WO2005102314A1

WO2005102314A1 - The use of butylphthalide homologues in preparing the drug of preventing and treating cerebral ischemic

Info

Publication number: WO2005102314A1
Application number: PCT/CN2005/000556
Authority: WO
Inventors: Zhentao Liu; Hongwu Zhang; Dongmin Shen; Xiaolong Feng; Rongduan Wang; Jingguo Sun
Original assignee: Shijiazhuang Pharma. Group Zhongqi Pharmaceutical Technology(Shijiazhuang) Co., Ltd.
Priority date: 2004-04-23
Filing date: 2005-04-22
Publication date: 2005-11-03
Also published as: CN100435792C; CN1689563A

Abstract

The invention disclosed the use of butylphthalide homologues in preparing the drug of preventing and treating cerebral ischemic and dementia resisting thrombus and thrombocyte agglutination. The butylphthalide homologues relate to the invention are 3-(3'-hydroxyl)butylphthalide and 3-hydroxyl-3-butylphthalide.

Description

Butylphthalide homologs in the preparation of prevention and treatment

Application in medicine for cerebral ischemic diseases

The present invention relates to butylphthalide homologues, specifically 3- (3'-hydroxy) butylphthalide,

Application of 3-hydroxy-3-butylphthalide in the preparation of medicines for the prevention and treatment of cerebral ischemic disease, anti-platelet aggregation and anti-thrombosis, and treatment and prevention of dementia. Background technique

Butylphthalide, also known as celeryme, was originally found in celery seeds, and its pharmacodynamic characteristics covered many aspects: 1. Improving ischemic brain energy metabolism; 2. Significantly reducing ischemic cerebral infarction in rats Area, improve neurological deficits; 3. improve cerebral edema caused by ischemia; 4. significantly improve local cerebral blood flow and pia mater circulation in the ischemic area; 5. significantly improve memory impairment caused by ischemia. The side effects are small. In Chinese Patent Application No. 93117148.2, the application of apigenin in the preparation of a medicament for preventing and treating diseases caused by cerebral ischemia in mammals or humans is disclosed. In addition, in Chinese Patent Application No. 98125618.X, the application of L-butylphthalide in the preparation of antithrombotic and antiplatelet aggregation drugs is clearly shown that this product has the function of regulating the NOS-NO-cGMP system and the brain Role of Arachidonic Acid Metabolism in Nerve Cells after Ischemia

Wang Chunhua's research shows (Wang Chunhua et al .: Studies on metabolites of butylphthalide in rats 1997, 32 (9): 641-646), butylphthalide is mainly converted into two metabolites 3- (3 '-Hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide (as shown below). The ratio of the prototype to the metabolites in the urine is about 9:91, and the ratio of the metabolites to the prototype drug in the brain is about 56:44, but the metabolites in the brain are only M l and no M Π. At present The pharmacodynamic effects of the two metabolites are not clear. From the structure of the above two compounds, they both belong to the butylphthalide homologues.

3- (3'-hydroxy) butylphthalide (Metabolite I, M I for short)

3-Hydroxy-3-butylphthalide (Metabolite II)

The object of the present invention is to provide new applications of butylphthalide homologues 3- (3'-hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide, so that it can be used in the development of future drugs as A new active ingredient is used in pharmaceutical preparations. Through a series of animal experiments in the following examples, the inventors found that 3- (3'-hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide, which are homologs of butylphthalide, have the following pharmacological effects:

1. Significantly improve the neurological symptoms caused by cerebral ischemia in rats due to brain trauma;

2. Improve the memory impairment caused by cerebral ischemia in rats;

3. Reduce cerebral edema caused by cerebral ischemia in rats;

4. Reduce stroke caused by cerebral ischemia in rats;

5. Improve the energy metabolism disorder caused by cerebral ischemia in rats; 6. Moon blood in the ischemic area of Zenglikou: ^;

7. Reduce the cerebral infarct area and reduce the symptoms of neurological deficits in rats with focal cerebral ischemia;

8. Anti-platelet aggregation and anti-thrombosis;

9. Prevention and treatment of dementia.

Based on the above findings, the inventors of the present application provided the following uses of 3- (3'-hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide, which are homologs of butylphthalide.

In one aspect, the present invention provides butylphthalide homologs 3- (3'-hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide in preparations for preventing and treating cerebral ischemia. Use in disease-causing drugs.

