Traditional Chinese medicine composition for preventing, treating and / or improving brain diseases directly related to brain microvascular endothelial cells and application of traditional Chinese medicine composition

Abstract

The invention provides a traditional Chinese medicine composition for preventing, treating and / or improving brain diseases directly related to brain microvascular endothelial cells and application of the traditional Chinese medicine composition, and belongs to the technical field of traditional Chinese medicine compositions. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: ginseng under forest for more than 10 years, fructus lycii, fructus rosae laevigatae, rhizoma polygonati, semen cuscutae, wine-processed glossy privet fruit, radix paeoniae alba, herba epimedii, honey-fried licorice root, honey-fried astragalus membranaceus, fried malt and honey. The honey-fried licorice root is prepared from wild licorice root; the radix astragali preparata is prepared by processing high-quality organic radix astragali. According to the traditional Chinese medicine composition, the brain microvascular endothelial cell protection activity of the traditional Chinese medicine composition can be effectively improved by improving the source and grade of key medicinal materials of the traditional Chinese medicine composition; the traditional Chinese medicine composition disclosed by the invention is more outstanding in the aspects of relieving oxidative damage, improving the cell survival state and reducing oxidative stress and apoptosis.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine composition technology, and particularly relates to a traditional Chinese medicine composition for the prevention, treatment and / or improvement of brain diseases directly related to brain microvascular endothelial cells and its application. Background Technology

[0002] Microvascular endothelial cells are a crucial barrier for maintaining the stability of the brain's microenvironment. Once damaged by oxidation, their adhesion and morphological stability decrease, and cell death increases, often amplifying local inflammation and metabolic disorders, ultimately affecting the normal function of brain tissue. Therefore, structural and functional disorders of brain microvascular endothelial cells are significant pathogenic factors leading to various brain diseases, such as cerebrovascular diseases and neurodegenerative diseases. Thus, enhancing the protective activity of brain microvascular endothelial cells can help prevent, treat, and improve brain diseases directly related to brain microvascular endothelial cells.

[0003] Currently, Chinese patent CN 101564494 B discloses a traditional Chinese medicine composition that nourishes the kidneys and replenishes essence, and tonifies the brain and calms the mind. It has the effect of treating cerebral arteriosclerosis with fundus arteriosclerosis, but its effect of enhancing the protective activity of cerebral microvascular endothelial cells needs to be further improved. Summary of the Invention

[0004] The purpose of this invention is to provide a traditional Chinese medicine composition and its application for the prevention, treatment and / or improvement of brain diseases directly related to brain microvascular endothelial cells, which can effectively enhance the protective activity of brain microvascular endothelial cells.

[0005] This invention provides a traditional Chinese medicine composition for the prevention, treatment, and / or improvement of brain diseases directly related to cerebral microvascular endothelial cells. The composition is made from the following raw materials in parts by weight: 2-5 parts ginseng, 6-9 parts wolfberry, 6-9 parts rosehip pulp, 6-9 parts polygonatum, 6-9 parts dodder seed, 6-9 parts wine-processed privet fruit, 6-9 parts white peony root, 12-22 parts epimedium, 2-5 parts prepared licorice root, 6-9 parts prepared astragalus root, 3-8 parts roasted malt, and 25-35 parts honey. The ginseng is forest-grown ginseng aged 10 years or more; the prepared licorice root is obtained from wild licorice; and the prepared astragalus is obtained from high-quality organic astragalus.

[0006] Preferably, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 3-4 parts ginseng, 7-8 parts wolfberry, 7-8 parts rosehip pulp, 7-8 parts polygonatum, 7-8 parts dodder seed, 7-8 parts wine-processed privet fruit, 7-8 parts white peony root, 15-20 parts epimedium, 3-4 parts prepared licorice root, 7-8 parts prepared astragalus root, 4-7 parts stir-fried malt, and 25-35 parts honey.

[0007] The present invention also provides the use of the traditional Chinese medicine composition described above in the preparation of medicaments for the prevention, treatment and / or improvement of brain diseases directly related to brain microvascular endothelial cells.

[0008] Preferably, the brain diseases directly related to brain microvascular endothelial cells include at least one of cerebrovascular diseases and neurodegenerative diseases.

[0009] Preferably, the cerebrovascular disease includes at least one of cerebral infarction, cerebral ischemia, cerebral hemorrhage, cerebral hypoperfusion, and atherosclerotic small vessel disease.

[0010] Preferably, the neurodegenerative disease includes at least one of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neurocognitive impairment, and brain trauma.

[0011] Preferably, the prevention, treatment, and / or improvement of brain diseases directly related to brain microvascular endothelial cells are achieved by enhancing the protective activity of brain microvascular endothelial cells.

[0012] Preferably, the enhanced protective activity against cerebral microvascular endothelial cells includes at least one of the following: 1) Alleviates oxidative damage to cerebral microvascular endothelial cells; 2) Improves the survival status of brain microvascular endothelial cells; 3) Inhibits oxidative damage and apoptosis in brain microvascular endothelial cells; 4) Reduce the level of ROS in brain microvascular endothelial cells.

[0013] The present invention also provides a traditional Chinese medicine preparation for the prevention, treatment and / or improvement of brain diseases directly related to brain microvascular endothelial cells, wherein the active ingredients include the traditional Chinese medicine composition described in the above-mentioned scheme.

[0014] Preferably, the dosage form of the traditional Chinese medicine preparation is an oral preparation; the oral preparation includes tablets, capsules, oral liquids, pills, or granules.

