Application of phthalide dimer in preparation of medicine for treating or preventing nervous system diseases

Abstract

The invention provides an application of a phthalide dimer in preparation of a medicine for treating or preventing nervous system diseases. Wherein the phthalide dimer is formed by carrying out [4 + 2] or [2 + 2] cycloaddition on two phthalide monomers, the phthalide dimer comprises 1-2 spiro atoms, and the chemical formula structure of the phthalide monomers is as shown in the following formula I. The # imgabs0 # phthalide dimer compound can reduce the volume of cerebral infarction of a rat suffering from cerebral ischemia reperfusion injury (MCAO / R), improve the injury of cortex and hippocampal neurons and improve the cerebral infarction activity of the rat. The neuron cell morphology is basically recovered to normal, the cranial nerve function is improved, and the effect of preventing and treating cerebral arterial thrombosis is achieved. Meanwhile, the compound can relieve neurological impairment caused by chronic cerebral ischemia (2VO bilateral common carotid artery ligation model rats), improve the spatial learning ability of the model rats, improve the learning cognition level and protect neurons.

Description

Technical Field

[0001] The present invention relates to the technical field of cerebral ischemic diseases, and in particular to the use of a phthalide dimer in the preparation of a drug for treating or preventing nervous system diseases. Background Art

[0002] Cerebral ischemic disease refers to a disease caused by a variety of reasons that leads to widespread insufficient blood supply to the brain, causing cerebral ischemia and hypoxia, resulting in a series of clinical manifestations of brain dysfunction. It is also a neurological disease. Clinically, cerebral ischemia is divided into acute cerebral ischemia (also known as "(acute) ischemic stroke", "acute cerebral ischemia-reperfusion injury") and chronic cerebral ischemia (also known as "chronic cerebral hypoperfusion"). According to statistics, the incidence of cerebral ischemic disease in my country is relatively high. 70% of people over 60 years old have varying degrees of cerebral ischemia, and the proportion of cerebral ischemic disease in people over 80 years old reaches 80%, and the disability and mortality rates are high, which seriously threatens the patient's physical health and quality of life.

[0003] The pathogenesis of cerebral ischemia is complex, mainly caused by cerebrovascular lesions, resulting in thrombosis and vascular obstruction, which leads to brain tissue hypoxia, ischemic necrosis, and ultimately leads to clinical symptoms such as neurological dysfunction. Among them, chronic cerebral ischemia is a state of cerebral hypoperfusion before brain tissue damage occurs. Long-term cerebral hypoperfusion will further cause brain tissue damage. The cerebral hypoperfusion state can be reversed and restored under certain intervention measures. Clinical drug treatment is often based on anticoagulation (such as warfarin) and antiplatelet aggregation (such as aspirin) to improve cerebral microcirculation. Acute cerebral ischemia is brain tissue damage caused by acute cerebral ischemia or prolonged ischemia, and its clinical treatment is mainly thrombolysis. However, thrombolytic therapy after excessive ischemia or prolonged ischemia may further aggravate the damage to nerve cells and aggravate brain tissue damage. Therefore, the methods for treating cerebral ischemia in clinical practice are very limited, and the terminal result of cerebral ischemia is brain tissue damage. Therefore, it is very necessary to develop drugs to protect brain tissue damage or neuroprotection.

[0004] It should be emphasized here that cerebral ischemia is mainly caused by thrombosis and vascular obstruction. Its treatment methods are mainly anticoagulation or thrombolysis, which activates fibrinogen in the blood, and then dissolves fibrin to dissolve the formed thrombus, promote the recanalization of blocked blood vessels, restore blood flow, and reduce brain tissue damage. Although vasodilation can also restore cerebral blood flow, without anticoagulation or thrombolysis, a sudden increase in cerebral blood flow may further form thrombi, while releasing a large number of reactive oxygen free radicals, causing inflammation and oxidative stress. In addition, restoring cerebral blood flow in acute cerebral ischemia may aggravate brain tissue damage and be irreversible. Therefore, vasodilation is equivalent to further damaging brain tissue, and direct vasodilation is generally not used clinically.

[0005] Due to the complex pathogenesis of cerebral ischemia, the limited duration of drug action, and the short empty window period, the efficacy of the above-mentioned drugs is very limited. Moreover, there is currently no effective preparation for treating damaged neurons after ischemia on the market. Therefore, there is an urgent need to search for and develop effective drugs for treating nerve damage after ischemia, that is, drugs for nervous system diseases. Patent application document CN111018876A discloses the use of phthalide dimers in the preparation of vasodilating drugs. Although this application document discloses that some of these compounds have the function of dilating blood vessels by antagonizing the Cav1.2 receptor, there are also some dimers (such as chuanxiongdiolide R5, chaxiongnolide D, chuanxiongnolide B, etc.) that do not have the activity of dilating blood vessels. And the paper "Discovery of Novel Phthalides from Chuanxiong Caulis et Folium with Vasorelaxant Activity against Thoracic Aorta" shows that the antiplatelet aggregation and anticoagulant activities of phthalide dimers are weak. Therefore, whether phthalide dimer compounds can be used as neuroprotective drugs for treating acute and chronic cerebral ischemia injury has not been reported yet, and it is also impossible to simply predict the function of such compounds in treating cerebral ischemia diseases. Summary of the Invention

[0006] The main object of the present invention is to provide an application of a phthalide dimer in the preparation of a drug for treating or preventing nervous system diseases, so as to provide a new drug capable of treating or preventing nervous system diseases.

[0007] To achieve the above object, according to the first aspect of the present invention, there is provided an application of a phthalide dimer, its salt, its stereoisomer, its prodrug or its metabolite in the preparation of a drug for treating or preventing nervous system diseases. The phthalide dimer is formed by [4 + 2] or [2 + 2] cycloaddition of 2 phthalide monomers, and the phthalide dimer includes 1 - 2 spiro atoms. The chemical formula structure of the phthalide monomer is shown in the following formula I:

[0008]

[0009] Wherein, in formula I, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 are each independently selected from any one of the following: hydrogen, ORa, OCOORa, OCONRaRb, OSO 2 Ra or OSO 3 Ra;

[0010] Ra and Rb are each independently selected from any one of the following: hydrogen, substituted or unsubstituted C 1 -C 12alkyl, substituted or unsubstituted C 1 -C 12 alkenyl, substituted or unsubstituted C 1 -C 12 alkynyl, substituted or unsubstituted C 6 -C 30 aryl or substituted or unsubstituted C 1 -C 30 heteroaryl;

[0011] represents a single bond or a double bond.

[0012] Furthermore, each phthalide monomer in the phthalide dimer is independently selected from any one of the following:

[0013] Furthermore, the phthalide dimer includes:

[0014] Furthermore, the phthalide dimer is extracted from natural medicinal plants or chemically synthesized.

[0015] Furthermore, the natural medicinal plants include Umbelliferae plants.

[0016] Furthermore, the Umbelliferae plants include Ligusticum chuanxiong, Angelica sinensis and Ligusticum sutchuenense.

[0017] Furthermore, the nervous system diseases include: acute cerebral ischemia and chronic cerebral ischemia.

[0018] Furthermore, the characteristic manifestations of the nervous system diseases include: nerve function damage.

[0019] Furthermore, the characteristic manifestations of the nervous system diseases include: brain tissue damage.

