A composition containing phthalide extract from the stems and leaves of Ligusticum chuanxiong and its application.

Abstract

This invention discloses a composition containing a phthalide extract from the stems and leaves of Ligusticum chuanxiong and its application, specifically relating to the field of pharmaceutical technology. The main active ingredient of the composition is the phthalide extract from the stems and leaves of Ligusticum chuanxiong; the phthalide extract comprises 3-butenylphthalide, ligustilide, neo-ostrichylide, ligustilide I, ligustilide H, ligustilide A, and angelicin A, with the total amount of active ingredients not less than 50%. This invention successfully prepared a phthalide extract from the non-medicinal parts of Ligusticum chuanxiong, its stems and leaves, and applied it to the treatment of ischemic stroke. This invention discovers an anti-ischemic stroke drug from the traditionally discarded stems and leaves of Ligusticum chuanxiong, opening a new avenue for the development of ischemic stroke drugs.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a composition containing phthalide extract from the stems and leaves of Ligusticum chuanxiong and its application. Background Technology

[0002] Ischemic stroke is caused by insufficient blood supply to the brain tissue due to thrombosis or embolism, leading to ischemia and hypoxia of nerve cells, impaired function, and clinical symptoms of neurological dysfunction. It is characterized by rapid onset, high incidence, high mortality / disability rates, and high recurrence rates, accounting for approximately 87% of all stroke cases. Clinically, the main treatments for ischemic stroke are thrombolysis or interventional thrombectomy, but their application is limited due to their narrow treatment window (<4.5 hours) and high risk of bleeding. To date, there are no ideal drugs for treating ischemic stroke. Furthermore, reperfusion can further aggravate brain tissue damage, known as cerebral ischemia-reperfusion injury (I / R). Therefore, there is an urgent need to find new drugs for treating ischemic stroke.

[0003] Based on the advantages of the "one body, multiple uses" principle in drug resource discovery of traditional Chinese medicine, the discovery of anti-ischemic stroke drugs from the stems and leaves of *Ligusticum chuanxiong* (known for its role in treating dizziness) could open a new avenue for the development of drugs for ischemic stroke. Given the universality and scientific validity of the "one body, multiple uses" theory in traditional Chinese medicine, new medicinal parts can be discovered by comparing their chemical composition, pharmacological efficacy, and clinical uses with traditional non-medicinal parts. *Ligusticum chuanxiong*, as a commonly used bulk commodity, is not only in demand for clinical decoction pieces but is also a major raw material for many traditional Chinese medicine preparations. As a major byproduct of *Ligusticum chuanxiong* production, its stems and leaves have abundant biomass (self-tested by the research group: fresh weight 62%, dry weight 52%, see...). Figure 1 However, its application is limited, leading to serious resource waste. According to statistics, the domestic annual output of Ligusticum chuanxiong reached 17,000 tons in 2022 (source: Tiandi Yuntu Traditional Chinese Medicine Industry Big Data), with its stems and leaves exceeding 8,000 tons annually, indicating abundant resources. The suitable harvesting period for Ligusticum chuanxiong stems and leaves is from early May to mid-May each year. This not only ensures the harvest of stems and leaves but also does not affect the yield and quality of the medicinal parts of Ligusticum chuanxiong. The stems and leaves of Ligusticum chuanxiong were anciently known as *miwu*, and their use has a long history. Records in *Mingyi Bielu*, *Bencao Jing Jizhu*, and *Bencao Gangmu* state that *miwu* is effective for treating chronic wind-related ailments, long-term wind-related headaches, and dizziness. Currently, in the main Ligusticum chuanxiong producing areas of Dujiangyan and Pengzhou in Sichuan, there is a common practice of consuming Ligusticum chuanxiong stems and leaves, often used in folk medicine to prevent cardiovascular and cerebrovascular diseases.

[0004] The term "wind-dizziness" first appeared in Chao Yuanfang's "Treatise on the Causes and Symptoms of Various Diseases" during the Sui Dynasty, describing sudden dizziness as the initial and primary symptom. The renowned traditional Chinese medicine scholar, Academician Wang Yongyan, considered wind-dizziness to be a case of deficiency in the root and excess in the branch, belonging to the category of stroke-like disorders (Western medicine's cerebrovascular accident).

[0005] Ligusticum chuanxiong is a traditional Chinese medicine whose rhizome is used medicinally. However, our research group has made a series of advances in discovering the chemical value of Ligusticum chuanxiong's stems and leaves. For the first time, we have elucidated the metabolic profiles and functional differences of different parts of Ligusticum chuanxiong, revealing that phthalide compounds are the main components of each part, and phthalide polymers are the main components that distinguish the stems and leaves from the medicinal parts, the rhizome. These phthalide polymers may be the material basis for the "treating dizziness and vertigo" properties of Ligusticum chuanxiong's stems and leaves. The maximum daily dose of Ligusticum chuanxiong's stems and leaves is 560 times the adult daily dose, with low acute toxicity and good safety, indicating great potential for drug development. Summary of the Invention

[0006] Therefore, the present invention provides a composition containing phthalide extract of Ligusticum chuanxiong stems and leaves and its application, in order to solve the problem of insufficient utilization of Ligusticum chuanxiong stems and leaves in existing methods.