[0015] This invention provides a traditional Chinese medicine composition for the prevention, treatment, and / or improvement of brain diseases directly related to cerebral microvascular endothelial cells. The composition is made from the following raw materials in parts by weight: 2-5 parts ginseng, 6-9 parts wolfberry, 6-9 parts rosehip pulp, 6-9 parts polygonatum, 6-9 parts dodder seed, 6-9 parts wine-processed privet fruit, 6-9 parts white peony root, 12-22 parts epimedium, 2-5 parts prepared licorice root, 6-9 parts prepared astragalus root, 3-8 parts roasted malt, and 25-35 parts honey. The ginseng is forest-grown ginseng aged 10 years or more; the prepared licorice root is obtained from wild licorice; and the prepared astragalus is obtained from high-quality organic astragalus. The effects of traditional Chinese medicine (TCM) compound prescriptions often stem from the synergistic action of multiple components and multiple processes. Therefore, when the source, harvesting period, or part of the key medicinal material changes, even if the prescription name and compatibility structure remain consistent, the composition and proportion of chemical components that can enter the body and ultimately act on cells may shift, resulting in different strengths and stability of biological effects. This invention, by improving the "origin, part, and grade" of the key medicinal materials in the TCM composition, can effectively enhance the protective activity of the TCM composition against cerebral microvascular endothelial cells. Compared with TCM compositions of the same formula from conventional sources and of the same grade, the TCM composition of this invention shows more outstanding performance in reducing oxidative damage, improving cell survival, and reducing oxidative stress and apoptosis, thus effectively enhancing its biological effects and application value. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The effect of different concentrations of H2O2 treatment for 24 h on the viability of bEnd.3 cells; Figure 2 The bright field diagrams of each group of cells are representative of the effects of drug-containing serum on the morphology and cell viability of bEnd.3 cells after H2O2 damage. The scale bar is 200 μm. Figure 3 The effect of drug-containing serum on the morphology and viability of bEnd.3 cells after H2O2 damage was investigated using CCK-8 assays to measure relative cell viability; data are presented in the form of... Represented as (n=5). Comparison with the model group: P <0.01, P <0.05, P <0.01; Figure 4The effect of drug-containing serum intervention on the viability and toxicity of bEnd.3 cells after H2O2 injury (Calcein-AM / PI) is represented by Calein-AM / PI double staining (green: live cells; red: dead cells), with a scale bar of 200 μm. Figure 5 To quantify the proportion of viable cells in bEnd.3 cells after H2O2 injury by measuring the effect of drug-containing serum intervention on cell viability and cytotoxicity (Calcein-AM / PI); data are presented in... Representing (n=3), compared with the model group: P <0.01, P <0.01; Figure 6 Annexin V-FITC / PI double staining flow cytometry scatter plots of each group in the effect of drug-containing serum intervention on apoptosis of bEnd.3 cells after H2O2 damage; Figure 7 To quantify the apoptosis rate in bEnd.3 cells after H2O2 injury, the effect of drug-containing serum intervention was investigated; data were presented as follows: Represented as (n=3). Comparison with the model group: P <0.01; Figure 8 The effect of drug-containing serum intervention on intracellular ROS levels in bEnd.3 cells after H2O2 damage (DCFH-DA) is represented by the DCFH-DA fluorescence diagram. Figure 9 The results of quantitative analysis of ROS fluorescence intensity in bEnd.3 cells after H2O2 damage, using drug-containing serum intervention, are shown in the DCFH-DA study. The data are presented as follows: Represented as (n=3). Comparison with the model group: P <0.01, P <0.05, P <0.01. Detailed Implementation

[0018] This invention provides a traditional Chinese medicine composition for the prevention, treatment, and / or improvement of brain diseases directly related to cerebral microvascular endothelial cells. The composition is made from the following raw materials in parts by weight: 2-5 parts ginseng, 6-9 parts wolfberry, 6-9 parts rosehip pulp, 6-9 parts polygonatum, 6-9 parts dodder seed, 6-9 parts wine-processed privet fruit, 6-9 parts white peony root, 12-22 parts epimedium, 2-5 parts prepared licorice root, 6-9 parts prepared astragalus root, 3-8 parts roasted malt, and 25-35 parts honey. The ginseng is forest-grown ginseng aged 10 years or more; the prepared licorice root is prepared from wild licorice; and the prepared astragalus is prepared from high-quality organic astragalus.

[0019] In this invention, the ginseng is forest-grown ginseng that is over 10 years old. Compared with ordinary ginseng, forest-grown ginseng has a longer growth cycle and an ecological environment that is closer to the wild state, which is more conducive to the continuous accumulation of secondary metabolites such as saponins and the improvement of the richness of the component spectrum.

[0020] In this invention, the roasted licorice is prepared from wild licorice; the roasted astragalus is prepared from premium grade organic astragalus. In this invention, processed licorice root and processed astragalus root play a "synergistic and harmonizing" role in the traditional Chinese medicine composition. The grade and source of these raw materials can affect the overall effect of the composition. Taking licorice root as an example, its source and processing conditions affect the content and ratio of glycyrrhizic acid and polyphenols. In compound prescriptions, licorice root may participate in protection through anti-inflammatory and antioxidant effects, and also affect the overall "tolerance and synergistic effect" of the prescription, making the effects of other medicinal materials more easily manifested at the cellular level. In this invention, astragalus root is related to immune regulation and antioxidant defense. Its polysaccharides and saponins / carotenoids may work together to improve the cell's ability to cope with oxidative stimuli. Therefore, when the source of processed licorice root and processed astragalus root is upgraded from "conventional" to "premium grade," in addition to "higher content" of active ingredients, their batch-to-batch stability and effective ingredient ratio are more controllable, which is more conducive to forming repeatable biological effects.