[0020] Furthermore, the brain tissue damage includes: neuron damage.

[0021] To achieve the above object, according to the second aspect of the present invention, there is provided a composition for treating or preventing nervous system diseases, the composition comprising: (a) a phthalide dimer as described in the above application; and optionally (b) a pharmaceutically acceptable carrier.

[0022] Applying the technical solution of the present invention, the phthalide dimer compound of the present application can reduce the cerebral infarction volume (such as the cerebral infarction volume in the acute ischemic stroke MCAO / R rat model), improve the damage of neurons in the cerebral cortex and hippocampus of rats, make the morphology of neuron cells basically return to normal, improve the cranial nerve function, and play a role in preventing and treating cerebral ischemia diseases. At the same time, the phthalide dimer compound can reduce neuronal apoptosis by inhibiting the expression of pyroptosis proteins and play its neuroprotective role.

[0023] The phthalide dimer compound can also reduce the neurological deficits caused by chronic cerebral ischemia (2VO bilateral common carotid artery ligation model rats), improve the spatial learning ability of the model rats, improve the learning and cognitive level, and protect neurons. It can be seen that the phthalide dimer compound has a relatively positive impact on the improvement of neurological diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The specification drawings forming a part of the present application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0025] Figure 1 The figure shows the results of TTC staining of brain tissue showing the effect of ligustilide A on cerebral infarction in MCAO / R rats in Example 1; among them, a is the control group, b is the sham operation group, c is the model group, d is the ligustilide A-L group, e is the ligustilide A-H group, and f is the butylphthalide group;

[0026] Figure 2 The figure shows the results of HE staining showing the effect of ligustilide A on the pathology of the cerebral cortex and hippocampal CA1 region in MCAO / R rats in Example 1; among them, a is the sham operation group, b is the model group, c is the ligustilide A-L group, d is the ligustilide A-H group, and e is the butylphthalide group;

[0027] Figure 3 The figure shows the staining observation diagram of the effect of ligustilide A on TUNEL-positive cell apoptosis in MCAO / R rats in Example 1; among them, a is the sham operation group, b is the model group, c is the ligustilide A-L group, d is the ligustilide A-H group, and e is the butylphthalide group;

[0028] Figure 4 The figure shows the statistical chart of the effect of different dosages of ligustilide A on the survival rate of OGD / R (oxygen-glucose deprivation injury) PC12 cells in Example 2;

[0029] Figure 5 The figure shows the flow cytometry detection results and statistical chart of the effect of different dosages of ligustilide A on apoptosis of OGD / R PC12 cells in Example 2;

[0030] Figure 6 The Western blot result diagram showing the effects of different dosages of Angelol A in Example 2 on the protein expressions of Caspase-1, GSDMD-N, and mature IL-1β in OGD / R PC12 cells undergoing pyroptosis;

[0031] Figure 7 The result diagram of TTC staining of brain tissues showing the effects of Riligustilide in Example 3 on the cerebral infarction in MCAO / R rats; among them, a is the sham operation group, b is the model group, c is the Riligustilide-L group, d is the Riligustilide-H group, and e is the butylphthalide group;

[0032] Figure 8 The HE staining result diagram (HE staining, ×200) showing the effects of Riligustilide in Example 3 on the pathological results of the hippocampal CA1 region in MCAO / R rats; among them, a is the sham operation group, b is the model group, c is the Riligustilide-L group, d is the Riligustilide-H group, and e is the butylphthalide group;

[0033] Figure 9 The Nissl staining result diagram (Nissl staining, ×200) showing the effects of Riligustilide in Example 3 on the pathological results of the hippocampal CA1 region in MCAO / R rats; among them, a is the sham operation group, b is the model group, c is the Riligustilide-L group, d is the Riligustilide-H group, and e is the butylphthalide group;

[0034] Figure 10 The representative experimental pictures showing the effects of Angelol A on the spatial exploration of 2VO rats based on the Morris water maze experiment in Example 4; among them, a is the sham operation group, b is the model group, c is the Angelol A-L group, d is the Angelol A-H group, and e is the ginkgo biloba extract group;

[0035] Figure 11 The HE staining result diagram (HE staining, ×200) showing the effects of Angelol A on the pathological results of the hippocampal region in 2VO rats in Example 4; among them, a is the sham operation group, b is the model group, c is the Angelol A-L group, d is the Angelol A-H group, and e is the ginkgo biloba extract group;

[0036] Figure 12The figure shows the Nissl staining results of the effect of ligustilide A on the pathological results in the hippocampus of 2VO rats in Example 4 (Nissl staining, ×200); among them, a is the sham operation group, b is the model group, c is the ligustilide A-L group, d is the ligustilide A-H group, and e is the Ginkgo biloba extract group;

[0037] Figure 13 The figure shows the HE staining results of the effect of senkyunolide O on the pathological results in the hippocampus of 2VO rats in Example 5 (HE staining, ×200); among them, a is the sham operation group, b is the model group, c is the senkyunolide O-L group, d is the senkyunolide O-H group, and e is the Ginkgo biloba extract group;

[0038] Figure 14 The figure shows the Nissl staining results of the effect of senkyunolide O on the pathological results in the hippocampus of 2VO rats in Example 5 (Nissl staining, ×200); among them, a is the sham operation group, b is the model group, c is the senkyunolide O-L group, d is the senkyunolide O-H group, and e is the Ginkgo biloba extract group;

[0039] Figure 15 The figure shows a schematic diagram of the pyroptosis pathway;

[0040] Figure 16 The figure shows a schematic diagram of the inhibition of the pyroptosis pathway of neurons by ligustilide A of the present application. Detailed implementation manners

[0041] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments may be combined with each other. The present invention will be described in detail below with reference to the drawings and in combination with the embodiments.

[0042] Glossary:

[0043] Middle cerebral artery occlusion / reperfusion (MCAO / R) rat model: This type of rat model is used to simulate acute ischemic stroke and can detect the therapeutic and preventive effects of phthalide dimers on acute ischemic stroke.

[0044] Two-vessel occlusion ischemia (2VO) rat model: This type of rat model is used to simulate chronic cerebral ischemia diseases and can detect the therapeutic and preventive effects of phthalide dimers on chronic cerebral ischemia diseases.

[0045] The pyroptosis pathway: (as Figure 15 shown)

[0046] K released by cerebral ischemia-reperfusion injury or chronic hypoperfusion + 、Ca 2+ 、DNA fragments bind to pattern recognition receptors on adjacent cells, activating and initiating the inflammasome pathway. Inflammasome receptors (NLRP3, AIM2, NLRC4, etc.), adaptor protein ASC, and effector protein caspase-1 assemble into an inflammasome. Activated caspase-1 or caspase-8 activates IL-1β and IL-18, and simultaneously cleaves the GSDMD-FL protein to form GSDMD-NT (classical pathway). The N-terminal fragment self-oligomerizes to the plasma membrane to form pores, leading to cell swelling, cytoplasmic efflux, and pyroptosis. Activated caspase-11 can also directly induce GSDMD-NT to cause pore formation (non-classical pathway).