In the above aspect, the disease caused by cerebral ischemia is selected from neurological symptoms caused by cerebral ischemia, memory disorders caused by cerebral ischemia, cerebral edema caused by cerebral ischemia, stroke caused by cerebral ischemia, and brain deficiency Impaired energy metabolism caused by blood, cerebral infarction caused by cerebral ischemia and loss of nerve function

In another aspect, the present invention provides butylphthalide homologues 3- (3'-hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide in preparation for improving cerebral ischemic brain Use of blood flow in medicine. 'In yet another aspect, the present invention provides butylphthalide homologs 3- (3'-hydroxy) butylphthalide, 3-hydroxy-3-butylphthalide in the preparation of anti-platelet aggregation and antithrombotic Use in medicine.

In yet another aspect, the present invention provides butylphthalide homologs 3- (3'-hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide in the preparation of a medicament for treating and preventing dementia. Use.

The beneficial effect of the present invention is to clarify the therapeutic effect of butylphthalide homologues 3- (3'-hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide on diseases caused by cerebral ischemia. detailed description

The following further describes the present invention with reference to the examples, but these examples are merely illustrative and do not have any limitation on the essence and scope of the present invention. The true shield and scope of the invention is only limited by the appended claims.

The experimental methods hereinafter are all conventional methods known to those skilled in the art, in which the butylphthalide homologue 3- (3'-hydroxy) butylphthalide tube is called MI, 3-hydroxy-3-butylbenzene The phthalate cartridge is called M II.

Example 1. Effects of M1 and ΜΠ on neurological symptoms caused by traumatic brain injury in rats Experimental animals: Male wistar rats

Actual-check method: A cone-shaped metal object weighing 220g was dropped freely from a height of 30cm, and hit the cranial parietal bone of the left side of the left coronary suture of the rat, resulting in traumatic brain injury in rats. Oral administration after 15 minutes: M l and MΠ ( 20, 40, 80 mg / kg), and the behavior changes were scored after 24 hours.

Neurobehavioral score: Behavioral observation was performed 24 hours after ischemia. Lift the rat's tail about 1 foot away from the ground and observe the condition of the two forelimbs; push the rat's shoulders and observe whether there is a difference in resistance between the two sides; place the rat on the ground and observe its walking. Scoring according to the following criteria: 0 points: Both forelimbs are strong and symmetrically extend to the ground. Both shoulders have a resistance of 4 and the walking is normal.

1 point: Internal contralateral shoulder rotation, adduction of forelimbs; uniform shoulder resistance and normal walking. 2 points: Internal contralateral shoulder rotation, adduction of forelimbs; When pushing shoulders, resistance to contralateral surgery decreases; walking is normal.

3 points: Internal contralateral shoulder rotation, adduction of forelimbs; When pushing both shoulders, the resistance of the opposite side of the operation decreases; walking around the circle.

4 points: Internal contralateral shoulder rotation, adduction of forelimbs; no spontaneous activity.

The higher the score, the more severe the animal's behavioral disorder. Experimental grouping: operation group (solvent control), group A (MI 20mg / kg), group B (MI 40mg / kg), group C (MI 80mg / kg), group D (MI II 20mg / kg), group E ( Mll40mg / kg), F group (Mll80mg / kg), G is MK801 O.lmg / kg group (injection), the other groups were administered orally.

Experimental results: The rats in the blank group had no abnormal behavior, with a neurobehavioral score of 0. Most of the animals in the solvent control group had a neurological score of 2 and showed contralateral forelimb internal rotation, and lateral compression of the contralateral extensor muscles weakened. . A small number of animals developed contralateral circle symptoms, which were rated as 3 points. Individual animals had mild symptoms and only showed contralateral forelimb internal rotation, with a score of 1; or severe symptoms, lack of autonomous activity, and scored 4 points. The mean nerve score in the solvent control group was 2.6 ± 0.3. After 15 minutes of ischemia, oral administration of MI 40 and 80 mg / kg can significantly improve their neurological symptoms (1.4 points, cadaveric <0.01 and 1.1 points, <0.001), and oral administration of ΜΠ40 and 80 mg / kg can significantly improve their neurological symptoms. (1.5 points, <0.05 and 1.0 points, <0.001), MK801 had the most significant effect (0.8 points, <0.001). It is shown that the two homologues of butylphthalide Ml and M II can improve the neurological symptoms caused by cerebral ischemia caused by brain trauma.

Example 2. Effects of MI and Mil on memory impairment caused by ischemia Experimental equipment: Shuttle box, the same as in Example 1

Experimental animals same as in Example 1

Practical method:

(1) Acquiring learning and memory training in rats:

(2) Cerebral artery ligation surgery: Rats were subjected to cerebral artery ligation surgery according to the Tamura method. After 24 hours, the experiment of measuring the learning and memory of rats was repeated.