[0021] Based on the logic of "medicinal materials—components—effect direction," this invention yields a modified traditional Chinese medicine composition that exhibits a more stable and significant improvement trend across multiple cellular endpoints. In other words, by rationally optimizing key medicinal materials, the effective substance input into the body through the traditional Chinese medicine composition is more sufficient or better matched, thereby more effectively intervening in key stages of oxidative damage. Compared with using ordinary raw materials, the traditional Chinese medicine composition of this invention can further reduce the level of oxidative stress in brain microvascular endothelial cells, reduce the oxidative damage burden on membrane structures and key intracellular molecules, thereby reducing the probability of cell death being triggered and improving cell survival. Therefore, the traditional Chinese medicine composition of this invention has a stronger comprehensive regulatory ability on the continuous chain of "oxidative stress—brain microvascular endothelial cell death—cell state recovery."

[0022] In one embodiment, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: ginseng 3-4 parts, wolfberry 7-8 parts, rosehip pulp 7-8 parts, polygonatum 7-8 parts, dodder seed 7-8 parts, wine-processed privet fruit 7-8 parts, white peony root 7-8 parts, epimedium leaf 15-20 parts, prepared licorice root 3-4 parts, prepared astragalus root 7-8 parts, stir-fried malt 4-7 parts, and honey 25-35 parts.

[0023] The present invention also provides the use of the traditional Chinese medicine composition described above in the preparation of medicaments for the prevention, treatment and / or improvement of brain diseases directly related to brain microvascular endothelial cells.

[0024] In one implementation, the brain diseases directly related to the brain microvascular endothelial cells include at least one of cerebrovascular diseases and neurodegenerative diseases.

[0025] In one embodiment, the cerebrovascular disease includes at least one of cerebral infarction, cerebral ischemia, cerebral hemorrhage, cerebral hypoperfusion, and atherosclerotic small vessel disease.

[0026] As one implementation, the neurodegenerative disease includes at least one of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, HIV-related neurocognitive impairment, and brain trauma.

[0027] In one implementation, the prevention, treatment, and / or improvement of brain diseases directly related to brain microvascular endothelial cells are achieved by enhancing the protective activity of brain microvascular endothelial cells.

[0028] As one implementation, the enhanced protective activity of cerebral microvascular endothelial cells includes at least one of the following: 1) Alleviates oxidative damage to cerebral microvascular endothelial cells; 2) Improves the survival status of brain microvascular endothelial cells; 3) Inhibits oxidative damage and apoptosis in brain microvascular endothelial cells; 4) Reduce the level of ROS in brain microvascular endothelial cells.

[0029] The present invention also provides a traditional Chinese medicine preparation for the prevention, treatment and / or improvement of brain diseases directly related to brain microvascular endothelial cells, wherein the active ingredients include the traditional Chinese medicine composition described in the above-mentioned scheme.

[0030] In one embodiment, the dosage form of the traditional Chinese medicine preparation is an oral preparation; the oral preparation includes tablets, capsules, oral liquids, pills, or granules.

[0031] In one embodiment, the oral preparation is an oral liquid; the preparation method of the oral liquid is the same as the preparation method of the oral liquid preparation of traditional Chinese medicine composition disclosed in CN101564494 B, and the specific steps include: 1. Extract ginseng with water or ethanol 2-4 times, each time for 1-3 hours, filter, combine the filtrates to obtain extract A; extract other Chinese medicinal materials besides ginseng with water 2-4 times, each time for 1-3 hours, filter, combine the filtrates to obtain extract B; 2. The extract B is concentrated using a vacuum method, and the relative density of the solution is controlled at 1.05~1.15 g / ml at 70~80℃. The solution is then filtered to obtain a concentrated solution. 3. After cooling the concentrated solution, stir continuously and slowly add an ethanol aqueous solution with a volume concentration of 95% to adjust the pH of the solution to 8-9, until the ethanol volume concentration is 50%-80%. Let it stand for precipitation for 48-72 hours, take the supernatant and filter to obtain the filtrate. 4. Add 0.3% by weight of activated carbon to the filtrate obtained in step 3, then heat to boiling. Filter the resulting decolorized solution and concentrate it to a relative density of 1.10~1.20 g / ml at 20℃ to obtain a concentrated solution. 5. Add extract A to the concentrate obtained in step 4, mix well, and then add honey after decolorization and decarbonization; then make an oral liquid using conventional processes.