[0047] In the first typical embodiment of the present application, there is provided an application of a phthalide dimer, its salt, its stereoisomer, its prodrug or its metabolite in the preparation of a drug for treating or preventing nervous system diseases. The phthalide dimer is formed by [4+2] or [2+2] cycloaddition of 2 phthalide monomers, and the chemical formula structure of the phthalide monomer is shown in Formula I below:

[0048]

[0049] Among them, in Formula I, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 are each independently selected from any one of the following: hydrogen, ORa, OCOORa, OCONRaRb, OSO 2 Ra or OSO 3 Ra; Ra and Rb are each independently selected from any one of the following: hydrogen, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C 1 -C 12 alkenyl, substituted or unsubstituted C 1 -C 12 alkynyl, substituted or unsubstituted C 6 -C 30 aryl or substituted or unsubstituted C 1 -C 30 heteroaryl; represents a single bond or a double bond.

[0050] The phthalide dimer of the present application can be extracted and isolated from traditional Chinese medicines such as Ligusticum chuanxiong Hort., Angelica sinensis (Oliv.) Diels or Ligusticum jeholense Nakai et Kitag. of the Umbelliferae family, and has the characteristics of definite curative effect, few side effects, rich treatment targets, etc. It can also be formed by [4+2] or [2+2] cycloaddition of two single phthalides. Phthalide dimers have functions such as vasodilation, anti-tumor, inhibition of spleen tyrosine kinase (Syk) activity and alleviation of Alzheimer's disease in existing reports, but there is no report on the function of being able to treat acute and chronic cerebral ischemia injury.

[0051] It should be emphasized that there is no necessary connection between vasodilation and the treatment of cerebral ischemia diseases. Cerebral ischemia diseases are mainly caused by thrombosis / vascular occlusion, and thrombosis / vascular occlusion is caused by abnormal blood components in the blood vessels. For example, when the vascular endothelium is damaged, thrombin activates platelets, platelets aggregate, and fibrin "bridges" to form a thrombus, causing vascular occlusion; imbalance of coagulation factors and anticoagulation factors caused by diseases or genetic factors, and lack of anticoagulation factors. Therefore, the treatment methods for cerebral ischemia diseases are anticoagulation, antiplatelet aggregation and thrombolysis. If vasodilation treatment is carried out, it will cause the thrombus to move to other parts with the blood flow, resulting in more serious embolism. At the same time, the increase in cerebral blood flow will release a large amount of reactive oxygen free radicals, triggering inflammation and oxidative stress, exacerbating brain tissue damage. While failing to achieve the purpose of treating acute cerebral ischemia injury, it may also pose a serious threat to life.

[0052] In a preferred embodiment, each phthalide monomer in the phthalide dimer is independently selected from any one of the following:

[0053]

[0054] In a preferred embodiment, the phthalide dimer includes:

[0055] Among them, ligustilide can polymerize to form levistilide A (molecular weight: 380.48, molecular formula: C 24 H 28 O 4 , the molecular structural formula is shown in Formula A) and its stereoisomer riligustilide (the molecular structural formula is shown in Formula B); ligustilide can also polymerize with its isomer to form senkyunolide O (molecular weight: 380.48, molecular formula: C 24 H 28 O 4 , the molecular structural formula is shown in Formula C).

[0056] Among them, in formula A, the Z,Z'-6,6',7,3'a positions of two monomers are connected to form a bridged ring, and the formed bridged ring shares a spiro atom with the five-membered ring of a single phthalide. In formula B, the Z,Z'-6,8',7,3' of two monomers are connected to form a bridged ring, and the formed bridged ring shares a spiro atom with the five-membered ring of a single phthalide. In formula C, the E,Z'-6,6',7,3'a of two monomers are connected to form a bridged ring, and the formed bridged ring shares a spiro atom with the five-membered ring of a single phthalide. Formulas A, B, and C belong to bridged-ring compounds and each contains 1 spiro atom.

[0057] Any means capable of obtaining the above phthalide dimers is applicable to this application. In a preferred embodiment, the phthalide dimers are extracted from natural medicinal plants or chemically synthesized. Any plant capable of extracting the above phthalide dimers is applicable to this application. In a preferred embodiment, the natural medicinal plants include Umbelliferae plants. In a preferred embodiment, the Umbelliferae plants include Ligusticum chuanxiong, Angelica sinensis, and Chaoxiong.

[0058] Any neurological disease in which any phthalide dimer can play a role is applicable to this application, including neurological diseases caused by cerebral ischemia. In a preferred embodiment, the neurological diseases include acute cerebral ischemia and chronic cerebral ischemia.

[0059] Any neurological disease that may be caused by cerebral ischemia is applicable to this application. In a preferred embodiment, the characteristic manifestations of the neurological diseases include: neurological function impairment. In a preferred embodiment, the characteristic manifestations of the neurological diseases include: brain tissue damage. Among them, neuron damage / white matter damage can lead to learning and memory dysfunction. White matter is composed of the axons of neurons and glial cells, and neurons transmit signals through white matter. Therefore, if neurons or white matter are damaged, the mediated nerve signals will be blocked, and thus the learning and memory function will be impaired.

[0060] Among them, when cells are infected or stimulated by injury, the inflammasome is activated, Caspase-1 will be recruited and activated by the inflammasome, and then Caspase-1 can cleave the GSDMD (gasdermin D) protein to generate the GSDMD-N fragment, and this fragment will translocate to the cell membrane to form pores, promoting the release of cell contents including inflammatory mediators, and ultimately leading to pyroptosis. At the same time, Caspase-1 can also promote the maturation and release of IL-1β (interleukin-1β).

[0061] Any disease related to reduced cognitive function is applicable to this application. In a preferred embodiment, brain tissue damage includes: neuronal damage. Nissl bodies can reflect the functional state of neurons. The number, shape, and distribution of Nissl bodies can reflect the functional state of neurons. When neurons are damaged or over-fatigued, the Nissl bodies will decrease. During the recovery process of injury or fatigue, the Nissl bodies gradually increase to the normal level, and can serve as a marker of the functional state of neurons.

[0062] In the second typical embodiment of this application, a composition for treating or preventing nervous system diseases is provided. The composition includes: (a) a phthalide dimer as in the above application; and optionally (b) a pharmaceutically acceptable carrier.

[0063] The following further describes this application in detail with specific embodiments, and these embodiments should not be construed as limiting the scope claimed in this application.

[0064] The butylphthalide used in the following embodiments of this application is the Butylphthalide Soft Capsules of Enbipu. Ligustilide A can be extracted and separated from the medicinal material of Ligusticum chuanxiong. In this application, it is purchased from Chengdu Refines Biological Technology Co., Ltd. (batch number: O002006004) and extracted, separated, and purified from the ethyl acetate part of Ligusticum chuanxiong. Riligustilide is purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (batch number: DI6GB171470), and Senkyunolide O is purchased from Beijing MedChemExpress Co., Ltd. (batch number: M044841)

[0065] Example 1 Effects of Phthalide Dimer Ligustilide A on Neuroprotection in MCAO / R Rats

[0066] 1. Experimental Animals

[0067] SPF-grade SD rats (male, body weight 280 ± 20 g). The experimental rats are provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SYXK (Jing) 2020-0033. They are raised in the Experimental Animal Center of Beijing University of Chinese Medicine.

