Traditional Chinese medicine composition with functions of softening hard mass and dredging collaterals as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention provides a traditional Chinese medicine composition with functions of softening hardness and dredging collaterals as well as a preparation method and application of the traditional Chinese medicine composition. The traditional Chinese medicine composition with the functions of softening hardness and dredging collaterals is prepared from the following raw material medicines in parts by weight: 20-40 parts of turtle shells, 20-40 parts of radix paeoniae alba, 14-22 parts of sedum sarmentosum, 9-21 parts of teasel roots, 9-21 parts of rhizoma acori graminei, 9-21 parts of astragalus membranaceus, 14-22 parts of ligusticum wallichii and 3-15g of honey-fried licorice roots. The traditional Chinese medicine composition has the advantages that the traditional Chinese medicine composition adopts a treatment method of softening hardness to dissipate stagnation, dredging collaterals and relieving spasm, and can improve clinical symptoms such as cognitive impairment related to small cerebral vasculopathy of a patient, and / or anxiety and depression, and / or sleep disorder and the like. In-vitro experiments prove that the traditional Chinese medicine composition has a protection effect on human brain microvascular endothelial cell injury caused by hypoxia and reoxygenation, and can promote cell proliferation and inhibit cell apoptosis. Therefore, the traditional Chinese medicine composition can be applied to prevention and / or treatment of clinical symptoms related to small cerebral vasculopathy under the pathological mechanism of pulse stagnation and collateral arthralgia.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and relates to a traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals, its preparation method and application. Background Technology

[0002] Cerebral small vessel disease (CSVD) is a group of clinical, imaging, and pathological syndromes closely related to brain aging, primarily affecting small blood vessels in the brain, including arterioles with a diameter <400 μm and their distal branches, microarteries, and capillaries. Currently, the diagnosis of CSVD mainly relies on its imaging features, including recent subcortical small infarcts, presumed vascular lacunar infarcts, presumed vascular white matter hyperintensity (WMH), presumed vascular perivascular space (PVS), cerebral microbleeds, cortical iron deposition, cerebral hemorrhage and other hemorrhagic markers, and brain atrophy. Studies have found that the onset of CSVD is closely related to age. Resting lacunar infarcts and WMH account for up to 30% of cases in healthy individuals. CSVD symptoms are diverse, sometimes presenting only as the aforementioned imaging abnormalities, and clinically manifesting as long-term headaches, dizziness, sleep disturbances, anxiety, depression, and other emotional abnormalities. As the disease progresses, it gradually affects the patient's gait, memory, bowel and bladder function, and may even lead to stroke and dementia. It is worth noting that CSVD accounts for 25% of stroke causes and 50% of recurrent stroke causes in Chinese people, and is associated with 45% of cognitive impairment and decline in independent living function, making it an important factor that accelerates brain aging.

[0003] Currently, there are no specific and effective drugs for treating CSVD. Treatment methods are still based on those for large vessel diseases, mainly including antihypertensive, antiplatelet aggregation, and lipid-lowering therapies. CSVD carries the risk of both ischemia and hemorrhage, which greatly limits the effectiveness of antiplatelet therapy alone. Moreover, atherosclerosis is not the main pathological change in CSVD, and lipid-lowering drugs are not very effective in treating CSVD clinically.

[0004] Modern medicine believes that CSVD initially manifests as endothelial dysfunction leading to increased permeability of the blood-brain barrier, allowing toxic substances to enter the brain parenchyma, damaging brain cells and resulting in cerebrovascular disease. Traditional Chinese medicine believes that CSVD primarily affects small blood vessels smaller than 400 μm, similar to collateral vessels. Collateral vessels branch off from meridians, crisscrossing and spreading throughout the body. Brain collateral vessels are diffused throughout the brain, with a fine structure remarkably similar to the lesions affected by CSVD. Academician Wang Yongyan also proposed the theory of "toxic damage to brain collateral vessels" to guide clinical treatment of cerebrovascular diseases. Therefore, Traditional Chinese Medicine can treat CSVD by addressing the collateral vessels. Collateral vessels possess physiological characteristics of transportation, substance exchange, and nourishment. Furthermore, their small size and slow blood flow make them prone to stagnation, congestion, and accumulation. Obstruction or impaired perfusion of collateral vessels leads to stagnation of collateral qi, obstruction of collateral vessels, spasm of collateral vessels, and stagnation of heat toxins within the collateral vessels. This refers to the imbalance of Qi and the disordered metabolism of Qi, blood, and body fluids, which is equivalent to the small blood vessel spasm caused by poor microcirculation in modern medicine. The onset of hypertension is closely related to small blood vessel spasm to a certain extent, and the most common consequence of microcirculatory disorders is blood stasis. In addition, phlegm and turbidity are another common pathological factor in vascular diseases. Mucus and fat deposits are traditionally considered "phlegm," while fibrous and connective tissue hyperplasia is considered "hardness." Furthermore, vessel wall hyperplasia is also a major key factor in vascular diseases. Diseases of the collateral vessels are often accompanied by diseases of the brain, internal organs, and meridians throughout the body, thus the affected areas are widespread. Diseases of the collateral vessels and their surrounding structures can further lead to changes in the brain and internal organs, affecting the brain and the entire body's mental functions, resulting in forgetfulness, decreased attention and executive function, anxiety, depression, apathy, irritability, and behavioral disorders. When brain disease affects all internal organs, the clinical symptoms are widespread, manifesting as hemiplegia, dysarthria, gait abnormalities, frequent falls, and urinary abnormalities.