[0007] This invention provides a method for preparing a phthalide extract from the stems and leaves of Ligusticum chuanxiong, and characterizes its chemical components using UHPLC / Q-Orbitrap. The content of seven representative phthalide components was determined, and the efficacy of this drug composition against ischemic stroke was studied using a rat MCAO model. Exploring the structurally diverse phthalides from the stems and leaves of Ligusticum chuanxiong may be a shortcut to discovering drugs against ischemic stroke. This not only helps to realize the development of Ligusticum chuanxiong medicinal resources, making the most of resources and turning waste into treasure, but also benefits the quality and efficiency of the Ligusticum chuanxiong industry.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] According to a first aspect of the present invention, a composition containing a phthalide extract of Ligusticum chuanxiong stems and leaves is provided, wherein the main active ingredient of the composition is the phthalide extract of Ligusticum chuanxiong stems and leaves; the phthalide extract of Ligusticum chuanxiong stems and leaves includes 3-butenylphthalide, ligustilide, neo-spathololide, ligustilide I, ligustilide H, ligustilide A and angelicalide A, and the total amount of active ingredients is not less than 50%.

[0010] Furthermore, the extraction method of the phthalide extract from the stems and leaves of Ligusticum chuanxiong includes: naturally air-drying the stems and leaves of Ligusticum chuanxiong, pulverizing and sieving them, adding them to a percolation tank, soaking them in 95% ethanol at room temperature and then percolating to extract them, collecting the eluent, concentrating it under reduced pressure to obtain an extract, dispersing it with water, extracting it with ethyl acetate, concentrating the ethyl acetate extract under reduced pressure, eluting the highly polar non-phthalide components with methanol using X-5 macroporous resin, eluting it with 90% ethanol, collecting the eluent, concentrating it under reduced pressure to obtain an extract, and freeze-drying it to obtain the phthalide extract from the stems and leaves of Ligusticum chuanxiong.

[0011] The application of a composition containing phthalide extract of Ligusticum chuanxiong stem and leaves in the preparation of a drug for the prevention and treatment of stroke, according to a second aspect of the present invention.

[0012] According to a third aspect of the present invention, a medicament for preventing and treating stroke includes a composition containing an extract of phthalide from the stems and leaves of Ligusticum chuanxiong.

[0013] Furthermore, the drug also contains pharmaceutically acceptable carriers or excipients.

[0014] Furthermore, the pharmaceutically acceptable carrier or excipient includes one or more solid, semi-solid, or liquid excipients.

[0015] Furthermore, the drug is formulated into a pharmaceutically acceptable preparation.

[0016] Furthermore, the pharmaceutically acceptable formulation includes tablets, capsules, granules, injections, pills, syrups, powders, or ointments.

[0017] The present invention has the following advantages:

[0018] (1) This invention utilizes the stems and leaves of Ligusticum chuanxiong, which is not commonly used as a medicinal herb, to extract phthalide-free components and characterize them. This fully utilizes the stems and leaves of Ligusticum chuanxiong, which is not commonly used, and provides a theoretical basis for the use of the whole plant of Ligusticum chuanxiong as a medicinal herb.

[0019] (2) This invention prepares a simple and efficient pharmaceutical composition from phthalide extracts (3-butenylphthalide, ligustilide, neo-spathololide, ligustilide I, ligustilide H, ligustilide A and angelicalide A) from the stems and leaves of Ligusticum chuanxiong, thus enriching the medicinal parts of Ligusticum chuanxiong.

[0020] (3) The pharmacodynamic results of the present invention show that the drug composition can improve neurological deficits caused by ischemia-reperfusion, reduce cerebral infarction area, reduce brain water content and edema rate; indicating that the drug composition has a better effect in preventing and treating ischemic stroke, and is more effective than a single commercially available drug. Attached Figure Description

[0021] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0022] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0023] Figure 1 This is a diagram showing the biomass percentage of different parts of Ligusticum chuanxiong resource, provided in the background art of this invention;

[0024] Figure 2 The total ion chromatogram of the phthaloyl phthalate extract of Ligusticum chuanxiong stem and leaf in positive ion mode provided in Example 1 of the present invention;