[0068] 2. Experimental Methods

[0069] 2.1 Grouping and Administration of Experimental Animals

[0070] Male Sprague-Dawley rats were randomly divided into a sham operation group, a model group, a low-dose ligustilide A group (20 mg / kg), a high-dose ligustilide A group (40 mg / kg), and a positive drug group of butylphthalide soft capsules (63 mg / kg, clinical equivalent dose). Ligustilide A and butylphthalide were both dissolved in 0.5% CMC-Na. The sham operation group and the model group were given an intragastric administration of the corresponding volume of 0.5% CMC-Na. 30 minutes after administration, surgery was performed to construct the MCAO / R model (fasting for 12 hours before modeling without water restriction), and cerebral ischemia-reperfusion injury was performed 1 hour and 30 minutes after modeling. Samples were taken 22 hours after reperfusion.

[0071] 2.2 Establishment of the MCAO / R rat model

[0072] 30 minutes after intragastric administration, the rat MCAO / R model was constructed using the suture method. The sham operation group did not insert the suture, and the other operations were the same. The rats were anesthetized by intraperitoneal injection of sodium pentobarbital, and then the rats were fixed supine on the operating table. After shaving, iodophor was applied. A longitudinal incision was made in the midline of the neck (at 2.5 cm), and the right common carotid artery (CCA) and external carotid artery (ECA) of the rat were separated, and the proximal ends of these two arteries were ligated. The internal carotid artery (ICA) was clamped with a microarterial clamp. A small incision was made with an ophthalmic scissors at the distal end of the CCA ligation (about 5 cm from the bifurcation of the common carotid artery), and a suture was inserted. The ICA artery clamp was released, and the force and direction of the suture were quickly adjusted so that it entered the ICA from the CCA until the starting area of the anterior cerebral artery. When encountering resistance, it was inserted with a little more force and then stopped (the black dot marked on the suture stopped at the cross boundary of the CCA, ECA, and ICA). After suturing the wound, an electric blanket was used to keep the rat's body temperature at 37°C. After 1 hour and 30 minutes of ischemia, the suture was pulled out to the marked black dot. 22 hours after reperfusion, the neurological deficit function was scored using the modified Zea Longa method.

[0073] 2.3 Neurological function scoring

[0074] According to the Bederson grading method, the Zea Longa scoring standard was used to score the neurological function of the animals 24 hours after cerebral ischemia-reperfusion injury. 0 points: no neurological injury; 1 point: the contralateral forelimb adducted and flexed when the tail was lifted; 2 points: rotated to the contralateral side when crawling; 3 points: fell to the contralateral side when standing or crawling; 4 points: no spontaneous activity accompanied by consciousness disorder. The higher the score, the more severe the neurological deficit of the animal. The condition was observed, scored, and recorded 22 hours after reperfusion. The modeling was considered successful if the score was above 1 point except for the sham operation group. Rats that died or the modeling was unsuccessful were excluded.

[0075] 2.4 Brain tissue sampling and determination of cerebral infarction area

[0076] After neurobehavioral function scoring, the rats were anesthetized, blood was taken from the abdominal aorta, and cold saline was perfused from the apex of the heart. Immediately, the rats were decapitated to remove the brain (removing the cerebellum and olfactory bulb), the moisture of the brain tissue was blotted dry with filter paper, and then it was frozen in a -20°C refrigerator for 10 min. After taking it out and placing it on a brain mold, it was sectioned, and the brain was coronally cut into 5 slices along the optic chiasm and 2 mm from the optic nerve. The sections were placed in 0.2% TTC at 37°C for light-avoiding staining, with 15 min for positive staining and 15 min for counterstaining. Then they were taken out and fixed in 4% tissue fixative, photographed after 24 h, the infarct area of the brain tissue was measured with Image J, and the percentage of the infarct area was calculated, and the data was recorded. The calculation formula for the percentage of the brain infarct area is shown in Equation 1.

[0077] Equation 1: Infarction rate = sum of the areas of the white ischemic regions of each slice / sum of the areas of each brain slice × 100%

[0078] 2.5 Brain tissue sampling and HE staining, TUNEL immunofluorescence staining

[0079] The brain tissues of each group of rats were collected (the same as in 2.4), fixed in 4% tissue fixative for 24 h, then paraffin-embedded, and coronally sectioned at about 3.5 μm. Then the sections were successively immersed in xylene for 20 min, replaced with xylene and immersed for 20 min, absolute ethanol for 5 min, replaced with absolute ethanol and immersed for 5 min, 75% ethanol for 5 min, and washed with tap water. The sections were stained with hematoxylin solution for 3 - 5 min, washed with tap water, differentiated with the differentiating solution, washed with tap water, blued with the bluing solution, and rinsed with running water. Then the sections were successively dehydrated in 85% and 95% gradient ethanol for 5 min each, stained with eosin solution for 5 min. Then the sections were successively placed in absolute ethanol for 5 min, replaced with absolute ethanol and immersed for 5 min, replaced with absolute ethanol again and immersed for 5 min, xylene for 5 min, replaced with xylene and immersed for 5 min, and sealed with neutral gum. Microscopic examination was performed, and image acquisition and analysis were carried out.

[0080] The sections were dewaxed, repaired, inactivated for enzymes, repaired, equilibrated at room temperature, added with TdT enzyme reaction solution, reacted at 37°C in the dark, added with Streptavadin-HRP, and reacted at 37°C in the dark. DAB method was used for color development, hematoxylin was used for counterstaining, xylene was used for transparency treatment, and sealed with gum. Photographs were taken under a 200-fold microscope to observe the apoptosis of nerve cells and calculate the TUNEL positive cell rate. The cell nuclei were stained blue with DAPI nuclear stain, and the TUNEL positive cells showed green fluorescence. The Image J software was used to calculate the number of TUNEL-stained positive cells in the field of view and calculate the ratio of positive cells.

[0081] 2.6 Data processing

[0082] SPSS software was used for data statistics, and the independent samples T-test method was used to analyze the data for inter-group comparison. The data of each group were expressed as (Mean ± standard deviation), P < 0.05 indicates a statistically significant difference, and P < 0.01 indicates a highly statistically significant difference. (vs control group, ## P < 0.01; vs model group, *P < 0.05, **P < 0.01).

[0083] 3. Experimental results

[0084] 3.1 Effects of ligustilide A on the neurological function of MCAO / R rats

[0085] The neurological function scores of rats in each group are shown in Table 1. Compared with the model group, both the high-dose and low-dose ligustilide A groups could improve the neurological behavior function of MACO / R rats to varying degrees after administration, and the improvement degree in the high-dose group was significant.

[0086] Table 1 Effects of ligustilide A on the neurological function scores of MCAO / R rats ( n = 8)

[0087]

[0088] 3.2 Effects of ligustilide A on the cerebral infarction in MCAO / R rats

[0089] The statistical results of the infarction conditions and infarction areas of the brain tissues of rats in each group are shown in Figure 1 and Table 2 respectively (where a is the control group, b is the sham operation group, c is the model group, d is the ligustilide A-L group, e is the ligustilide A-H group, and f is the butylphthalide group). The brain tissue staining of the control group and sham operation rats was uniform, and no infarcted tissue was found. Compared with the sham operation group, white infarcted areas of different sizes were visible in the brain tissues of rats in the cerebral ischemia-reperfusion injury model group, indicating that the model caused large-area infarction in the rats' brains. The infarction areas in the ischemic regions of the rats in the ligustilide A administration groups of 20 and 40 mg / kg were significantly reduced, and the cerebral infarction rate was significantly decreased (P < 0.01), showing a dose-dependent relationship.