[0005] In summary, cerebral small vessel disease is mainly based on endothelial dysfunction, poor microcirculation, and small vessel spasm. Poor microcirculation in small vessels means that the flow of Qi and blood in the brain's collaterals is obstructed, leading to stagnation and the formation of "nodules" on the vessel walls. Therefore, traditional Chinese medicine diagnosis of small vessel disease can start from "collateral stagnation" and "nodules," focusing on softening and dispersing nodules, while also promoting blood circulation, removing blood stasis, and relieving spasms.

[0006] Traditional Chinese medicine (TCM) has conducted research and developed prescriptions for treating cognitive decline, sleep disorders, anxiety, and depression, but it lacks research on their comorbid mechanisms and effective prescriptions that can treat different diseases with the same approach. Currently, there are no TCM combinations for treating cerebral small vessel disease. Summary of the Invention

[0007] Based on the shortcomings of the existing technology, the purpose of this application is to provide a traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals, its preparation method and application.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: One of the technical solutions of the present invention provides a traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals, which is made from the following raw materials in parts by weight: 20-40 parts of turtle shell, 20-40 parts of white peony root, 14-22 parts of creeping sedum, 9-21 parts of teasel root, 9-21 parts of acorus tatarinowii rhizome, 9-21 parts of astragalus root, 14-22 parts of chuanxiong rhizome, and 3-15 parts of prepared licorice root.

[0009] Preferably, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 25-35 parts of turtle shell, 25-35 parts of white peony root, 16-20 parts of creeping sedum, 12-18 parts of teasel root, 12-18 parts of acorus tatarinowii rhizome, 12-18 parts of astragalus root, 16-20 parts of chuanxiong rhizome, and 6-12 parts of prepared licorice root.

[0010] More preferably, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 30 parts of turtle shell, 30 parts of white peony root, 18 parts of creeping sedum, 15 parts of teasel root, 15 parts of acorus tatarinowii rhizome, 15 parts of astragalus root, 18 parts of chuanxiong rhizome, and 9 parts of prepared licorice root.

[0011] More preferably, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 35 parts of turtle shell, 30 parts of white peony root, 20 parts of creeping sedum, 18 parts of teasel root, 12 parts of acorus tatarinowii rhizome, 18 parts of astragalus root, 16 parts of chuanxiong rhizome, and 12 parts of prepared licorice root.

[0012] More preferably, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 30 parts of turtle shell, 40 parts of white peony root, 22 parts of creeping sedum, 21 parts of teasel root, 12 parts of acorus tatarinowii, 18 parts of astragalus root, 18 parts of chuanxiong rhizome, and 9 parts of prepared licorice root.

[0013] More preferably, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 40 parts of turtle shell, 20 parts of white peony root, 12 parts of creeping sedum, 12 parts of teasel root, 12 parts of acorus tatarinowii rhizome, 15 parts of astragalus root, 20 parts of chuanxiong rhizome, and 12 parts of prepared licorice root.

[0014] More preferably, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 30 parts of turtle shell, 25 parts of white peony root, 16 parts of creeping sedum, 12 parts of teasel root, 12 parts of acorus tatarinowii rhizome, 18 parts of astragalus root, 20 parts of chuanxiong rhizome, and 12 parts of prepared licorice root.

[0015] The second technical solution of the present invention provides a method for preparing a traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals, the specific steps of which are as follows: (1) Take each raw material according to the above proportions: turtle shell, white peony root, creeping sedum, teasel root, acorus tatarinowii, astragalus root, chuanxiong rhizome, and prepared licorice root, and soak in water for 0.2-2 hours; there is no requirement for the amount of water required for soaking, generally just enough to touch all the medicinal materials; (2) Add 2-11 times the amount of water of the medicinal materials, decoct for 0.5-3.5 hours, and pour out the decoction after decoction; (3) Repeat step (2) at least twice to combine the medicinal solutions, which contain a traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals. The medicinal solutions may be further supplemented with pharmaceutically acceptable adjuvants to prepare clinically acceptable tablets, capsules, granules, oral liquids, mixtures or syrups.

[0016] The third technical solution of the present invention provides the application of a traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals in the preparation of drugs for the prevention / treatment of cerebral small vessel disease.