[0032] As one embodiment, the steps of the preparation method of the oral liquid are as follows: 1. Ginseng was extracted with ethanol three times, each time for 2 hours. After filtration, the filtrates were combined to obtain extract A. Other Chinese medicinal materials, excluding ginseng, were extracted with water twice, for 2 hours and 1.5 hours respectively. After filtration, the filtrates were combined to obtain extract B. 2. The extract B is concentrated using a vacuum method, and the relative density of the solution is controlled at 1.05~1.15 g / ml at 70~80℃. The solution is then filtered to obtain a concentrated solution. 3. After cooling the concentrated solution, stir continuously and slowly add an ethanol aqueous solution with a volume concentration of 95% to adjust the pH of the solution to 8-9, until the ethanol volume concentration is 60%-75%. Let it stand for precipitation for 48-72 hours, take the supernatant and filter to obtain the filtrate. 4. Add 0.3% by weight of activated carbon to the filtrate obtained in step 3, then heat to boiling. Filter the resulting decolorized solution. The medicinal juice is a brownish-red liquid with a slightly bitter taste, but it must not have an ethanol taste. Concentrate it to a relative density of 1.10~1.20 g / ml at 20℃ to obtain a concentrated solution. 5. Add extract A to the concentrate obtained in step 4, mix well, and then add honey after decolorization and decarbonization; add 3‰ (g / g) benzoic acid or 0.25‰ (g / g) paraben to adjust the pH to 4.0~6.0 and the relative density to 1.10~1.25g / ml; boil the solution for 30 minutes to sterilize, and then fill it into bottles to obtain the final product.

[0033] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a traditional Chinese medicine composition for the prevention, treatment, and / or improvement of brain diseases and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1: A traditional Chinese medicine oral liquid for the prevention, treatment, and / or improvement of brain diseases directly related to cerebral microvascular endothelial cells. Improved raw material system (hereinafter referred to as "high-end version"): The main raw materials are wild ginseng (≥10 years old), wild roasted licorice root, Epimedium brevicornu, and premium-grade organic Astragalus membranaceus. The remaining medicinal materials (white peony root, wolfberry, privet fruit (processed), dodder seed, Rosa laevigata pulp, roasted malt, and Polygonatum sibiricum (processed)) all meet the requirements of first-class products. See Table 1 for details. The formula is as follows: The Chinese herbal medicine composition is made from the following raw materials in parts by weight: ginseng 3 parts, wolfberry 8 parts, Rosa laevigata pulp 8 parts, Polygonatum sibiricum 8 parts, dodder seed 8 parts, wine-processed privet fruit 8 parts, white peony root 8 parts, Epimedium brevicornu 20 parts, roasted licorice root 4 parts, roasted Astragalus membranaceus 8 parts, roasted malt 7 parts, and honey 35 parts.

[0035] Table 1 Improved Raw Material System

[0036] Preparation method: 1. Ginseng was extracted with ethanol three times, each time for 2 hours. After filtration, the filtrates were combined to obtain extract A. Other Chinese medicinal materials, excluding ginseng, were extracted with water twice, for 2 hours and 1.5 hours respectively. After filtration, the filtrates were combined to obtain extract B. 2. The extract B is concentrated using a vacuum method, and the relative density of the solution is controlled at 1.05~1.15 g / ml at 70~80℃. The solution is then filtered to obtain a concentrated solution. 3. After cooling the concentrated solution, stir continuously and slowly add an ethanol aqueous solution with a volume concentration of 95% to adjust the pH of the solution to 8-9, until the ethanol volume concentration is 60%-75%. Let it stand for precipitation for 48-72 hours, take the supernatant and filter to obtain the filtrate. 4. Add 0.3% by weight of activated carbon to the filtrate obtained in step 3, then heat to boiling. Filter the resulting decolorized liquid. The medicinal juice is a brownish-red liquid with a slightly bitter taste, but it must not have an ethanol taste. Concentrate it to a relative density of 1.10~1.20 g / ml at 20℃ to obtain a concentrated solution. 5. Add extract A to the concentrate obtained in step 4, mix well, and then add honey after decolorization and decarbonization; add 3‰ (g / g) benzoic acid or 0.25‰ (g / g) paraben to adjust the pH to 4.0~6.0 and the relative density to 1.10~1.25g / ml; boil the solution for 30 minutes to sterilize, and then fill it into bottles to obtain the final product.

[0037] Comparative Example 1 Compared with Example 1, the raw material system was replaced with the following common raw material system, and the proportions and preparation methods were the same as in Example 1.

[0038] The standard raw material system (hereinafter referred to as "standard version") uses ordinary cultivated ginseng, ordinary processed licorice root, ordinary epimedium, and standard quality astragalus root as the main raw materials. The remaining medicinal materials (white peony root, wolfberry fruit, privet fruit [processed], dodder seed, rosehip kernel, roasted malt, and polygonatum [processed]) all meet the requirements for first-class products. See Table 2 for details.

[0039] Table 2 Common Raw Material System

[0040] Experimental Example 1 1. Materials and Methods 1.1 Materials 1.1.1 Reagents and Instruments Reagents: Comparative Example 1: standard version of traditional Chinese medicine preparation; Example 1: high-end version of traditional Chinese medicine preparation. Main cell experimental reagents included H2O2, CCK-8 assay kit, DCFH-DA reactive oxygen species probe, Annexin V-FITC / PI apoptosis detection kit, DMEM (high glucose) cell culture medium, fetal bovine serum (FBS), etc. Consumables: Animal blood collection tubes, serum separation tubes, 0.22 µm filter membranes, 6-well plates, 24-well plates, 96-well plates. Instruments: CO2 incubator, biosafety cabinet, inverted fluorescence microscope, microplate reader, flow cytometer, etc.

[0041] 1.1.2 Animals and Cells Animals: Adult male SD rats aged 7-8 weeks and weighing 180-200 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Cells: bEnd.3 mouse cerebral angioendothelioma cells were purchased from Wuhan Pronoss Biotechnology Co., Ltd.