[0090] Table 2 Effects of ligustilide A on the cerebral infarction area in MCAO / R model rats n = 8)

[0091]

[0092] 3.3 Effects of ligustilide A on the pathological changes of the brain tissues of MCAO / R rats (HE staining)

[0093] The HE staining results of the brain tissues of rats in each group are shown in Figure 2As shown (a: sham operation group, b: model group, c: Ligustilide A-L group, d: Ligustilide A-H group, e: Butylphthalide group), in the model group, the brain tissue sections of rats showed cell necrosis and pyknosis, and the cell nuclei shrank, indicating extensive necrosis of brain tissue cells in MCAO / R model rats. The high-dose Ligustilide A group could significantly reduce nerve cell necrosis and maintain the normal state of cells.

[0094] 3.4 Effect of Ligustilide A on neuronal apoptosis in MCAO / R rats

[0095] The apoptotic conditions of nerve cells in each group of rats were as Figure 3 (a: sham operation group, b: model group, c: Ligustilide A-L group, d: Ligustilide A-H group, e: Butylphthalide group) and shown in Table 3. No TUNEL-positive cells were seen in the sham operation group, and no cell apoptosis was observed. Compared with the sham operation group, the number of TUNEL-positive cells in the infarcted cortex of MCAO / R rats increased significantly, indicating obvious cell apoptosis after modeling. Compared with the model group, the number of TUNEL-positive cells in the Ligustilide A administration groups of 20 and 40 mg / kg decreased significantly (P<0.01), indicating that Ligustilide A has the effect of inhibiting neuronal apoptosis in MCAO / R rats.

[0096] Table 3 Effect of Ligustilide A on TUNEL-positive cell apoptosis in MCAO / R model rats ( n = 8)

[0097]

[0098] Example 2 Study on the neuroprotective effect and mechanism of Ligustilide A

[0099] 1. Experimental methods

[0100] 1.1 PC12 cell culture

[0101] PC12 cells: The cells were cultured in DMEM medium containing 10% fetal bovine serum (containing 10 U / mL of penicillin and streptomycin), and placed in an incubator at 37°C and 5% CO 2 with saturated humidity for culture. When the cells were in the logarithmic growth phase, they were used for research.

[0102] Cell resuscitation: Take out the cryopreserved cells from the liquid nitrogen tank and quickly dissolve them in a 37°C water bath. Put them into the pre-prepared complete medium (containing glucose, glutamine, amino acids, vitamins, NaCl, KCl, anhydrous CaCl 2 and anhydrous MgSO 4 and Na 2 HCO 3 and NaHCO 3 and Na 2 HPO4 Gently mix in (including HEPES), and centrifuge at 1000 rpm / min for 5 min. Discard the supernatant, add complete medium and mix well, then inoculate into a 25×25 cm 2 culture flask. Culture in a 5% CO 2 incubator.

[0103] Cell passage: When the cells grow to 80 - 90%, aspirate and discard the old medium, wash once with PBS, add 2.5% trypsin and digest until the cells become round. Add 3 mL of complete medium to terminate the digestion, pipette the cells in the culture flask evenly, transfer them to a 15 mL centrifuge tube, centrifuge at 1000 rpm / min for 5 min, collect the cell pellet, add fresh complete medium, and transfer to a new culture flask at a ratio of 1:5.

[0104] Cell cryopreservation: Digest and centrifuge (1000 rpm / min, 3 min) the PC12 cells in good growth state, and collect the cell pellet. Add an appropriate amount of cell cryopreservation solution pre-cooled at 4°C (90% fetal bovine serum, 10% DMSO), gently mix the cells, adjust the cell density to 4×10 6 cells / mL, aliquot 1 mL into a cell cryopreservation tube, place it in a programmable cryo box, immediately put it into an -80°C refrigerator, and then transfer it to liquid nitrogen.

[0105] 1.2 OGD / R model establishment

[0106] PC12 cells in the logarithmic growth phase are divided into: control group (cultured in complete medium without OGD / R treatment); model group (OGD / R); drug treatment group; drug treatment + OGD / R group. Each group has 6 replicate wells.

[0107] Establishment of the OGD / R cell model: Take PC12 cells in the logarithmic growth phase, seed them at a density of 1×10 5 cells / mL in a 96-well plate, and culture in a 5% CO 2 incubator for 24 h. Wash 3 times with PBS pre-warmed at 37°C, add hypoxic and hypoglycemic medium (containing NaCl, KCl, anhydrous CaCl 2 , anhydrous MgSO 4 , Na 2 HCO 3 , NaHCO 3 , Na 2 HPO 4 and HEPES). Place it in a cell hypoxic culture system (1% O 2 , 5% CO 2 , 94% N 2 ) and equilibrate for 30 min, then culture under hypoxic and hypoglycemic conditions in a 37°C incubator for 6 h. After that, replace the hypoxic and hypoglycemic medium with the corresponding drug-containing complete medium and continue to culture for 19 h.

[0108] 1.3 Detection of the effect of ligustilide A on the survival rate of OGD / R cell model by CCK-8 method

[0109] Take OGD / R PC12 cells and administer ligustilide A at a series of concentration gradients of 0, 1.56, 3.12, 6.25, 12.50, 25.00, 50.00 μmol / L for 19 h. Then add 100 μL of complete medium containing 10% CCK-8 and incubate in a 37 °C, 5% CO 2 Incubate in a saturated humidity incubator for 2 h. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value of each plate at a wavelength of 450 nm. Calculate the effect of ligustilide A on the survival rate of PC12 cells induced by OGD / R.

[0110] 1.4 Detection of apoptosis rate

[0111] After hypoxia for 5 h according to item 1.2, add complete medium containing ligustilide A (0, 3.12, 6.25, 12.50 μmol / L) to PC12 cells and continue to culture for 19 h. Collect the cell supernatant and digest the cells with trypsin without EDTA until the cells become round. Centrifuge at 1500 rpm / min for 5 min to collect the cells in the supernatant, and centrifuge at 1000 rpm / min for 5 min to collect the adherent cells. Mix the supernatant and cell precipitate, and resuspend the cells once with pre-cooled PBS. Add 100 μL of Loading Buffer to resuspend the cells. Add 5 μL of Annexin V-FITC and mix gently, and react in the dark for 15 min to bind to phosphatidylserine on the cell surface. Then add 5 μL of PI and mix gently to stain necrotic or late apoptotic cells. After filtration, use a flow cytometer to detect and calculate the apoptosis rate of PC12 cells. Detect Annexin V-FITC at Ex / Em = 488 / 525 nm and detect PI at Ex / Em = 488 / 630 nm.