[0017] Furthermore, the pathogenesis of the aforementioned cerebral small vessel disease is stagnation and obstruction of the blood vessels.

[0018] Furthermore, the clinical manifestations of the aforementioned small vessel disease include cognitive decline and / or anxiety, depression, and / or sleep disorders.

[0019] Furthermore, the imaging manifestations of the cerebral small vessel lesions include lacunar foci and / or newly developed subcortical small infarcts and / or white matter hyperintensities and / or microhemorrhages, or combined with enlarged perivascular spaces.

[0020] The fourth technical solution of the present invention provides a drug for the prevention / treatment of cerebral small vessel disease, the drug comprising: the above-mentioned traditional Chinese medicine composition with the function of softening and unblocking collaterals, and pharmaceutically acceptable pharmaceutical excipients; The dosage form of the drug is tablets, capsules, granules, oral liquid, mixture, or syrup.

[0021] The pharmaceutical excipients include, but are not limited to: lactose, starch, pregelatinized starch, mannitol, dextrin, powdered sugar, sodium carboxymethyl cellulose, hydroxypropyl cellulose, gelatin, sucrose, sorbitol, honey, flavoring, or sodium benzoate.

[0022] Compared with the prior art, this application has at least the following improvements and beneficial effects: (1) The traditional Chinese medicine composition of this invention uses softening and dispersing nodules, unblocking collaterals and relieving spasms as treatment methods, which can improve the clinical symptoms of patients with cerebral small vessel disease, such as cognitive impairment, and / or anxiety, depression, and / or sleep disorders. In vitro experiments have shown that this traditional Chinese medicine composition has a protective effect against cerebral microvascular endothelial cell damage caused by hypoxia-reoxygenation, and can promote cell proliferation and inhibit cell apoptosis. Therefore, this traditional Chinese medicine composition can be used for the prevention and / or treatment of clinical symptoms of cerebral small vessel disease under the pathological mechanism of "pulse stagnation and collateral obstruction".

[0023] (2) The present invention selects each raw material and their compatibility to achieve the effect of softening hardness and unblocking collaterals. It fully utilizes the therapeutic characteristics of the synergistic effect of multiple components, multiple pathways and multiple targets of traditional Chinese medicine, which can improve a variety of clinical symptoms related to cerebral small vessel lesions and achieve the same treatment for different diseases. It has great value for promotion and application.

[0024] (3) In this formula, turtle shell is the principal ingredient. It is salty and cold in nature and plays a role in softening hardness and dispersing nodules, nourishing yin and suppressing yang. White peony root is sour and sweet and nourishes yin. It works together with turtle shell to nourish yin and blood. Sedum sarmentosum is sweet and bland in taste and cool in nature. It has the effect of clearing heat and detoxifying. It also clears dampness and detoxifies. Ligusticum chuanxiong is pungent and warm and invigorates blood and qi. It is a qi medicine in blood. It warms and unblocks blood vessels, invigorates blood and removes blood stasis. It also has the effect of guiding blood to the brain. Astragalus membranaceus is sweet and warm and tonifies the spleen and qi. When used with Ligusticum chuanxiong, it can also counteract the coldness of the principal ingredient and protect the spleen and stomach. Dipsacus asperoides tonifies the liver and kidneys and takes into account the common origin of the liver and kidneys. Acorus tatarinowii has the effect of opening the orifices and awakening the mind. The two are used as adjuvant medicines to protect the kidney qi and awaken the mind. Licorice can not only harmonize the medicines, but its sweet and mild nature can also relieve pain. When all the medicines are used together, they work together to soften hardness and dissipate nodules, unblock the meridians and relieve spasms. With the nodules dispersed and the meridians unblocked, the meridians are nourished, the qi and blood are replenished, the brain and spirit are nourished, and the mental function can be restored. Attached Figure Description

[0025] Figure 1 The effects of traditional Chinese medicine composition on the proliferation and migration of H / R-HBMECs cells; Figure 2 The effects of traditional Chinese medicine composition on mitochondrial membrane potential and apoptosis in H / R-HBMECs; Figure 3 The effect of traditional Chinese medicine composition on secretory factors of H / R-HBMECs. Detailed Implementation

[0026] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0027] Unless otherwise specified, all raw materials used in this invention are not subject to any particular restriction on their source; they may be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0028] Example 1: A traditional Chinese medicine composition with the function of softening hard masses and unblocking collaterals (I) 30 parts turtle shell, 30 parts white peony root, 18 parts creeping sedum, 15 parts teasel root, 15 parts acorus tatarinowii, 15 parts astragalus root, 18 parts chuanxiong rhizome, and 9 parts prepared licorice root.

[0029] Example 2: A traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals (II) 30 parts turtle shell, 30 parts white peony root, 14 parts creeping sedum, 9 parts teasel root, 9 parts acorus tatarinowii, 9 parts astragalus root, 14 parts chuanxiong rhizome, and 3 parts prepared licorice root.

