin 5mg / kg body weight + rhodioloside 2.5mg / kg body weight + catalpol 7.5mg / kg body weight), administered via tail vein injection, administration volume 2 mL / kg, tail vein administration; (4) the nasal drops in Example 24 (administration dose: baicalin 15mg / kg body weight + rhodioloside 7.5mg / kg body weight + catalpol 22.5mg / kg body weight), administered via nasal drops, administration volume 0.2 mL / kg, nasal drops administration.

[0178] Administration method: Administer the drugs and dosages according to the instructions for each group immediately after cerebral ischemia.

[0179] 3. Inspection indicators and standards 3.1 Neurobehavioral science: Same as 3.1 in the experimental method of Example 28.

[0180] 3.2 Brain water content: Same as 3.2 in the test method of Example 28.

[0181] 3.3 Scope of cerebral infarction: Same as 3.3 in the test method of Example 28.

[0182] 4. Statistical methods: GraphPad Prism software was used for data analysis. Data are expressed as mean ± standard deviation. One-way ANOVA was used to compare multiple groups of data. P <0.05 indicates a statistically significant difference. ，P <0.01 indicates a statistically significant difference.

[0183] Test results 1. Effects on neurological function 24 hours after administration, compared with the sham surgery group, the model group showed a significant increase in neurobehavioral scores. P <0.01); Compared with the model group, the neurobehavioral scores of rats in the injection group of Example 20 and the nasal drop group of Example 24 were significantly reduced ( P <0.05, P <0.01). The results are shown in Table 12.

[0184] Table 12: Effects on neurological and behavioral scores in the cerebral ischemia-reperfusion model ( ±SD)

[0185] Note: Compared with the sham surgery group: Compared with the model group, # P <0.05, ## P <0.01 2. Effects on brain water content 24 hours after administration, compared with the sham-operated group, the brain water content in the model group was significantly increased. P <0.01); Compared with the model group, the brain water content of the injection group in Example 20 and the nasal drop group in Example 24 was significantly reduced ( P <0.05). The results are shown in Table 13.

[0186] Table 13: Effects on brain water content in the cerebral ischemia-reperfusion model ( ±SD)

[0187] Note: Compared with the sham surgery group: Compared with the model group, # P <0.05, ## P <0.01 3. Impact on the area of ​​cerebral infarction 24 hours after administration, compared with the sham surgery group, the infarct area in the model group was significantly increased. P <0.01); Compared with the model group, the infarct area was significantly reduced in the injection group in Example 20 and the nasal drop group in Example 24 (<0.01); P <0.05, P <0.01). The results are shown in Table 14.

[0188] Table 14: Effects on the infarct area in the cerebral ischemia-reperfusion model ( ±SD)

[0189] Note: Compared with the sham surgery group: Compared with the model group, # P <0.05, ## P <0.01 Experimental conclusions The herbal compound of the present invention, whether in injection or nasal drops, can effectively treat ischemic cerebral edema, improve the neurobehavioral behavior of rats with cerebral ischemia, and reduce the brain water content and cerebral infarction area of ​​rats with cerebral ischemia.

[0190] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A complex of traditional Chinese medicine components, wherein the complex comprises baicalin, rhodioloside, and catalpol.

2. The herbal ingredient complex according to claim 1, characterized in that, By weight, the herbal compound contains 5-200 parts of baicalin, 5-150 parts of rhodioloside, and 5-550 parts of catalpol.

3. The herbal ingredient complex according to claim 1, characterized in that, By weight, the compound of traditional Chinese medicine ingredients contains 50-200 parts of baicalin, 25-150 parts of rhodioloside, and 75-400 parts of catalpol.

4. The herbal ingredient complex according to claim 1, characterized in that, The mass ratio of baicalin, rhodioloside, and catalpol in the compound of traditional Chinese medicine components is 1:(0.25-0.75):(1-2).

5. The herbal ingredient complex according to any one of claims 1-4, characterized in that, The compound of traditional Chinese medicine ingredients also includes one or more of tanshinone, gastrodin, and geniposide.

6. A pharmaceutical composition comprising a complex of traditional Chinese medicine ingredients according to any one of claims 1-5.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition also contains a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition is either a gastrointestinal or non-gastrointestinal dosage form.

9. The use of the traditional Chinese medicine component complex according to any one of claims 1-5 or the pharmaceutical composition according to any one of claims 6-8 in any of the following aspects: To prepare drugs for treating cerebral edema; To prepare drugs for treating cerebral ischemia; To prepare drugs that reduce the infarct area in patients with cerebral ischemia; To prepare drugs that reduce the water content of brain tissue in patients with cerebral ischemia; To prepare drugs that improve neurological deficits in patients with cerebral ischemia.

10. The application according to claim 9, characterized in that, The cerebral edema mentioned is ischemic cerebral edema.

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