[0025] Figure 3 This is a high-performance liquid chromatogram of the mixed reference standard and the extract of Ligusticum chuanxiong stem and leaf provided in Example 1 of the present invention; wherein, A: test solution at 280 nm; B: test solution at 230 nm; C: mixed reference solution at 280 nm; D: mixed reference solution at 230 nm; in the figure, 1-Ligusticum chuanxiong lactone I; 2-Ligusticum chuanxiong lactone H; 3-Ligusticum chuanxiong lactone A; 4-Z-Ligustilide; 5-Angelicainolone A; 6-Neo-Cnidium monnieri lactone; 7-3-Butenylphthalide;

[0026] Figure 4 The pharmacodynamics of the phthalyl extract of Ligusticum chuanxiong stem and leaf provided in Example 2 of this invention for the prevention and treatment of ischemic stroke includes: A: Schematic diagram of drug administration; B: Infarct volume; C: Statistical graph of infarct volume; D: Neurological dysfunction score; E: Cerebral edema rate; F: Brain water content.

[0027] Figure 5 The image shows the HE staining and Nissl staining results of the rat brain tissue cortex region provided in Example 2 of the present invention;

[0028] Figure 6 The levels of inflammatory factors in the ischemic brain region provided in Embodiment 2 of the present invention; wherein, A: level of inflammatory factor IL-1β; B: level of inflammatory factor IL-18; C: level of inflammatory factor TNF-α;

[0029] Figure 7 The phthalide extract of Ligusticum chuanxiong stem and leaf provided in Example 2 of this invention regulates CASR expression; wherein, A: CASR immunofluorescence; B: CASR Western blot expression; C: CASR immunofluorescence quantitative analysis; D: CASR protein Western blot expression quantitative analysis;

[0030] Figure 8 The phthalide extract from the stems and leaves of Ligusticum chuanxiong provided in Example 2 of this invention blocks the oligomerization of NLRP3 inflammasomes and inhibits the inflammatory response; wherein, A: NLRP3 immunofluorescence; B: inflammasome Western blot expression; C: quantitative analysis of NLRP3 immunofluorescence; D: quantitative analysis of inflammasome protein Western blot expression.

[0031] Figure 9The phthalide extract from the stems and leaves of Ligusticum chuanxiong provided in Example 2 of this invention reduces the disruption of the blood-brain barrier; wherein, A: Evans blue staining; B: Evans blue leakage; C: Western blot expression of tight junction protein; D: quantitative analysis of Western blot expression of tight junction protein. Detailed Implementation

[0032] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Instruments and reagents

[0034] 1. Instruments

[0035] Ultimate3000RSLC ultra-high performance liquid chromatograph (Thermo Fisher Scientific); QExactive high resolution mass spectrometer (Thermo Fisher Scientific); Agilent 1260 high performance liquid chromatograph (Agilent Technologies); MCAO line plug (Shenzhen Ruiward Life Science Technology Co., Ltd.); BSA224S electronic balance and BP211D electronic balance (Beijing Sartorius Scientific Instruments Co., Ltd.)

[0036] 2. Medicinal materials and reagents

[0037] The stems and leaves of Ligusticum chuanxiong were collected in Meishan City, Sichuan Province in 2020 and identified as Ligusticum chuanxiong Hort. plants by Professor Hu Changjiang of Chengdu University of Traditional Chinese Medicine. The stems and leaves were crushed and passed through a No. 2 sieve for later use. Ligusticum chuanxiong lactone I (2203185901), Ligusticum chuanxiong lactone H (2203185801), and Neo-Synostemma pentaphyllum lactone (220326479) were purchased from Sichuan Hengcheng Zhiyuan Biotechnology Co., Ltd.; Angelica sinensis lactone A (PRF7110726) and Ligusticum chuanxiong lactone A (PRF8031301) reference standards were purchased from Chengdu Purifa Technology Development Co., Ltd.; Z-ligustilide (G010-190105) reference standard was purchased from Chengdu Ruifensi Biotechnology Co., Ltd.; and 3-butenylphthalide (CFS201801) was purchased from ChemFaces. Chloral hydrate was purchased from Chengdu Kelong Chemical Co., Ltd., and meloxicam injection was purchased from Qingdao Kangdian Animal Pharmaceutical Co., Ltd.; acetonitrile and methanol were of chromatographic grade, the experimental water was ultrapure water, and the remaining reagents were of analytical grade.

[0038] Example 1

[0039] 1. Preparation of crude phthalide extract from the stems and leaves of Ligusticum chuanxiong:

[0040] The stems and leaves of Ligusticum chuanxiong were naturally air-dried, pulverized and passed through a No. 2 sieve, and added to a percolation tank. After soaking in 8 times the amount of 95% ethanol at room temperature for 24 hours, the extract was percolated. The eluent was collected, concentrated under reduced pressure to obtain an extract, dispersed in water, and extracted 6 times with ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure, and the highly polar non-phthalide components were eluted with 20% methanol and 90% ethanol using X-5 macroporous resin. The eluent was collected, concentrated under reduced pressure to obtain an extract, and freeze-dried to obtain the phthalide extract of Ligusticum chuanxiong stems and leaves.