[0112] 1.5 Protein immunoblot analysis (Western blot)

[0113] After hypoxia for 5 h according to item 1.2, PC12 cells were respectively added with complete medium containing ligustilide A at 0, 3.125, 6.25, and 12.5 μmol / L and continued to be cultured for 19 h. After washing with pre-cooled PBS, protein lysate was added and allowed to act on ice for 30 min. Then, PC12 cells were collected with a cell scraper into a 1.5 mL EP tube and centrifuged at 12000 rpm / min at 4 °C for 5 min, and the supernatant was the extracted protein. Protein quantification was performed according to the instructions of the BCA kit. The protein samples were successively subjected to electrophoresis, electrotransfer, blocking, primary antibody incubation, secondary antibody incubation, and chemiluminescence steps, and the expression levels of the immunoreactive proteins cleaved Caspase-1, GSDMD-N, and mature IL-1β in cells were detected with enhanced chemiluminescence (ECL) reagent, exposed using Chemi-Doc XRS+ TM The chemiluminescence intensity was analyzed by ImageLab software. The ratio of each protein to the internal reference was calculated for semi-quantitative analysis.

[0114] 1.6 Data processing

[0115] Same as the content in "2.6 Data processing" in Example 1.

[0116] 2. Experimental results

[0117] 2.1 Ligustilide A inhibits OGD / R-induced nerve cell death

[0118] The protective effect of ligustilide A on hypoxia and glucose deprivation injury of PC12 cells was studied by OGD / R experiment, as Figure 4 shown. The results showed that ligustilide A had a protective effect on OGD / R-induced PC12 cell injury. When the concentration of ligustilide A was 1.56, 3.12, 6.25, and 12 μmol / L, it had neuroprotective activity and rescued 64.94% ± 7.47% (P > 0.05), 73.75% ± 6.80% (P < 0.05), 77.72% ± 9.12% (P < 0.05), and 77.69% ± 15.55% (P < 0.05) of PC12 cells, respectively.

[0119] 2.2 Ligustilide A inhibits OGD / R-induced nerve cell apoptosis

[0120] Whether ligustilide A could inhibit OGD / R-induced nerve cell apoptosis was further studied by flow cytometry, and the results were as Figure 5As shown in the figure. Compared with the control group, the apoptosis rate of OGD / R-induced PC12 cells increased by 10.73% (P<0.001); compared with the OGD / R group, when the concentration of ligustilide A was 3.12, 6.25, and 12.5 μmol / L, the apoptosis rate decreased by 6.93% (P<0.01), 8.40% (P<0.01), and 10.47% (P<0.01), respectively. When the concentration of ligustilide A was 12.5 μmol / L, the damaged PC12 cells were restored to the normal level. Ligustilide A could inhibit OGD / R-induced neuronal apoptosis in a dose-dependent manner.

[0121] 2.3 Ligustilide A inhibits OGD / R-induced neuronal pyroptosis

[0122] The results of the expression of proteins related to pyroptosis are as Figure 6 shown. Compared with the control group, after OGD / R modeling, the protein expressions of cleaved Caspase-1, GSDMD-N, and mature IL-1β in PC12 cells were significantly increased, indicating that both models could induce neuronal pyroptosis. Compared with the model group, ligustilide A at concentrations of 3.12, 6.25, and 12.5 μmol / L could significantly inhibit the protein expression levels of the pyroptosis-related proteins Caspase-1, GSDMD-N, and mature IL-1β in neuronal cells PC12 in a dose-dependent manner. The results suggest that ligustilide A can inhibit the cleavage of Caspase-1 protein in damaged neuronal cells, further reduce the activation of GSDMD protein, thereby inhibiting the translocation of the N-terminal of the activated GSDMD protein to the cell membrane and inhibiting the occurrence of neuronal pyroptosis (as Figure 16 shown).

[0123] Example 3 Effects of phthalide dimer Riligustilide on neuroprotection in MCAO / R rats

[0124] 1. Experimental animals

[0125] The same as in Example 1.

[0126] 2. Experimental methods

[0127] 2.1 Grouping, dosing of experimental animals, and establishment of MCAO / R model

[0128] Except that the dosing groups were divided into the low-dose Riligustilide group, the high-dose Riligustilide group, and the positive drug butylphthalide group, the other contents were the same as those in "2.1 Grouping and dosing of experimental animals" and "2.2 Establishment of MCAO / R rat model" in Example 1.

[0129] 2.2 Neurological function scoring

[0130] The operation is the same as that in Example 1.

[0131] 2.3 Brain tissue sampling, determination of cerebral infarction area, and HE staining

[0132] The operation is the same as that in Example 1.

[0133] 2.3 Nissl staining of brain tissue sections

[0134] For the routine dewaxing of paraffin sections of brain tissue to water: sequentially place the sections in xylene I for 20 min, xylene II for 20 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 75% alcohol for 5 min, and rinse with distilled water 3 times; stain the sections with toluidine blue: stain the sections with toluidine blue for 5 min, wash with distilled water, and control the degree of differentiation with 1% glacial acetic acid, wash the dye with distilled water, and dry the sections in an oven; clear and mount the sections: make them transparent with xylene and mount them with neutral balsam. Finally, perform image scanning and acquisition with an optical microscope.

[0135] 2.4 Data processing

[0136] Same as the content in "2.6 Data processing" in Example 1.

[0137] 3. Experimental results

[0138] 3.1 Effect of Riligustilide on the neurological function of MCAO / R rats

[0139] The neurological function scores of rats in each group are shown in Table 4. Compared with the sham operation, the evaluation score of neurological function injury in MCAO / R rats increased, indicating damage to the nervous system of the model group rats; compared with the model group, the neurological function scores of the high-dose Riligustilide group and the positive drug butylphthalide group decreased significantly (P<0.05), and the effects were comparable, indicating that Riligustilide can improve the neurological deficit in MCAO / R rats.

[0140] Table 4 Results of the effect of Riligustilide on the neurological function score of MCAO / R rats ( n = 8)

[0141]

[0142] 3.2 Effect of Riligustilide on the cerebral infarction in MCAO / R rats

[0143] The infarction conditions and statistical results of the infarction area of brain tissues of rats in each group are as Figure 7As shown in Table 5, infarcts occurred in different parts of the brain tissue of the rats in the model group. Compared with the model group, both the low- and high-dose Riligustilide groups and the positive drug butylphthalide group could reduce the infarct area in the right cerebral tissue. The high-dose Riligustilide group and the butylphthalide group could significantly reduce the cerebral infarct area (P<0.01), and could be reduced to 47.71% and 64.43% respectively.

[0144] Table 5 Effects of Ligustilide A on cerebral infarct volume in MCAO / R rats ( n = 8)

[0145]

[0146] 3.3 Effects of Riligustilide on pathological changes in the brain tissue of MCAO / R rats (HE staining)

[0147] The results of HE staining were as Figure 8 shown. The morphological structure of neurons in the sham operation group of rats was normal, evenly dispersed, and the nucleoli were clearly visible, without obvious pathological damage. Compared with the sham operation group, most of the cells in the cerebral cortex of the model group of rats were shrunk, and some neurons were lost. In the low- and high-dose Riligustilide groups and the positive drug butylphthalide group, the number of normal cells increased and the degree of cell damage decreased.

[0148] 3.4 Effects of phthalide dimer Riligustilide on pathological changes in the brain tissue of MCAO / R rats (Nissl staining)

[0149] The results of Nissl staining were as Figure 9 shown. The morphological structure of neurons in the cerebral cortex of the sham operation group of rats was normal, the cell contours were clear, and the number of Nissl bodies was relatively large. Compared with the sham operation group, the Nissl bodies in the model group of rats were significantly reduced, showing damage such as nuclear pyknosis. By comparing with the model group, the number of Nissl bodies in each administration group could be increased to varying degrees.