[0030] Example 3: A traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals (Part 3) 30 parts turtle shell, 25 parts white peony root, 18 parts creeping sedum, 15 parts teasel root, 9 parts acorus tatarinowii, 9 parts astragalus root, 14 parts chuanxiong rhizome, and 3 parts prepared licorice root.

[0031] Example 4: A traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals (IV) 25 parts of turtle shell, 25 parts of white peony root, 16 parts of creeping sedum, 18 parts of teasel root, 12 parts of acorus tatarinowii rhizome, 12 parts of astragalus root, 16 parts of chuanxiong rhizome, and 9 parts of prepared licorice root.

[0032] Example 5: A traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals (V) 35 parts of turtle shell, 30 parts of white peony root, 20 parts of creeping sedum, 18 parts of teasel root, 12 parts of acorus tatarinowii rhizome, 18 parts of astragalus root, 16 parts of chuanxiong rhizome, and 12 parts of prepared licorice root.

[0033] Example 6: A traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals (VI) 25 parts of turtle shell, 25 parts of white peony root, 22 parts of creeping sedum, 12 parts of teasel root, 9 parts of acorus tatarinowii, 9 parts of astragalus root, 14 parts of chuanxiong rhizome, and 3 parts of prepared licorice root.

[0034] Example 7: A traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals (VII) 30 parts of turtle shell, 40 parts of white peony root, 22 parts of creeping sedum, 21 parts of teasel root, 12 parts of acorus tatarinowii, 18 parts of astragalus root, 18 parts of chuanxiong rhizome, and 9 parts of prepared licorice root.

[0035] Example 8: A traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals (VIII) 40 parts of turtle shell, 20 parts of white peony root, 12 parts of creeping sedum, 12 parts of teasel root, 12 parts of acorus tatarinowii rhizome, 15 parts of astragalus root, 20 parts of chuanxiong rhizome, and 12 parts of prepared licorice root.

[0036] Example 9: A traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals (IX) 30 parts of turtle shell, 25 parts of white peony root, 16 parts of creeping sedum, 12 parts of teasel root, 12 parts of acorus tatarinowii rhizome, 18 parts of astragalus root, 20 parts of chuanxiong rhizome, and 12 parts of prepared licorice root.

[0037] Example 10: A traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals (X) 25 parts of turtle shell, 25 parts of white peony root, 14 parts of creeping sedum, 9 parts of teasel root, 15 parts of acorus tatarinowii rhizome, 15 parts of astragalus root, 18 parts of chuanxiong rhizome, and 9 parts of prepared licorice root.

[0038] Examples 1-10 employ different dosages of traditional Chinese medicine combinations, implemented according to the individual patient's constitution and treatment focus. Example 1 serves as the baseline for this compound, suitable for most patients with "pulse stagnation and collateral obstruction" type cerebral small vessel disease; Example 2 reduces the dosage of assistant and adjuvant herbs, primarily suitable for patients with milder symptoms; Example 3 reduces the dosage of qi-tonifying herbs, focusing on clearing heat and promoting blood circulation; Example 4 primarily reduces the dosage of the principal herb, making it more balanced, and increases the dosage of adjuvant herbs, resulting in an overall balanced constitution; Example 5 increases both tonifying and softening effects; Example 6 focuses on clearing heat, with a relatively mild overall effect; Example 7 enhances blood-nourishing effects while also clearing heat, promoting blood circulation, and tonifying qi; Example 8 emphasizes softening and breaking up masses; Example 9 focuses on promoting blood circulation and qi flow, while also tonifying qi; Example 10 focuses on resolving phlegm and opening the orifices, while also softening masses. These 10 examples demonstrate the individualization of traditional Chinese medicine compound treatment and prove the universality and practicality of the claimed formulation range.

[0039] Example 11 Preparation of granules of traditional Chinese medicine composition with the function of softening hardness and unblocking collaterals Take any one of the traditional Chinese medicine compositions described in Examples 1-10, add 8-10 times the amount of water, decoct for 3 hours, and filter out the liquid. Add another 10 times the amount of water, decoct for 2.5 hours, filter out the liquid, combine the two decoctions, let stand, filter out the supernatant, concentrate, cool, add 2 times the amount of alcohol to the concentrate, stir and let it precipitate overnight. Take the supernatant, concentrate to a thick extract; add appropriate pharmaceutical excipients such as pre-crosslinked starch, granulate, dry, granulate, and package into 20g / bags.

[0040] Example 12 Preparation of tablets / capsules of traditional Chinese medicine composition with softening and unblocking function Take any one of the traditional Chinese medicine compositions described in Examples 1-10, add 9-11 times the amount of water, decoct for 2-3.5 hours, and filter out the liquid. Add another 9 times the amount of water, decoct for 2.5 hours, filter out the liquid, combine the two decoctions, let stand, filter out the supernatant, concentrate, cool, add 3 times the amount of alcohol to the concentrate, stir and let it precipitate overnight. Take the supernatant, concentrate to a thick extract; add the pharmaceutical excipient pre-crosslinked starch, vacuum dry, pulverize and granulate, and compress into tablets or capsules.