[0041] 2. Characterization by UHPLC-MS

[0042] 2.1 Sample Preparation

[0043] Accurately weigh the phthalide extract from the stems and leaves of *Ligusticum chuanxiong*, place it in a 50 mL volumetric flask, and dilute to the mark with methanol. Filter through a 0.22 μm organic phase microporous membrane to prepare a 15 mg / mL sample solution. Accurately weigh Z-ligustilide, ligustilide A, 3-butenylphthalide, ligustilide I, ligustilide H, and angelicalide A reference standards, and dissolve them in methanol to prepare a 1 mg / mL standard solution. Store all prepared samples at 4 °C until analysis by UHPLC / Q-Orbitrap MS.

[0044] 2.2 Chromatographic conditions

[0045] The chromatograph was a UPLC-DAD3000 model, with an ACQITYBEHC18 column (17 μm, 21 × 100 mm, Waters UK); the mobile phase was 0.1% formic acid water (A) – acetonitrile (B), with gradient elution: 0–3 min, 20%–30% B; 37 min, 30%–40% B; 7–10 min, 40%–50% B; 10–17 min, 50%–60% B; 17–19 min, 60%–85% B; 19–25 min, 85%–95% B; 25–30 min, 95% B; column temperature: 25℃; flow rate set to 0.2 μL / min; injection volume 2 μL.

[0046] 2.3 Mass Spectrometry Conditions

[0047] Spray ionization (ESI) was used for data acquisition in Full MS / dd-MS2 mode, with simultaneous scanning of positive and negative ions. The capillary temperature was 300℃, the vaporization temperature was 250℃, the spray voltage was 3.5kV, and the spray current was 18μA. The capillary voltages for positive and negative ion modes were 3.5kV and 3.0kV, respectively. Both the sheath gas and the auxiliary gas were high-purity nitrogen. The sheath gas flow rate was 35arb, the auxiliary gas flow rate was 10arb, and the sweep gas flow rate was 0arb. The MS1 full scan range was 100–1200 Da, the scan time was 0.2 s, and the detection time was 30 min. The MS1 full scan resolution was 70,000, and the MS2 resolution was 13,500.

[0048] 2.4 Component Identification and Characterization

[0049] Raw mass spectrometry data obtained from UHPLC / Q-Orbitrap MS were imported into Compound Discover v3.1 software for initial screening using the software's online database and an imported self-built database. Based on chromatographic retention time, quasi-molecular ion peaks, and fragment ions, MS1 and MS2 mass spectra of the quasi-molecular ion peaks were extracted. More accurate characterization was performed based on the mass spectrometric fragmentation patterns of phthalide standards and phthalide analogs, as well as comparisons with literature mass spectrometry fragments. Forty-two phthalide components present in the prepared pharmaceutical composition were identified. (See [link to relevant documentation]). Figure 2 Table 1.

[0050] Table 1. Component information of phthalide extract from the stems and leaves of Ligusticum chuanxiong.

[0051]

[0052]

[0053]

[0054] Among them, numbers 7, 10, 21, 26, 27, and 32 are comparisons with reference standards, while the others are comparisons with existing literature.

[0055] 3. HPLC content determination

[0056] The content of seven phthalide components in the drug composition was determined using 3-butenylphthalide, ligustilide, neo-spathololide, ligustilide I, ligustilide H, ligustilide A and angelicalide A as reference standards.

[0057] 3.1 Chromatographic conditions and system suitability test

[0058] Chromatographic column Dikma C 18(250mm×4.6mm, 5μm); Column temperature: 32℃; Detection wavelength: Z-ligustilide, ligustilide A, angelicalide A, ligustilide I, and ligustilide H are detected at 280nm, neo-spathololide and 3-butenylphthalide are detected at 230nm; Flow rate: 1mL / min; Injection volume: 10μL; Mobile phase: water (A)–methanol (B); Gradient elution: 0–5min, 50%B; 5–10min, 50%–65%B; 10–15min, 65%–80%B; 15–20min, 80%–90%B; 20–25min, 90%–95%B. Under the above chromatographic conditions, the solutions from sections "3.2" and "3.3" were injected for analysis. The theoretical plate number of each analyte peak was not less than 4000, and the resolution between each peak was greater than 1.5. The chromatogram is shown below. Figure 3 .

[0059] 3.2 Preparation of mixed reference solution

[0060] Weigh appropriate amounts of 3-butenylphthalide, Z-ligustilide, neo-sericulture lactone, ligustilide A, angelica lactone A, ligustilide I, and ligustilide H standards respectively, accurately weigh them, place them in a 5 mL volumetric flask, dissolve them in methanol and dilute to the mark, shake well, filter through a 0.45 μm microporous membrane, and prepare a mixed reference solution containing 0.836, 1.064, 0.370, 0.992, 0.900, 0.600, and 0.520 mg / mL of 3-butenylphthalide, Z-ligustilide, neo-sericulture lactone, ligustilide A, angelica lactone A, ligustilide I, and ligustilide H respectively, as the stock solution.