[0150] Example 4 Improvement of phthalide dimer Ligustilide A in 2VO rats

[0151] 1. Experimental animals

[0152] Same as in Example 1.

[0153] 2. Experimental methods

[0154] 2.1 Establishment of chronic cerebral ischemia model

[0155] The experimental rats were adaptively fed for 5 days before the operation. The bilateral common carotid artery ligation method (2VO) was used to establish the model. The rats were fasted for 14 h before the operation and anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg / kg). They were fixed in the supine position, the neck was shaved and prepared for skin, disinfected with iodophor, and a longitudinal incision of about 2 cm was made along the neck skin. The subcutaneous muscles were bluntly dissected to expose the bilateral common carotid arteries. In the model group, the bilateral common carotid arteries were ligated with 3-0 surgical thread, and the neck skin was closed and sutured layer by layer. In the sham operation group, only the bilateral common carotid arteries were exposed. Animal warming was noted throughout the operation, and the animals were placed in the animal cage after waking up. The animal status and wound healing were observed every day after the operation. Two weeks after modeling, the learning and memory of the model rats were detected by the step-down test. Compared with the sham operation group, the rats with significantly reduced memory ability were identified as successfully modeled VCI rats (qualified after screening).

[0156] 2.2 Grouping and administration of experimental animals

[0157] The model rats qualified by the step-down test were randomly divided into a model group, a low-dose ligustilide A dimer group (10 mg / kg), a high-dose ligustilide A group (20 mg / kg), and a ginkgo biloba extract group (25 mg / kg, clinical equivalent dose), with 10 rats in each group. Two weeks after modeling, the rats in each group were continuously administered by gavage for four weeks to intervene and evaluate the drug efficacy.

[0158] 2.3 Step-down test

[0159] Two weeks after modeling, the step-down test was used to evaluate the success of the model. Specifically: on the first day, the learning ability of the rats was detected, and on the second day, the memory maintenance and consolidation ability of the rats was tested. The experimental operations on the two days were the same. First, the rats were placed in the box (length: 20 cm, width: 20 cm, height: 29 cm) to freely adapt for 2 - 3 min, and then they were placed on a circular platform (diameter: 9.5 cm, height: 7 cm). At the same time, the instrument was turned on to energize the bottom of the copper grid at the bottom of the box. After the trained animals received an electric shock, they jumped back to the safe platform. The rats were trained repeatedly many times, and the time for the rats to jump off the platform and the number of mistakes of repeatedly getting off the platform (the actual number of times the rats jumped from the insulated step-down platform to the power grid within a certain time) were recorded, which was regarded as the learning score of the rats; 24 h later, the rats in each group were tested during the test period, and the time for the animals to jump off the platform for the first time and the number of mistakes within 5 min were recorded, which was used as the memory score of the rats. Based on the performance during the training period and the test period, the learning and memory ability of the rats was evaluated. Among them, compared with the sham operation group, a significant increase in the number of mistakes in the model group was considered a successful model.

[0160] 2.4 Morris water maze experiment

[0161] On the 36th - 42nd day of the experiment (i.e., two weeks after modeling, the last week during the four - week drug administration period), the Morris water maze test was conducted to evaluate the spatial learning and memory ability of rats. The water maze consisted of a circular pool (diameter: 160 cm, height: 50 cm, water depth: 20 cm). The circular pool was divided into four quadrants. The escape platform (diameter: 12 cm) was placed at the mid - point of the first quadrant and was preferably 1 - 2 cm below the water level. During the experiment, the water temperature was controlled at 22 ± 2 °C to ensure the reliability of the experimental results. On the 6th day of the water maze experiment, the platform was removed to examine the spatial exploration ability of rats. The rats were placed in the third quadrant, and the number of times each rat crossed the platform within 60 s and the swimming time in the target quadrant were captured in real - time. The swimming paths of the rats were analyzed using SupeMze (XR - Xmaze, China) software to obtain the spatial exploration data of the target rats.

[0162] 2.5 Brain tissue sampling, HE staining, and Nissl staining

[0163] The operation was the same as that in Example 3.

[0164] 3. Experimental results

[0165] 3.1 Evaluation of the improvement of ligustilide A on the learning and memory ability of 2VO rats based on the step - down test

[0166] The detection results of the memory stage of rats are shown in Table 6. Compared with the sham - operation group, the latency (the time of the first jump off the platform within 5 min) of the model group was significantly reduced (P < 0.01), and the number of errors increased significantly (P < 0.01). The first jump - off time of the sham - operation group was about 1.89 times that of the model group, and the number of times of jumping off the platform was about 0.35 times that of the model group. Compared with the model group, the high - dose ligustilide A group and the Ginkgo biloba extract group could extremely significantly prolong the latency time (P < 0.01), and could increase the first jump - off time by about 37.19% and 45.86% respectively, and reduce the number of errors of repeatedly jumping off the platform by about 45.04% and 48.58% (P < 0.01 or P < 0.05); in addition, compared with the model group, the low - dose ligustilide A group could also significantly reduce the number of errors by 38.65% (P < 0.05). The experimental results show that ligustilide A can improve the memory level of 2VO rats.

[0167] Table 6 Latency and number of error values of rats in each group during the test stage n = 8

[0168]

[0169]

[0170] 3.2 Evaluation of the improvement of cognitive level of ligustilide dimer osthole in 2VO rats based on the water maze experiment

[0171] The positioning navigation trajectories and results of the spatial exploration experiment are as Figure 10 shown in Table 7. Compared with the sham operation group, the number of times of crossing the platform and the residence time in the target quadrant in the model group were significantly reduced (P<0.01, P<0.05). After 4 weeks of drug intervention treatment, both the osthole group and the Ginkgo biloba extract group could increase the number of times of crossing the platform and the residence time in the target quadrant to varying degrees. Compared with the model group, the high-dose osthole group and the Ginkgo biloba extract group could increase the number of times of crossing the platform by about 150% and 156% respectively (P<0.05), and significantly extend the swimming time in the quadrant where the platform is located by 37.54% and 37.79% (P<0.05). It shows that osthole can improve the behavioral cognitive level of 2VO rats after intervention.

[0172] Table 7 Results of the spatial exploration experiment in rats (x±s, n=8)

[0173]

[0174] 3.3 Improvement of the pathological results in the hippocampus of 2VO rats by osthole (HE staining)

[0175] The results of HE staining to evaluate the pathological changes in the hippocampus of chronic cerebral ischemia rats are as Figure 11 shown. In the sham operation group, the neuronal cell structures in the hippocampal CA1, CA3, and DG regions were normal, arranged neatly, the cell structures were relatively complete, the cytoplasm was full, the cell boundaries were obvious, the nuclear membrane and nucleolus were visible, and no neuronal degeneration or necrosis changes occurred; in the model group, the neuronal cells were arranged disorderly, the cell layers were uneven, and the cell nuclei were deeply stained and condensed; in the low- and high-dose osthole groups and the Ginkgo biloba extract group, the neuronal cells in each hippocampal region were relatively complete in arrangement, the cell morphology improved, the structure was relatively complete, the cell membrane was clear, and occasional cell damage was seen.