[0041] Example 13 Preparation of Traditional Chinese Medicine Composition / Oral Liquid / Syrup with Softening and Unblocking Functions Take any one of the traditional Chinese medicine compositions described in Examples 1-10, add 8-11 times the amount of water, decoct for 3 hours, and filter out the liquid. Add another 8 times the amount of water, decoct for 3 hours, filter out the liquid, combine the two decoctions, let stand, filter out the supernatant, concentrate, cool, add 3.5 times the amount of alcohol as the concentrate, stir and let it precipitate overnight. Take the supernatant, concentrate it to a thick extract; add appropriate pharmaceutical excipients sucrose and sodium benzoate to prepare a compound, oral liquid or syrup.

[0042] Example 14 Cell Experiment 1. Experimental Materials and Instruments 1.1 Experimental Cells Human brain microvascular endothelial cells (HBMECs) were purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd.

[0043] 1.2 Reagents Table 1 Main Reagents 1.3 Instruments Table 2 Main Instruments 2. Methods 2.1 Preparation of freeze-dried powder Take 30 g of turtle shell, 30 g of white peony root, 18 g of creeping sedum, 15 g of teasel root, 15 g of acorus tatarinowii rhizome, 15 g of astragalus root, 18 g of chuanxiong rhizome, and 9 g of prepared licorice root. Soak the above herbs in double-distilled water for 20 minutes, then decoct twice, adding water equal to 10 times the amount of herbs each time, and decocting for 1 hour each time. Place the decoction at -40 °C and -20 °C respectively, and then freeze-dry it to produce a freeze-dried powder of turtle shell and softening herb decoction. In subsequent use, dissolve the prepared freeze-dried powder of turtle shell and softening herb decoction in DMSO to make a solution. For example, a concentration of 1 mg / mL means that 1 mg of freeze-dried powder is dissolved in 1 mL of DMSO. This solution can be stored at -20 °C for later use.

[0044] 2.2 Cell Culture Human brain microvascular endothelial cells (HBMECs) were cultured in DMEM medium (containing 10% FBS and 1% penicillin / streptomycin) at 5% CO2 and 37 °C. To create hypoxic conditions, an anaerobic incubator was used, with nitrogen replacing oxygen to reduce the oxygen concentration to 1% (1% O2, 5% CO2, 94% N2). Cells were cultured in the anaerobic incubator for 12 h, followed by 8 h in a normoxic incubator.

[0045] 2.3 Cell viability assay Log-grown cells were seeded into 96-well plates and incubated overnight. After adhesion, different concentrations of turtle shell and soft vein decoction (final concentrations of lyophilized turtle shell and soft vein decoction were 0, 0.1, 0.5, and 1 μg / mL) were added, and the plates were incubated in an anoxic incubator for 12 h, followed by an oxygen-controlled incubator for 8 h. To assess cell viability, the original medium was replaced with medium containing 10% CCK8, and the cells were incubated for 1.5–2 h. The OD value was measured at 450 nm using a microplate reader. The calculation formula was: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0046] 2.4 Cell Scratch Assay Logarithmically grown HBMECs were inoculated into 12-well plates. After attachment, a straight line was drawn at the bottom of each well using a 10 μL pipette tip. The medium was then replaced with culture medium containing different concentrations of lyophilized turtle shell extract (0, 0.1, 0.5, 1 μg / mL). The plates were incubated in an anoxic incubator for 12 h, followed by an oxygen-controlled incubator for 8 h. Images were taken before and after the anoxic / reoxygenation treatment using an inverted microscope, and the area changes were statistically analyzed using ImageJ.

[0047] 2.5 Mitochondrial membrane potential detection Mitochondrial membrane potential was detected using the JC-1 kit to examine the effect of Biejia Ruanmai Decoction on HBMEC apoptosis. Cells were seeded in 12-well plates and, after adhesion, different concentrations of the drug were added, followed by H / R (ischemia / reperfusion) treatment. The old culture medium was discarded, and cells were slowly washed with PBS. JC-1 staining working solution (prepared according to the manufacturer's instructions) was added in the dark, and the cells were incubated at 37 °C for 20 min. Afterward, the cells were washed with JC-1 buffer (prepared according to the manufacturer's instructions), PBS was added, and images were taken using a high-content cell imaging system. Fluorescence was analyzed using ImageJ.

[0048] 2.6 Apoptosis Detection Apoptosis was detected using a kit (BD Biosciences). Cells were seeded in 6-well plates and allowed to adhere overnight. After treatment with drugs and H / R, the cells were digested with EDTA-free trypsin and centrifuged at 2000 rpm, 4°C, for 10 min. The cell pellet was washed with PBS, and FITC-binding buffer was added. FITC dye was added under light-protected conditions and incubated for 10 min. PI dye was added under the same conditions for 5 min. FITC-binding buffer was added before Beckman flow cytometry analysis. Soft and hard cell analysis results were obtained using the CytExpert system.