[0061] 3.3 Preparation of the test solution

[0062] Accurately weigh about 5 mg of the drug composition that has been freeze-dried to constant weight, place it in a 5 mL volumetric flask, dissolve and dilute it to the mark with methanol, shake well, and filter it through a 0.45 μm microporous membrane to obtain a test solution with a concentration of 1.526 mg / mL.

[0063] 3.4 Examination of Linear Relationships

[0064] Accurately pipette an appropriate amount of the mixed solution of the reference solution under section "3.2" and dilute with methanol to prepare a series of mixed reference solutions of different concentrations. Inject and determine the chromatographic conditions under section "3.1". Plot a standard curve with peak area (Y) as the ordinate and reference concentration as the abscissa (X, mg / mL) as the abscissa and perform linear regression. The results are shown in Table 2.

[0065] Table 2. Regression equations, correlation coefficients r, and linear ranges of the main components.

[0066]

[0067] 3.5 Precision Test

[0068] Accurately pipette the mixed reference solution from section "3.2" and inject it six times consecutively under the chromatographic conditions described in section "3.1". Record the peak areas and calculate the RSD values ​​of the peak areas for 3-butenylphthalide, Z-ligustilide, neo-spathololide, ligustilide A, angelicalide A, ligustilide I, and ligustilide H, which are 1.66%, 0.46%, 0.28%, 0.54%, 1.35%, 0.37%, and 0.53%, respectively. This indicates that the instrument has good precision.

[0069] 3.6 Stability Test

[0070] Take an appropriate amount of the test solution and inject it at 0, 2, 4, 8, 12, and 24 h according to the chromatographic conditions in section “3.1”. Record the peak areas and calculate the RSD values ​​of the peak areas of 3-butenylphthalide, Z-ligustilide, neo-spathololide, ligustilide A, angelicalide A, ligustilide I, and ligustilide H, which are 1.48%, 2.32%, 1.35%, 0.24%, 1.52%, 3.12%, and 0.45%, respectively, indicating that the test solution has good stability within 24 h.

[0071] 3.7 Repeatability Test

[0072] Six portions of the same batch of drug composition were taken, and six test solutions were prepared in parallel according to the method in section "3.3". The solutions were then injected and measured under the chromatographic conditions in section "3.1", and the peak areas were recorded. The calculated RSD values ​​of the peak areas were 1.54%, 2.45%, 1.79%, 1.65%, 1.24%, 1.48%, and 0.24%, respectively. This indicates that the method has good repeatability.

[0073] 3.8 Recovery Test

[0074] Take an appropriate amount of the drug composition with a determined content, make 6 portions, and add 100% of the contents of 3-butenylphthalide, Z-ligustilide, neo-orthosiphon, ligustilide A, angelica lactone A, ligustilide I, and ligustilide H in the phthalide extract sample of Ligusticum chuanxiong stem and leaf. Prepare the test solution according to the method in section "3.3", and determine the contents under the chromatographic conditions in section "3.1". The average recoveries of 3-butenylphthalide, Z-ligustilide, neo-orthosiphon, ligustilide A, angelica lactone A, ligustilide I, and ligustilide H were calculated to be 101.3%, 99.0%, 95.7%, 101.0%, 97.4%, 100.0%, and 101.7%, respectively. The RSD values ​​were 2.79%, 1.41%, 4.48%, 2.21%, 2.15%, 3.39%, and 3.72%, respectively. This indicates that the method used to determine the content of the above components has good recovery rate and high accuracy.

[0075] 3.9 Determination of sample content

[0076] Weigh an appropriate amount of the extract of the pharmaceutical composition, prepare the test solution according to the method in section "3.3", inject and determine the sample under the chromatographic conditions in section "3.1", record the peak area, and calculate the content of the main component in the sample using the external standard method based on the peak area. The calculated contents of 3-butenylphthalide, Z-ligustilide, neo-spathololide, ligustilide A, angelicalide A, ligustilide I, and ligustilide H in the sample are 365.6776, 96.9664, 29.7840, 22.0713, 15.9657, 8.4200, and 1.5424 mg / g, respectively. The total content of the main component phthalide in the pharmaceutical composition is 540.4274 mg / g.

[0077] Example 2

[0078] This embodiment provides the effect of phthalide extract from the stems and leaves of Ligusticum chuanxiong in preventing ischemic stroke.