[0042] 1.2 Methods 1.2.1 Preparation and processing of drug-containing serum Prior to the experiment, rats were acclimatized in the laboratory for one week in the barrier environment experimental animal room of the Tsinghua University Animal Center. They were housed individually in a quiet environment with strictly controlled conditions: temperature maintained at 22±1℃, relative humidity at 60%, and a 12-hour / 12-hour light / dark cycle (8:00 AM to 8:00 PM). Sterile drinking water and standardized feed were provided throughout the experiment. After acclimatization, SD rats were randomly divided into three groups: a control group, a standard drug-containing serum group, and a premium drug-containing serum group. Each treatment group was administered the drug by gavage at a clinically equivalent dose, while the control group was administered an equal volume of distilled water by gavage. Drug administration continued until the prescribed cycle. One hour after the last administration, blood was collected via the abdominal aorta. After coagulation at room temperature, the blood was centrifuged at 3000 g for 10 min, and the supernatant serum was collected. The serum was sterilely filtered through a 0.22 μm filter and then aliquoted and stored at −80℃ for later use.

[0043] 1.2.2 bEnd.3 Establishment of the cellular oxidative damage model and experimental grouping bEnd.3 cells were cultured in DMEM high-glucose medium (containing 10% FBS) at 37 ℃ and 5% CO2. After digestion, cells in the logarithmic growth phase were seeded into different wells at appropriate densities. An H2O2-induced oxidative damage model was established: the concentration and duration of H2O2 exposure were determined through preliminary experiments to ensure the model's stability and reproducibility (using cell viability variation as a reference standard). In the formal experiment, a normal control group, a model group, and a serum treatment group were included.

[0044] 1.2.3 Drug-containing serum intervention regimen After successfully establishing the oxidative damage model, different volume fractions (5%, 10%, 15%, and 20%) of drug-containing serum were added to each group of cells for intervention, and the volume was made up with blank serum. After the intervention, cell viability, apoptosis, and oxidative stress levels were measured.

[0045] 1.2.4 Cell morphology observation After each group of cells was damaged and intervened, bright-field observation and image acquisition were performed using an inverted microscope to record cell adhesion, morphological changes, and floating cell status.

[0046] 1.2.5 Cell viability assay (CCK-8) Logarithmically growing bEnd.3 cells were harvested, routinely digested, counted, and seeded into 96-well plates. The plates were incubated at 37 ℃ in a 5% CO2 incubator until the cells were stably adherent. The original culture medium was discarded, and a concentration gradient of H2O2 (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μg / mL) was established, and incubated for 24 h. Subsequently, CCK-8 working solution (prepared according to kit instructions) was added to each well, and the cells were incubated in the dark for 1–2 h. The absorbance (OD) was then measured at 450 nm. 450 Cells without H2O2 treatment were used as normal controls (cell viability was 100%). The relative cell viability (%) after treatment with each concentration of H2O2 was calculated, and the final H2O2 concentration that brought cell viability close to 50% was selected as the modeling dose for the subsequent oxidative damage model. All subsequent experiments were conducted under the conditions of this modeling concentration and fixed treatment time.

[0047] After determining the modeling conditions, an oxidative damage model was established in bEnd.3 cells by inducing them with the final H2O2 concentration described above. After the damage was completed, the culture medium was replaced, and drug-containing serum was added for intervention. To control the influence of serum matrix and ensure that the total serum volume was consistent across all groups, the intervention culture medium for the treatment group was prepared using a ratio of "drug-containing serum + blank serum" to keep the total serum volume fraction constant at 20%. The specific settings were as follows: the standard drug-containing serum group consisted of 5% standard serum + 15% blank serum, 10% standard serum + 10% blank serum, 15% standard serum + 5% blank serum, and 20% standard serum; the modified (high-end) drug-containing serum group consisted of 5% high-end serum + 15% blank serum, 10% high-end serum + 10% blank serum, 15% high-end serum + 5% blank serum, and 20% high-end serum. A normal control group (with 20% blank serum added, no H2O2) and a model control group (with 20% blank serum added after H2O2 damage) were also set up. After the intervention, CCK-8 assays were performed using the same method as above, and relative cell viability was calculated.

[0048] 1.2.6 Cell viability and toxicity staining (Calcein-AM / PI) bEnd.3 cells were seeded in 96-well plates and subjected to H2O2 damage and drug-containing serum intervention according to groupings. Live / dead cells were then stained. After treatment, Calcein-AM / PI double staining working solution was added for staining. After incubation at 37 ℃ in the dark for approximately 30 min, images were observed and acquired under a fluorescence microscope: Calcein-AM showed green fluorescence in live cells (Ex / Em=494 / 517 nm), and PI showed red fluorescence in dead cells (Ex / Em=535 / 617 nm).

[0049] 1.2.7 Apoptosis Detection (Annexin V-FITC / PI) After damage and intervention, the culture medium was discarded, and cells were digested with EDTA-free trypsin and collected. After washing with pre-cooled PBS at 4 °C, cells were resuspended in 1× Binding Buffer. Annexin V-FITC and PI were added sequentially according to the kit instructions for staining in the dark, followed immediately by flow cytometry analysis. Voltage adjustment and compensation settings were performed using blank tubes and single-stained tubes. Data were collected by flow cytometry, and the proportions of early and late apoptotic cells were calculated.