[0049] 2.7 Detection of adhesion molecules and endothelial functional factors Cells were seeded in 96-well plates and treated with different concentrations of turtle shell decoction and H / R. The supernatant was collected, and the levels of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1), and endothelin-1 (ET-1) were detected according to the instructions.

[0050] 3. Results 3.1 Turtle shell and soft vein decoction can promote the proliferation and migration of HBMECs. To investigate the effects of Biejia Ruanmai Decoction on HBMECs under H / R conditions, HBMECs were treated with different concentrations of Biejia Ruanmai Decoction. CCK8 results showed that under H / R conditions, Biejia Ruanmai Decoction promoted HBMEC proliferation in a concentration-dependent manner. Figure 1 (A). To further investigate the effect of Biejia Ruanmai Decoction on the migration ability of HBMECs, HBMECs were treated with different concentrations of Biejia Ruanmai Decoction under H / R conditions. Similarly, the scratch assay showed that Biejia Ruanmai Decoction could promote the migration of HBMECs in a dose-dependent manner. Figure 1 (Chinese BC).

[0051] 3.2 Turtle shell and soft vein decoction can reduce mitochondrial membrane potential damage and inhibit cell apoptosis. A decrease in mitochondrial membrane potential is a hallmark event in the early stages of apoptosis. The JC-1 detection kit detects changes in mitochondrial membrane potential by detecting changes in fluorescence color; a high mitochondrial membrane potential produces red fluorescence, while a low mitochondrial membrane potential produces green fluorescence. The results indicate that Biejia Ruanmai Decoction at 0.5 and 1 μg / mL significantly reduced mitochondrial membrane potential damage. Figure 2 (AB). Considering the effect of Biejia Ruanmai Decoction on mitochondrial membrane potential, this invention further investigates the effect of Biejia Ruanmai Decoction on H / R-induced apoptosis of HBMECs under H / R conditions. Similarly, the results of this invention show that Biejia Ruanmai Decoction at concentrations of 0.5 and 1 μg / mL can alleviate H / R-induced apoptosis ( ). Figure 2 Medium CD).

[0052] 3.3 Turtle shell softening decoction can reduce the expression of adhesion molecules, chemokines, and endothelin. Damaged endothelial cells secrete adhesion molecules (ICAM-1, VCAM-1), chemokines (MCP-1), and endothelin, which in turn recruit neutrophils and monocytes to release inflammatory factors and other toxic substances, damaging brain tissue. To investigate the effects of turtle shell decoction on these secreted factors, this invention uses ELISA to detect their expression. The results showed that under H / R conditions, ICAM-1 (… Figure 3 (A), VCAM-1 ( Figure 3 (China B), MCP-1 ( Figure 3 (C) and ET-1 ( Figure 3 Both D and E were elevated to varying degrees, and the addition of different concentrations of Biejia Ruanmai Decoction was able to reduce their expression in a dose-dependent manner. This further indicates that Biejia Ruanmai Decoction can reduce the secretion of cytokines, thereby protecting endothelial cells from damage.

[0053] 4. Conclusion This traditional Chinese medicine composition can protect H / R-HBMECs by promoting cell proliferation and migration, while inhibiting apoptosis and reducing the expression of adhesion molecules.

[0054] Example 15 Clinical Trial 1. Materials 1.1 Research Subjects The 72 patients were from the neurology ward or outpatient department of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, affiliated with Shanghai University of Traditional Chinese Medicine.

[0055] 1.2 Diagnostic criteria Western medical imaging diagnosis refers to the 2013 Neuroimaging Vascular Changes Criteria Reporting Panel criteria, which include the presence of lacunar foci, and / or new subcortical small infarcts, and / or white matter hyperintensity, and / or microbleeds, or enlarged perivascular spaces. Based on the TCM syndrome differentiation and diagnosis, and with reference to "Traditional Chinese Medicine Diagnostics" and Zhu Wenfeng's "Syndrome Differentiation", the diagnosis is pulse stagnation and collateral obstruction.

[0056] 1.3 Inclusion Criteria (1) Meets the diagnostic criteria of Western medicine imaging: The cranial magnetic resonance imaging shows one of the following: recent subcortical small infarction, white matter hyperintensity (WMH), lacunar infarcts, microbleeds (CMB), or combined with enlarged perivascular spaces (EPVS); (2) Meets the TCM diagnostic criteria for "pulse stagnation and collateral obstruction" syndrome; (3) Age ≥ 30 years old and ≤ 70 years old, gender not limited; (4) Understand and agree to participate in this study and sign the informed consent form.