[0079] 1. Grouping and administration of experimental animals

[0080] SPF-grade male SD rats, weighing 280–300 g, were randomly divided into a sham-operated group, a model group, a positive control group (butylphthalide capsule control group, administered at 20 mg / kg according to clinical conversion), and a low-, medium-, and high-dose drug combination group (SLECX). Each group consisted of 12 animals. All experimental drugs were dissolved in 0.5% CMCNa. Rats in all groups were administered the drug by gavage at 9:00 AM daily for three days prior to surgery. Two nights before surgery, the rats were fasted but allowed free access to water. One hour after gavage administration on the third day, the MCAO / R model was established in all groups (except the sham-operated group).

[0081] 2. Rat model of cerebral ischemia-reperfusion

[0082] A rat model of MCAO was established using the suture occlusion method. Rats were anesthetized with 7% chloral hydrate, and meloxicam injection (5 mg / kg) was used for intraoperative analgesia. After fixation in the supine position, the neck was disinfected and prepared, and a right-sided cervical incision was made to separate the external carotid and common carotid arteries. An 18 mm suture occluded the right middle cerebral artery through an incision approximately 0.8 mm from the Y-shaped bifurcation of the external carotid artery. After 90 minutes of ischemia, the suture occluded the right middle cerebral artery, allowing blood to flow from the common carotid artery into the brain for reperfusion. In the sham surgery group, only the common carotid artery was dissected, and the wound was immediately sutured. Postoperatively, the rats were returned to their cages and given ample food and water until they naturally recovered. To prevent hypothermia, a heat lamp was used to maintain the rats' body temperature at approximately 37°C. Neurological function was evaluated 24 hours after reperfusion. (See schematic diagram.) Figure 4 A.

[0083] 3. The phthalide extract from the stems and leaves of Ligusticum chuanxiong reduced the infarct area in MCAO rats.

[0084] Rats were sacrificed 24 hours after reperfusion, and the brains were rapidly separated and placed in a -20°C refrigerator. After 20 minutes, brain tissue was removed at room temperature and dissected into 2 mm thick sections. Starting from the frontal pole, 2 mm coronal sections were prepared five times, discarding the cerebellum and olfactory bulb. Brain tissue sections were stained in 2.0% TTC solution at 37°C for 30 minutes, then washed three times with physiological saline. Brain tissue sections were fixed with 4% paraformaldehyde for 2 hours and captured with a digital camera. Infarct area was calculated using ImageJ software. The ischemic volume of cerebral infarction = infarct area * 2 mm / non-infarct area * 2 mm = infarct area of ​​each section * section thickness. In TTC staining, red areas represent non-infarct areas, and white areas represent infarct areas. The results showed that, compared with the model group, the phthalophthalic acid extract of Ligusticum chuanxiong stems and leaves significantly reduced the cerebral infarction volume in a dose-dependent manner. Figure 4 B, C).

[0085] 4. Phenylphthalide extract from the stems and leaves of Ligusticum chuanxiong improves neurological dysfunction in MCAO rats.

[0086] Twenty-four hours after reperfusion, the Zea Longa five-point scale was used to score the neurological deficits in each group of rats. Inability to fully extend the symmetrical forepaws scored 1 point; circling to the opposite side scored 2 points; tilting to the opposite side scored 3 points; inability to walk spontaneously and loss of consciousness scored 4 points. A score of 1 or higher was considered a successful model. Any rats that died, showed subarachnoid hemorrhage upon autopsy, or showed no neurological deficits (0 points) were considered to have failed the model. Rats scoring 4 points, indicating extremely severe impairment, were excluded from further study. Results are shown below. Figure 4 As shown in D, the prepared pharmaceutical composition can improve neurological deficits caused by ischemia-reperfusion in a dose-dependent manner.

[0087] 5. Phenylphthalide extract from the stems and leaves of Ligusticum chuanxiong improves cerebral edema in MCAO rats.

[0088] After assessing neurological function, rats were euthanized, and intact brain tissue was removed. The brain was then rapidly excised and cut in half along the sagittal suture. The ischemic and normal hemispheres were dried overnight at 100°C in a drying oven, and their weights were obtained before and after drying. Brain water content (%) = (wet weight - dry weight) / wet weight × 100%. Cerebral edema (%) = (ischemic brain water content - normal brain water content) / normal brain water content × 100%. The results showed ( Figure 4 E, F), administering different doses of the drug combination significantly reduced the increase in brain water content and edema on the ischemic side caused by ischemia-reperfusion.

[0089] 6. The phthalide extract from the stems and leaves of Ligusticum chuanxiong significantly improved I / R-induced neuronal damage in brain tissue.