[0050] 1.2.8 Intracellular ROS Level Detection (DCFH-DA) Fluorescence signals were collected after incubation with the DCFH-DA probe, and changes in ROS levels in each group were quantitatively analyzed. After the damage and intervention treatments were completed, the probe working solution was prepared according to the kit instructions. After discarding the culture medium, the DCFH-DA working solution was added to cover the cells, and the cells were incubated at 37 °C for 20 min. After incubation, the cells were washed three times with PBS to remove probes that had not entered the cells. Subsequently, images were acquired under a fluorescence microscope / laser confocal microscope (Ex / Em=488 / 525 nm), and the fluorescence intensity was quantitatively analyzed.

[0051] 1.2.9 Statistical Methods Experimental data The data is presented in the table. One-way ANOVA was used for comparisons among multiple groups, with post-hoc tests performed; non-normally distributed data were analyzed using nonparametric tests. P A value <0.05 was considered statistically significant.

[0052] 2 Results 2.1 Screening and Determination of Conditions for the Cellular H2O2 Oxidative Damage Model (bEnd.3) bEnd.3 cells were treated with different final concentrations of H2O2 (1–10 μg / mL) for 24 h, and the relative viability of the cells was detected using the CCK-8 assay (with the normal control group as 100%). The results showed that cell viability decreased in a dose-dependent manner with increasing H2O2 concentration. Figure 1(See Table 3). Considering both the degree of damage and model stability, the cell viability in the 5 μg / mL H2O2 group decreased to approximately 50% (51.00±0.37)%, meeting the requirements for modeling moderate-level damage. Therefore, 5 μg / mL H2O2 treatment for 24 h was selected as the subsequent bEnd.3 cell oxidative damage model condition. The results showed that hydrogen peroxide-induced endothelial cell damage consistently exhibited typical phenotypes: under bright field conditions, cells transitioned from a spread-out state to contraction and rounding, with increased intercellular spaces accompanied by shedding and increased debris. Quantitative analysis revealed decreased cell viability and a reduced proportion of live cells, while the proportion of apoptotic cells and intracellular oxidative stress levels significantly increased. This simultaneous deterioration of morphology, viability / survival, apoptosis, and oxidative stress suggests that the model not only caused a short-term decrease in cell activity but also involved damage to cell membrane integrity and the initiation of cell death processes, making it suitable for evaluating the protective effect of interventions on the overall state of endothelial cells.

[0053] Table 3 Effect of H2O2 concentration gradient treatment for 24 h on the relative viability of bEnd.3 cells (%) (n=5)

[0054] Note: Compared with the normal control group, P <0.05.

[0055] 2.2 Effect of drug-containing serum on bEnd.3 cell viability Bright-field observation showed that bEnd.3 cells in the normal control group adhered well to the wall, with predominantly polygonal / short spindle-shaped cells and relatively clear cell boundaries. They exhibited the typical cobblestone-like monolayer arrangement characteristic of endothelial cells, with relatively tight intercellular connections and a relatively uniform cell density within the field of view. The H2O2-damaged group showed significant morphological changes: the number of adherent cells decreased, some cells shrank and became rounded, exhibited increased refractive index, increased intercellular spaces, and a shift from dense to loose arrangement. Detached floating cells and cell debris / particulate matter were visible in the field of view, suggesting that oxidative stress impaired the adhesion and morphological stability of bEnd.3 cells.

[0056] After intervention with drug-containing serum, cell morphological damage was alleviated to varying degrees: compared with the model group, the number of adherent cells increased and the cell morphology was relatively extended in the higher concentration group of the ordinary drug-containing serum, the cell debris and floating cells decreased, and the intercellular connections were more continuous than before; the above improvements were more obvious after intervention with the modified drug-containing serum, and the trend of restoration of adherent cell density and morphology was more prominent with increasing concentration, and cell shrinkage and shedding were reduced.

[0057] Based on the clear indication of damage in the model from bright-field observation, the CCK-8 results further showed that intervention with drug-containing serum could improve cell viability and exhibited a certain dose-related relationship (Table 4). Figures 2-3 The standard version showed limited improvement at low concentrations (5%), but gradually increased cell viability within the range of 10%–20%. The improved version showed more pronounced viability recovery at lower concentrations (5%), and the increase was more continuous and stable as the concentration increased. Taking the highest dose point as an example, the improved version at 20% showed a greater recovery of cell viability (82.70±3.11)%, while the standard version at 20% showed approximately (68.16±2.52)%, indicating a difference in the protective strength of the two formulations under the same intervention system.

[0058] Table 4. Effects of drug-containing serum intervention on the relative viability of bEnd.3 cells after H2O2 injury (%) (n=5)

[0059] Note: Compared with the model group: P <0.05, P <0.01 2.3 Effects of drug-containing serum on cell viability and toxicity status Calcein-AM / PI double staining results showed that the normal control group cells were predominantly green fluorescent, with relatively intact cell adhesion and morphology, and fewer red PI-positive cells. The model group showed a significant decrease in green fluorescence, an increase in red PI-positive cells, and observed decreased cell density and cell debris, suggesting that H2O2 treatment induced significant cell death and impaired membrane integrity.