[0176] 3.4 Improvement of the pathological results in the hippocampus of 2VO rats by osthole (Nissl staining)

[0177] The results of Nissl pathological staining are as Figure 12As shown, in the sham operation group, the neurons in the CA1 and CA3 regions of the hippocampus of rats were arranged closely, with a complete structure and a rich number of Nissl bodies. The numbers of Nissl bodies in the CA1, CA3, and DG regions of the hippocampus of rats in the sham operation group were 57.0±4.0, 77.3±6.7, and 245.0±18.0, respectively; the numbers of Nissl bodies in the CA1, CA3, and DG regions of the hippocampus of rats in the model group could be reduced to 46.0±2.6, 47.7±5.0, and 173.7±8.1, and the numbers in each region were significantly reduced (P<0.01); the numbers of Nissl bodies in the CA1, CA3, and DG regions of rats in the low-dose ligustilide A group could be increased to 49.7±4.2, 62.7±1.5, and 183.3±9.1; the numbers of Nissl bodies in the CA1, CA3, and DG regions of rats in the high-dose ligustilide A group could be increased to 54.7±1.5, 66.0±5.3, and 197.3±7.5; the numbers of Nissl bodies in the CA1, CA3, and DG regions of the hippocampus of rats in the ginkgo biloba extract group were 55.0±1.7, 65.0±3.0, and 195.0±16.1. The results of the Nissl staining data showed that after intervention with ligustilide A, the number of Nissl bodies in the hippocampus of rats could be significantly increased, protecting neurons.

[0178] Example 5 Improvement of phthalide dimer senkyunolide O on 2VO rats

[0179] 1. Experimental animals

[0180] Same as Example 1.

[0181] 2. Experimental methods

[0182] 2.1 Establishment of chronic cerebral ischemia model, animal grouping and drug administration

[0183] Same as Example 3.

[0184] 2.2 Morris water maze experiment, tissue sampling of brain tissue, HE staining, and Nissl staining

[0185] Same as Example 3.

[0186] 3. Experimental results

[0187] 3.1 Evaluation of the improvement of senkyunolide O on the cognitive level of 2VO rats based on the water maze experiment

[0188] The results of the spatial exploration experiment are shown in Table 8. Compared with the sham operation group, the average number of times the 2VO model rats passed through the platform decreased by about 64%, and the average time in the target quadrant decreased by about 30%. Compared with the model group rats, the number of times the rats in the low-dose and high-dose ligustilide O groups and the ginkgo biloba extract group passed through the platform increased by about 112%, 104%, and 125% respectively, and the average time staying in the target quadrant increased by about 38%, 38%, and 25% respectively. The above results indicate that the phthalide dimer ligustilide O can improve the behavioral cognitive level of 2VO rats.

[0189] Table 8 Results of the spatial exploration experiment in rats (x±s, n = 8)

[0190]

[0191] 3.2 Improvement of the pathological results in the hippocampal region of 2VO rats by ligustilide O (HE staining)

[0192] The results of HE staining to evaluate the pathological changes in the hippocampal region of chronic cerebral ischemia rats are as Figure 13 shown. In the model group rats, neurons in the hippocampal CA1, CA3, and DG regions disappeared or the cell boundaries were missing. After administration of low-dose and high-dose ligustilide O, these changes in the hippocampal region were significantly reversed. In the ligustilide O and ginkgo biloba extract tablet administration groups, the outlines of nerve cells were clear, the structure was compact, and the nucleoli were intact.

[0193] 3.3 Improvement of the pathological results in the hippocampal region of 2VO rats by ligustilide O (Nissl staining)

[0194] The results of Nissl pathological staining are as Figure 14 shown. In the sham operation group rats, the neurons in the hippocampal CA1 and CA3 regions were arranged closely, the structure was complete, and the number of Nissl bodies was abundant. In the model group rats, Nissl bodies were lost, and Nissl bodies in the hippocampus and cortex were atrophied, with nuclear atrophy or pyknosis. Compared with the model group, after administration of ligustilide O and ginkgo biloba extract, the number of Nissl cells in the hippocampal CA1, CA3, and cortical regions increased significantly, and the neurons were significantly protected.

[0195] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: The phthalide dimer compound of the present application can reduce the infarct volume (such as the infarct volume in the acute ischemic stroke MCAO / R rat model), improve the damage of cortical and hippocampal neurons in rats, make the morphology of neuron cells basically return to normal, improve the cranial nerve function, and play a role in preventing and treating ischemic stroke. Moreover, the improvement effect of the high-dose group is better than that of the positive drug butylphthalide. At the same time, the phthalide dimer compound can reduce neuronal apoptosis by inhibiting the expression of pyroptosis proteins (Caspase-1, GSDMD-N, and IL-1β), and play its neuroprotective role.

[0196] Phthalide dimer compounds can also alleviate the neurological deficits caused by chronic cerebral ischemia (in rats with 2VO bilateral common carotid artery ligation model), improve the spatial learning ability of model rats, improve the learning and cognitive level, improve the neuronal damage in the hippocampal CA1, CA3 and DG regions and can reduce the number of lost Nissl bodies, protecting neurons. The therapeutic effect of the high-dose group is comparable to that of the positive drug Ginkgo biloba extract.

[0197] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A use of a phthalide dimer, a salt thereof, a stereoisomer thereof, a prodrug thereof or a metabolite thereof in the preparation of a drug for treating or preventing a nervous system disease, characterized in that: The phthalide dimer is formed by two phthalide monomers through [4+2] or [2+2] cycloaddition, and the phthalide dimer includes 1-2 spiro atoms. The chemical formula structure of the phthalide monomer is shown in the following formula I: Wherein, in Formula I, R1, R2, R3, R4, R5, R6, and R7 are each independently selected from any one of the following: hydrogen, ORa, OCOORa, OCONRaRb, OSO2Ra, or OSO3Ra; The Ra and Rb are each independently selected from any one of the following: hydrogen, substituted or unsubstituted C1-C 12 Alkyl, substituted or unsubstituted C1-C 12 Alkenyl, substituted or unsubstituted C1-C 12 Alkynyl, substituted or unsubstituted C6-C 30 Aryl or substituted or unsubstituted C1-C 30 heteroaryl; Indicates a single bond or a double bond.

2. The use according to claim 1, characterized in that: The phthalide monomers in the phthalide dimer are each independently selected from any one of the following:

3. The use according to claim 1, characterized in that: The phthalide dimer comprises:

4. The use according to claim 1, characterized in that: The phthalide dimer is extracted from natural medicinal plants or chemically synthesized.

5. The use according to claim 4, characterized in that: The natural medicinal plants include plants of the Umbelliferae family.

6. The use according to claim 5, characterized in that: The Umbelliferae plants include Chuanxiong, Angelica and Chaxiong.

7. The use according to claim 1, characterized in that: The nervous system diseases include acute cerebral ischemia and chronic cerebral ischemia.

8. The use according to claim 1, characterized in that: The characteristic manifestations of the nervous system diseases include: neurological damage.

9. The use according to claim 1, characterized in that: The characteristic manifestations of the neurological diseases include: brain tissue damage; Preferably, the brain tissue damage includes: neuronal damage.

10. A composition for treating or preventing nervous system diseases, characterized in that: The composition comprises: (a) the phthalide dimer for use according to any one of claims 1 to 9; and optionally (b) a pharmaceutically acceptable carrier.

Images