[0090] Brain tissue fixed in 40% neutral formaldehyde was dehydrated, embedded, and prepared into 4μm serial sections using a pathological sectioning machine for observation of the brain tissue pathological structure. HE staining results of the rat brain cortex showed ( Figure 5 In the model group, the cortical brain tissue of rats exhibited a loose structure and interstitial edema. Cell nuclei were shrunken, aggregated, and bluish-black in color. Nucleoli were absent or blurred, cells shrank into triangular shapes, and the cytoplasm showed diffuse vacuolation. The cytoplasm in the infarcted area was pale red. After intervention with phthalide extract from the stems and leaves of *Ligusticum chuanxiong*, low-dose treatment improved nuclear condensation, but did not significantly improve interstitial edema; however, it improved the vacuolation of the cytoplasm. In the medium-dose group, nuclear condensation in the ischemic cortex was significantly improved, neurons were neatly arranged, and intact cellular structures were present, with a reduction in necrotic cells. In the high-dose group, the ischemic side cortical structure tended to be similar to the normal group, with only a small amount of interstitial edema and vacuolation of the cytoplasm. Nissl staining results of the rat brain cortex showed (…). Figure 5 In group B), the I / R group showed severe neuronal damage on the infarcted side of the rat brain tissue, with irregular cell arrangement, atrophy, deep staining of neurons, lighter Nissl body staining, disappearance of some neurons, and increased intercellular spaces. Compared with the I / R group, the morphological changes of neurons in the brain tissue of rats treated with the Ligusticum chuanxiong stem and leaf phthalide extract group were alleviated, with more compact neuronal arrangement, more intact neuronal morphology, increased Nissl bodies, and a significantly increased number of normal neurons in a dose-dependent manner. These results indicate that the Ligusticum chuanxiong stem and leaf phthalide extract significantly alleviated the I / R-induced reduction of neurons and loss of Nissl bodies, thus reducing neuronal damage in brain tissue.

[0091] 7. The phthalide extract from the stems and leaves of Ligusticum chuanxiong significantly improved I / R-induced neuroinflammation in brain tissue.

[0092] To assess the inflammatory response, the levels of IL-β, IL-18, and TNF-α in tissue homogenates were measured using an ELISA kit according to the manufacturer's protocol. ELISA results showed ( Figure 6The IL-1β content in the ischemic area of ​​brain tissue in the model group was significantly higher than that in the I / R group, while the IL-1β content in the ischemic area of ​​brain tissue in all groups treated with Ligusticum chuanxiong stem and leaf phthalide extract was significantly lower than that in the model group. IL-18 levels decreased significantly in a dose-dependent manner under intervention with medium- and high doses of Ligusticum chuanxiong stem and leaf phthalide extract. TNF-α levels also showed an improving trend. Therefore, Ligusticum chuanxiong stem and leaf phthalide extract can inhibit neuroinflammation induced by cerebral ischemia-reperfusion.

[0093] 8. The phthalide extract of Ligusticum chuanxiong stem and leaf blocks CaSR-mediated NLRP3 inflammasome oligomerization and inhibits the inflammatory response.

[0094] Immunofluorescence staining was performed by embedding brain tissue in paraffin, cutting it into 5 mm sections, and mounting them on glass slides. The tissue was then dewaxed. Cells were fixed with 4% paraformaldehyde (PFA) at 37°C for 30 min. The tissue was blocked with 5% bovine serum albumin (BSA) for 30 min, then incubated overnight with primary antibody at 4°C. The next day, sections and the corresponding secondary antibody were incubated at 37°C for 1 hour. Cell nuclei were stained with DAPI, and cells were imaged under a microscope.

[0095] Western blot analysis was performed using fresh left rat brain tissue. The tissue was lysed on ice with RIPA lysis buffer, centrifuged at 12000 rpm for 5 min at 4°C, and the supernatant was collected to obtain total protein. Protein concentration was determined by BCA method. After adjusting the protein concentration, loading buffer was added, mixed, and boiled for 10 min to denature the protein. Proteins were separated by SDS-PAGE, transferred to PVDF membranes via semi-dry electrotransfer, blocked with 5% skim milk powder for 2 h, and incubated overnight at 4°C with the corresponding primary antibody. The membranes were washed with TBST washing buffer, incubated with the corresponding secondary antibody at room temperature for 1 h, and then developed using ECL chemiluminescence after TBST washing. Western blot analysis was performed to detect the expression of CaSR, NLRP3, Caspase-1, ASC, and tight junction proteins in the relevant treatment groups.

[0096] Immunofluorescence results of CASR showed that the CASR expression level in the model group was significantly increased compared with the I / R group. Compared with the I / R group, the phthalide extract of Ligusticum chuanxiong stems and leaves downregulated CASR expression in ischemic brain tissue. Figure 7 (A, C) Western blot analysis revealed that CASR protein expression was significantly downregulated after treatment with the extract.

[0097] Compared with the control group, the expression level of NLRP3 in the brain tissue of focal ischemic rats was significantly increased. Compared with the I / R group, the expression level of NLRP3 was downregulated by administration of 160 mg / kg Ligusticum chuanxiong stem and leaf phthalide extract. Figure 8 A, C). Western blot results showed that high-dose extracts of Ligusticum chuanxiong stems and leaves could reduce the protein expression of NLRP3, CASP1, and ASC to a certain extent and inhibit the overexpression of the inflammasome family.