[0060] After intervention with drug-containing serum, the green fluorescence in each treatment group increased compared to the model group, while the number of red PI-positive cells decreased, indicating varying degrees of alleviation of cell death phenotype. Overall, the modified intervention group showed more complete recovery of green fluorescence and less red signal, suggesting a more significant improvement in cell viability. Figure 4 Specifically, this can be discovered through quantitative analysis. Figure 5 The proportion of viable cells in the normal control group was (99.76±0.25)%, while it decreased to (62.24±2.12)% in the model group. After intervention with the standard 10% and 20% regimens, the proportion of viable cells recovered to (69.97±1.95)% and (77.46±4.13)%, respectively. In contrast, the proportions in the modified 10% and 20% regimens were (80.78±1.59)% and (87.69±0.68)%, respectively, indicating that under the same intervention conditions, the modified regimen improved cell viability to a greater extent.

[0061] In summary, both drug-containing sera improved cell status to some extent, but the modified version showed a more stable and pronounced protective trend: on the one hand, the modified version showed clearer morphological recovery and improved survival even at lower dosages; on the other hand, with increasing dosages, the modified version showed a more continuous improvement in cell viability and the proportion of live cells, while also showing a more significant reduction in apoptosis and intracellular oxidative stress levels. This characteristic of "improvement in multiple indicators in the same direction" is important because it suggests that the advantage of the modified version does not stem from the occasional fluctuations of a single detection indicator, but rather from a synergistic intervention effect at multiple stages of the cell damage chain.

[0062] 2.4 Effect of drug-containing serum on cell apoptosis levels Annexin V-FITC / PI dual staining flow cytometry results show ( Figure 6 In the normal control group, cells were mainly distributed in Annexin V. - / PI - In the first quadrant, the proportion of apoptotic cells was low. After H2O2 damage, the cell population significantly shifted towards the Annexin V-positive region, and Annexin V... + / PI - (Early apoptosis) and Annexin V + / PI + The increase in cells with late-stage apoptosis / secondary necrosis suggests that oxidative stress can significantly induce apoptosis in bEnd.3 cells. After intervention with drug-containing serum, the proportion of Annexin V positive cells decreased, and cell distribution reverted to the survival quadrant; the apoptosis rate in the 10% group of the ordinary version decreased to (52.33±17.43)%; the apoptosis rate in the 20% group of the ordinary version further decreased to (26.91±1.31)%. The apoptosis inhibition was more significant after intervention with the advanced version, with apoptosis rates of (22.12±3.55)% and (15.61±0.65)% in the 10% and 20% groups of the advanced version, respectively. This indicates that drug-containing serum can reduce the level of H2O2-induced apoptosis, and under the same intervention conditions, the modified version has a more prominent inhibitory effect on cell damage and apoptosis. Figure 7 ).

[0063] In summary, compared with the injury model, both drug-containing sera improved cell morphology and enhanced cell viability and survival status. P <0.05, while reducing apoptosis and alleviating intracellular oxidative stress levels ( P <0.05%. Compared to the regular version, the improved version shows a greater improvement at the same dosage. P <0.05%, and showed a clearer protective trend at lower dosages; at higher dosages, the improved version showed a more significant improvement in cell viability and survival status. P <0.05, with a more significant reduction in apoptosis and oxidative stress levels (P <0.05). Conclusion: Differences in the source and grade of key medicinal materials can affect the cell-protective effect of traditional Chinese medicine compositions. The improved version showed more outstanding performance in reducing oxidative damage, improving cell survival, and reducing oxidative stress and apoptosis, suggesting that optimizing the selection of key medicinal materials and preparation methods may enhance the biological effects and application value of the formulation.

[0064] It is evident that the improved version significantly reduces intracellular oxidative stress levels at the same dosage, while also more significantly downregulating the apoptosis rate, suggesting a more comprehensive intervention in the key process of "oxidative damage-cell death." The improved version achieves a stronger overall protective effect by enhancing the cell's own antioxidant defense capabilities, improving cellular energy metabolism, or mitigating the triggering of death signals.

[0065] 2.5 Effects of drug-containing serum on intracellular ROS levels DCFH-DA fluorescence results (see) Figure 8 and Figure 9 The results showed that the intracellular ROS fluorescence signal was weak in the normal control group; the fluorescence in the model group was significantly enhanced (43.97±2.97)%, indicating that the intracellular oxidative stress level increased after H2O2 treatment. After intervention with drug-containing serum, the intracellular fluorescence signal was weakened to varying degrees compared with the model group. Specifically, the fluorescence signals in the 10% and 20% groups of the ordinary version were (39.92±1.30)% and (31.39±0.80)%, respectively, showing a decreasing trend; while the fluorescence signals in the 10% and 20% groups of the modified version were (19.65±2.01)% and (16.96±1.70)%, respectively, showing a more significant decrease. This indicates that drug-containing serum can reduce the intracellular ROS level induced by H2O2, and the modified version has a more prominent effect.

[0066] In summary, this invention significantly enhances the protective effect of traditional Chinese medicine compositions on brain microvascular endothelial cells by optimizing the key medicinal material system. It is more prominent in reducing oxidative stress, decreasing cell death, and promoting cell recovery, laying a reliable foundation for further product optimization from the material and mechanistic levels.

[0067] Experimental Example 2 Quantitative analysis based on LC-QQQ-MS was performed on the conventional Chinese medicine preparation (Comparative Example 1) and the high-end Chinese medicine preparation prepared in Example 1, as well as their drug-containing serum. Solution preparation 1. Preparation of mixed reference solution Accurately weigh 1 mg each of rhodioloside, paeoniflorin, verbascoside, glycyrrhizin, hyperoside, privetin, ginsenoside Rg1, ginsenoside Re, astragaloside A, astragaloside B, astragaloside C, icariin, ginsenoside Rb1, astragaloside A, and glycyrrhizic acid reference standards, and prepare a 1 mg / mL stock solution. Then, accurately measure 10 μL of each reference standard stock solution and add 50 μL of methanol to prepare a mixed reference solution containing 50 μg / mL of each of the above reference standards. Dilute with methanol to prepare a standard dilution with a concentration of 1 μg / mL.