[0057] 1.4 Exclusion Criteria (1) Intracranial and peripheral vascular stenosis ≥50%; (2) Having visual or auditory impairments and being unable to cooperate with examinations and cognitive scale assessments; (3) Other neurological or mental illnesses with a clear cause previously diagnosed: mainly including stroke (excluding lacunar infarction), neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, etc.), white matter hyperintensity caused by other causes (including autoimmune, metabolic, poisoning-related intracranial lesions, etc.), hydrocephalus (excluding brain atrophy), and confirmed mental illnesses; (4) Previous diagnosis of cardiovascular events (including myocardial infarction, angina pectoris, previous carotid endarterectomy or carotid artery stenting, coronary artery stenting, coronary artery bypass surgery). (5) Suffering from other serious diseases (such as malignant tumors, heart failure, respiratory failure, renal failure and severe liver damage, severe hematological diseases or gastrointestinal bleeding); (6) Known allergy to any of the medications in the treatment regimen; (7) Pregnant or lactating women.

[0058] 1.5 Treatment Plan The control group (conventional Western medicine treatment + placebo of Biejia Ruanmai granules) and the experimental group (conventional Western medicine treatment + Biejia Ruanmai granules) were divided into two groups. The Biejia Ruanmai granules consisted of: 30 g of turtle shell, 30 g of white peony root, 18 g of Sedum sarmentosum, 15 g of Dipsacus asper, 15 g of Acorus tatarinowii, 15 g of Astragalus membranaceus, 18 g of Ligusticum chuanxiong, and 9 g of prepared licorice root. The Biejia Ruanmai granules were manufactured by Jiangyin Tianjiang Company (Jiangsu, China) on behalf of the inventor, with batch number 2108343. Patients took two packets twice daily, dissolved in 200 mL of warm water. The treatment course for both groups was 12 weeks.

[0059] 1.6 Observation Indicators (1) TCM syndrome scoring: A TCM syndrome scoring scale was developed based on the reference of "Traditional Chinese Medicine Diagnostics" and Zhu Wenfeng's "Syndrome Differentiation". The patient's symptoms, signs and other data were weighted and summed. Severe symptoms were scored by multiplying the weight by 1.5; moderate symptoms were scored by multiplying the weight by 1; mild symptoms were scored by multiplying the weight by 0.7. If the total weighted score is greater than 20 points, the patient can be diagnosed with the syndrome. (2) Neuropsychological scales: MoCA, MMSE, Auditory Vocabulary Learning Test (AVLT), Connect-the-Dots Test (TMT), Digit Span Test (DST), Stroop Color Word Test (Stroop), Complex Figure Test (CFT), Verbal Fluency Test (CVFT), Boston Naming Test (BNT), HAMD, HAMA, Pittsburgh Sleep Quality Index (PSQI); (3) Laboratory tests: complete blood count, liver and kidney function tests, blood lipids and blood glucose, etc.

[0060] 1.7 Clinical efficacy assessment (1) Clinical cure: The clinical symptoms and signs of traditional Chinese medicine have disappeared or basically disappeared, and the score has decreased by ≥95%; (2) Significant effect: Symptoms and signs are significantly improved, and the score is reduced by ≥70%; (3) Effective: Symptoms and signs improved, and the score decreased by ≥30%; (4) Ineffective: Symptoms and signs do not improve or even worsen, and the score decreases by less than 30%.

[0061] 2. Results 2.1 Comparison of general information between the two groups of patients The control group consisted of 16 males and 20 females, with a mean age of 58.33 ± 9.02 years; the treatment group consisted of 15 males and 21 females, with a mean age of 57.98 ± 9.23 years. There were no statistically significant differences in any baseline characteristics between the two groups. p >0.05, see Table 3.

[0062] Table 3 Comparison of general information between the two groups of patients 2.2 Comparison of Observation Indicators Table 4 shows the statistical analysis of MoCA, MMSE, Auditory Word Learning Test (AVLT), Connect-the-Dots Test (TMT), Digit Span Test (DST), Stroop Color Word Test, Complex Figure Test (CFT), Verbal Fluency Test (CVFT), Boston Naming Test (BNT), HAMD, HAMA, and Pittsburgh Sleep Quality Index (PSQI) before and after treatment in both groups. The results showed no statistically significant differences in any of the indicators before and after treatment in the control group. p >0.05), while in the experimental group, MoCA, HAMD, HAMA, and PSQI all showed significant reductions after treatment compared to before treatment, with statistically significant differences. p <0.05).

[0063] Table 4 Comparison of scale scores between the two groups of patients before and after treatment Note: Compared with before treatment,* p <0.05, ** p <0.01; compared with the control group, # p <0.05, ## p <0.01.