[0098] 9. The phthalide extract from the stems and leaves of Ligusticum chuanxiong significantly protects the integrity of the blood-brain barrier in brain tissue.

[0099] Evans blue staining was used to assess the integrity of the blood-brain barrier. Ten rats were randomly selected from each group and injected with 2% Evans blue solution via the tail vein at a dose of 4 mL / kg. One hour later, the rats were anesthetized, their hearts were exposed via thoracotomy, and 300 mL of 0.9% sodium chloride solution was rapidly perfused through the left ventricle-right atrial appendage pathway. The rats were then decapitated, and the ischemic hemisphere was weighed, homogenized, and three times the volume of 50% formamide solution was added. The mixture was incubated at 60℃ for 24 hours, followed by centrifugation at 3000 rpm for 15 minutes. The supernatant was collected, and the absorbance at 610 nm was measured using a microplate reader. The Evans blue content was then determined according to a standard curve.

[0100] The primary cause of blood-brain barrier disruption is uncontrolled inflammation following injury or disease. Major cytokines from systemic inflammation can also lead to conditions as severe as neurological disorders or injuries. Evans blue staining results showed leakage of Evans blue dye on the ischemic side of the brain in the model group, indicating that MCAO disrupts the integrity of the blood-brain barrier. Intervention with phthalide extract from the stems and leaves of Ligusticum chuanxiong significantly improved the disruption of the blood-brain barrier. Figure 9 A, B). Simultaneously, Western blotting results also showed that intervention with the extracts of Ligusticum chuanxiong stems and leaves increased the expression of tight junction proteins (A, B). Figure 9 C, D).

[0101] This invention successfully prepared a phthalide extract from the stems and leaves of Ligusticum chuanxiong, and identified 42 phthalide compounds using high-resolution mass spectrometry. Seven phthalide components were selected as indicator components for content determination. Using a rat MCAO model, the prepared drug composition was evaluated for its ability to prevent ischemic stroke based on infarct volume, neurological deficit, brain water content, and cerebral edema rate. Furthermore, it was found that the phthalide extract from the stems and leaves of Ligusticum chuanxiong can inhibit the expression of upstream CASR proteins in the MCAO, further mediating the blocking of downstream inflammasome NLRP3 family activation, reducing inflammatory responses (IL-1β, IL-18, TNF-α), thereby alleviating neuroinflammation in brain tissue and repairing blood-brain barrier damage. The study suggests that the phthalide extract from the stems and leaves of Ligusticum chuanxiong may alleviate ischemic brain injury by inhibiting CASR-mediated NLRP3 inflammasome activation.

[0102] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A composition containing phthalide extract from the stems and leaves of Ligusticum chuanxiong, characterized in that, The main active ingredient of the composition is a phthalide extract of Ligusticum chuanxiong stems and leaves; the phthalide extract of Ligusticum chuanxiong stems and leaves includes 3-butenylphthalide, ligustilide, neo-spathololide, ligustilide I, ligustilide H, ligustilide A and angelicalide A, and the total amount of active ingredients is not less than 50%; the extraction method of the phthalide extract of Ligusticum chuanxiong stems and leaves includes: naturally drying the stems and leaves of Ligusticum chuanxiong in the shade, crushing and sieving, adding them to a percolation tank, soaking them in 95% ethanol at room temperature and then percolating, collecting the eluent, concentrating it under reduced pressure to an extract, dispersing it with water, extracting it with ethyl acetate, concentrating the ethyl acetate extract under reduced pressure, eluting the highly polar non-phthalide components with methanol using X-5 macroporous resin, eluting it with 90% ethanol, collecting the eluent, concentrating it under reduced pressure to an extract, and freeze-drying it to obtain the phthalide extract of Ligusticum chuanxiong stems and leaves.

2. The use of the composition containing phthalide extract of Ligusticum chuanxiong stem and leaf as described in claim 1 in the preparation of a drug for the prevention and treatment of stroke.

3. A drug for preventing and treating stroke, characterized in that, Compositions comprising the phthalide extract of Ligusticum chuanxiong stems and leaves as described in claim 1.

4. The drug for preventing and treating stroke according to claim 3, characterized in that, The drug also contains pharmaceutically acceptable carriers or excipients.

5. The drug for preventing and treating stroke according to claim 4, characterized in that, The pharmaceutically acceptable carrier or excipient includes one or more solid, semi-solid, or liquid excipients.

6. The drug for preventing and treating stroke according to claim 5, characterized in that, The drug is formulated into a pharmaceutically acceptable preparation.

7. The drug for preventing and treating stroke according to claim 6, characterized in that, The pharmaceutically acceptable preparations are tablets, capsules, granules, injections, pills, syrups, powders, or ointments.

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