[0068] 2. Preparation of serum test solution Thaw serum samples at room temperature, place 100 μL of blank in a centrifuge tube, add 400 μL of methanol to precipitate proteins, vortex for 30 s, let stand for 10 min, then centrifuge at 13000 r / min for 15 min at 4 ℃. Take 200 μL of supernatant, dilute with deionized water 1:1, vortex, centrifuge at 13000 r / min for 15 min at 4 ℃ for 15 min, and inject 200 μL of supernatant for analysis.

[0069] 3. Preparation of the test solution Take 200 μL of the sample to be tested, place it in a centrifuge tube, add 1800 μL of methanol, vortex to mix evenly, sonicate for 30 min, cool, and filter through a 0.22 μm filter membrane to obtain the sample.

[0070] 4. Preparation of standard curve solutions Accurately pipette an appropriate amount of standard diluent and dilute it stepwise with methanol to obtain a reference solution with a concentration of 0.01~1000 ppb.

[0071] 5. Chromatographic conditions An ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) was used as the chromatographic column. The mobile phase was 0.1% formic acid aqueous solution (A)-acetonitrile (B), with gradient elution (0–2 min, 85% A; 2–3 min, 85%–75% A; 3–5 min, 75%–50% A; 5–9 min, 50%–20% A; 9–10 min, 20%–10% A; 10–11 min, 10% A; 11–11.1 min, 10%–85% A; 11.1–12 min, 85% A). The flow rate was 0.3 mL / min, the column temperature was 35 °C, and the injection volume was 5 μL.

[0072] 6. Mass spectrometry conditions Electrospray ionization (ESI) was used with curtain gas (CUR) at 40 psi, collision gas (CAD) at 9 psi, spray voltage (IS) at 5.5 kV, capillary temperature (TEM) at 550 °C, spray gas (GS1) at 35 psi, auxiliary heating gas (GS2) at 70 psi, and multiple reaction detection (MRM) scanning in both positive and negative ion modes. Ion pair acquisition information is shown in Table 5.

[0073] Table 5 Ion Pair Acquisition Information

[0074] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition for the prevention, treatment, and / or improvement of brain diseases directly related to cerebral microvascular endothelial cells, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: ginseng 2-5 parts, wolfberry 6-9 parts, rosehip pulp 6-9 parts, polygonatum 6-9 parts, dodder seed 6-9 parts, wine-processed privet fruit 6-9 parts, white peony root 6-9 parts, epimedium leaf 12-22 parts, prepared licorice root 2-5 parts, prepared astragalus root 6-9 parts, stir-fried malt 3-8 parts, and honey 25-35 parts; The ginseng in question is forest-grown ginseng that is over 10 years old. The roasted licorice was obtained by processing wild licorice. The roasted astragalus is prepared from premium-grade organic astragalus.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: ginseng 3-4 parts, wolfberry 7-8 parts, rosehip pulp 7-8 parts, polygonatum 7-8 parts, dodder seed 7-8 parts, wine-processed privet fruit 7-8 parts, white peony root 7-8 parts, epimedium leaf 15-20 parts, prepared licorice root 3-4 parts, prepared astragalus root 7-8 parts, stir-fried malt 4-7 parts, and honey 25-35 parts.

3. The use of the traditional Chinese medicine composition according to claim 1 or 2 in the preparation of medicaments for the prevention, treatment and / or improvement of brain diseases directly related to cerebral microvascular endothelial cells.

4. The application according to claim 3, characterized in that, The brain diseases directly related to brain microvascular endothelial cells include at least one of cerebrovascular diseases and neurodegenerative diseases.

5. The application according to claim 4, characterized in that, The cerebrovascular diseases mentioned include at least one of cerebral infarction, cerebral ischemia, cerebral hemorrhage, cerebral hypoperfusion, and atherosclerotic small vessel disease.

6. The application according to claim 4, characterized in that, The neurodegenerative diseases include at least one of the following: Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neurocognitive impairment, and brain trauma.

7. The application according to claim 3, characterized in that, The prevention, treatment, and / or improvement of brain diseases directly related to brain microvascular endothelial cells are achieved by enhancing the protective activity of brain microvascular endothelial cells.

8. The application according to claim 3, characterized in that, The enhanced protective activity against cerebral microvascular endothelial cells includes at least one of the following: 1) Alleviate oxidative damage to cerebral microvascular endothelial cells; 2) Improves the survival status of brain microvascular endothelial cells; 3) Inhibits oxidative damage and apoptosis in brain microvascular endothelial cells; 4) Reduce the level of ROS in brain microvascular endothelial cells.

9. A traditional Chinese medicine preparation for the prevention, treatment, and / or improvement of brain diseases directly related to cerebral microvascular endothelial cells, characterized in that, The active ingredients include the traditional Chinese medicine composition as described in claim 1 or 2.

10. The traditional Chinese medicine preparation according to claim 9, characterized in that, The dosage form of the traditional Chinese medicine preparation is an oral preparation; the oral preparation includes tablets, capsules, oral liquids, pills, or granules.

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