[0064] 2.3 Comparison of TCM syndrome differentiation and treatment efficacy As shown in Table 5, in the control group, 0 patients were basically cured, 0 showed significant improvement, 3 showed improvement (8.33%), and 33 showed no improvement (91.67%), with a total effective rate of 8.33%. In the experimental group, 0 patients were basically cured, 20 showed significant improvement (55.56%), 8 showed improvement (22.22%), and 8 showed no improvement (22.22%), with a total effective rate of 77.78%. The difference in the total efficacy of TCM syndromes between the groups was statistically significant. p <0.05).

[0065] Table 5. Comparison of the total therapeutic effect of TCM syndrome scores between the two groups of patients after treatment. Note: Compared with the control group, ***p <0.001.

[0066] 2.4 Safety Indicator Analysis As shown in Table 6, statistical analysis was performed on the safety indicators of liver and kidney function in the experimental group before treatment and after the 2nd and 12th weeks of treatment. The results showed that there were no statistically significant differences in any of the indicators. p >0.05).

[0067] Table 6 Comparison of liver and kidney function indicators in the experimental group before and after treatment 3. Conclusion The traditional Chinese medicine composition of this invention has the effect of protecting endothelial cells and improving clinical symptoms such as cognitive decline and / or anxiety, depression and / or sleep disorders associated with cerebral small vessel disease.

[0068] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition with the functions of softening hard masses and dredging collaterals, characterized in that, The traditional Chinese medicine composition is prepared from the following raw medicinal materials by weight: turtle shell 20-40 parts, white peony root 20-40 parts, sedum lineare 14-22 parts, rhizoma dangshen 9-21 parts, aconite 14-22 parts, radix paeoniae alba 3-15 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is prepared from the following raw medicinal materials by weight: turtle shell 25-35 parts, white peony root 25-35 parts, sedum lineare 16-20 parts, rhizoma dangshen 12-18 parts, aconite 12-18 parts, radix paeoniae alba 6-12 parts.

3. The traditional Chinese medicine composition according to claim 2, characterized in that, The traditional Chinese medicine composition is prepared from the following raw medicinal materials by weight: turtle shell 30 parts, white peony root 30 parts, sedum lineare 18 parts, rhizoma dangshen 15 parts, aconite 15 parts, radix paeoniae alba 9 parts.

4. The traditional Chinese medicine composition according to claim 2, characterized in that, The traditional Chinese medicine composition is prepared from the following raw medicinal materials by weight: turtle shell 35 parts, white peony root 30 parts, sedum lineare 20 parts, rhizoma dangshen 18 parts, aconite 12 parts, radix paeoniae alba 12 parts.

5. The traditional Chinese medicine composition according to claim 2, characterized in that, The traditional Chinese medicine composition is prepared from the following raw medicinal materials by weight: turtle shell 30 parts, white peony root 40 parts, sedum lineare 22 parts, rhizoma dangshen 21 parts, aconite 12 parts, radix paeoniae alba 9 parts.

6. The traditional Chinese medicine composition according to claim 2, characterized in that, The traditional Chinese medicine composition is prepared from the following raw medicinal materials by weight: turtle shell 40 parts, white peony root 20 parts, sedum lineare 12 parts, rhizoma dangshen 12 parts, aconite 12 parts, radix paeoniae alba 15 parts.

7. The traditional Chinese medicine composition according to claim 2, characterized in that, The traditional Chinese medicine composition is prepared from the following raw medicinal materials by weight: turtle shell 30 parts, white peony root 25 parts, sedum lineare 16 parts, rhizoma dangshen 12 parts, aconite 12 parts, radix paeoniae alba 12 parts.

8. The preparation method of the traditional Chinese medicine composition according to any one of claims 1-7, characterized in that, The specific steps are as follows: (1) according to any one of claims 1-7, the raw medicinal materials are taken in the following proportions: turtle shell, white peony root, sedum lineare, rhizoma dangshen, aconite, radix paeoniae alba, and water is added for soaking for 0.2-2 hours; (2) 2-11 times the amount of water is added, and decoction is performed for 0.5-3.5 hours, and the decoction is poured out after decoction; (3) step (2) is repeated at least twice, and the decoctions are combined, and the decoctions contain the traditional Chinese medicine composition having the functions of softening and dredging collaterals.

9. The use of the traditional Chinese medicine composition according to any one of claims 1-7 in the preparation of a drug for preventing / treating cerebral small vessel disease, characterized in that, The cerebral small vessel disease is pulse stagnation and collateral block.

10. A prophylactic / therapeutic drug for cerebral small vessel disease, characterized by comprising the compound or salt according to claim 1. The medicine comprises the traditional Chinese medicine composition according to any one of claims 1-7, and a pharmaceutically acceptable pharmaceutical adjuvant; the medicine is in any one of the following dosage forms: granules, tablets, capsules, oral liquid, mixture, or syrup; and the pharmaceutical adjuvant is selected from one or more of the following: lactose, starch, pre-gelatinized starch, mannitol, dextrin, sugar powder, sodium carboxymethyl cellulose, hydroxypropyl cellulose, gelatin, sucrose, sorbitol, honey, essence, or sodium benzoate.

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