(3) Grouping and administration: There are 8 groups, model group, sham operation group, group A (Ml 15mg / kg), group B (MI 30mg / kg), group C (Ml 60mg / kg), group D ( MlI 15 mg / kg), group E (Mll 30 mg / kg), and group F (MlI 60 mg / kg). Postoperative The drug was administered orally at 15 minutes, and the change of the latency of the active and passive avoidance responses to the rats was observed after 24 hours.

Experimental results: See Table 1. Compared with the sham operation group, the number of active avoidance in the model group was significantly less, and the active avoidance latency and the passive avoidance latency were prolonged significantly, suggesting that their conditional learning and memory ability was significantly reduced. Compared with the model group, the number of active avoidances in groups B, C, E, and F increased significantly (<0.05 and), and the latency of active avoidance and passive avoidance response was significantly reduced, indicating that M1 and ΜΠ improved local brain Effects of ischemic memory disorders

Table 1: Effects of MI and Mil on learning and memory abilities of rats undergoing cerebral artery ligation (± s)

Number of active avoidance groups Active avoidance latency Passive avoidance latency Model group 6.5 + 4.1 4.25 ± 1.43 4.05 + 3.10 Sham operation group 18 · 5 ± 3 · 1 ^ΔΔ 0 · 15 ± 0.31 ^ΔΔ 0 · 05 ± 0 · 41 ^ΔΔ

Group A 8.5 + 5.3 3.25 ± 0.43 3.12 ± 1.35

Group B 12.5 ± 6.6 '1.85 ± l ^ S ⁵ "0.59 ± 0.4

Group C 15.2 + 4.1 0.58 ± 1.490.05 0.05 0.50

Group D 9.1 ± 4.7 "^ 2.95 + 1.23 3.25 + 1.41

Group E 13.8 ± 3.9 1.64 ± 1.38 0.46 ± 0.56

Group F 15.09 ± 5.5 ^χ ^'58 0.63 ± 1.55

Compared with the sham operation group <0.05, cadaver <0.01; compared with the model group ※ cadaver <0.05, ^ 'cadin <0.01 Example 3, Effects of M I and Mil on cerebral vascular ligation in rats

Animal in danger as in Example 1

Experimental method: Rats were ligated with the right middle cerebral artery to cause cerebral edema. After 15 minutes, Ml and Mil (20, 40, 80 mg / kg) were orally administered, and they were sacrificed for 24 hours. The brain, called the left and right hemisphere wet weight. After baking at 100 ° C for 24 hours, the dry weight was measured, and the brain tissue water weight was calculated by the wet and dry method. The tissue was digested for 4 hours, the pH value was adjusted with HCI, and the ion-selective electrode was connected to an oxygen tissue analyzer to measure the Na + K + concentration in the brain tissue.

Real eyelid grouping: operation group, branch operation group, the rest of the groups are the same as in Example 1

Experimental results:

(1) Influence on the water content of brain tissue: See Table 2. Compared with the sham operation group, the water content of brain tissue in the model group increased significantly. Compared with the model group, the A B C D E F groups can reduce the brain tissue water content to a different degree or significantly, and the Ml and Mil groups have a concentration-dependent reduction of brain tissue water content.

Table 2: Effects of Ml and ΜΠ on brain water content in middle cerebral artery ligated (± s) group Brain water content Na + content (micromolecule K + content (micromolecule /

(%) / G dry brain tissue per gram dry brain tissue) model group 81.5 ± 0.21 348.3 ± 14.7 436.9 ± 11.3 sham operation group 76.5 ± 0.41 ^ΔΔ 265.1 ± 9 · 3 ^ΔΔ 485.3 ± 13.9 ^ΔΔ

Group A 80.14 ± 0.53 325.5 ± 23 · 2 '446.5 ± 13.3

Group B 79.58 + 0.66 ⁵⁸ 305.5 + 18.7 ^ 458.9 ± 12.8 ⁸⁵

Group C 78.25 ± 0.41 285.9 ± 29.2 ^s " ⁵⁸ 475.3 ± 9.5.

Group D 80.25 ± 0.69 333.6 ± 25.8 ^s "443.5 ± 8.9

Group E 79.44 ± 0.48 309.5 ± 21.1 455.4 ± 11. ό ⁵⁸

F group 78.39 ± 0.55 280.4 ± 24.4 ⁸⁵ 474.1 ± 10.5 '^ Compared with the sham operation group ^ P <0.05, ^ΔΔ <0.01; compared with the model group ※ cadaver <0.05, ※ cadaver <0.01

(2) Effect on Na + K + in brain tissue: See Table 2. Compared with the sham operation group, the Na + content in the brain tissue of the model group was significantly increased, and the K + content was significantly decreased. Groups A, B, C, D, E, and F can improve the Na + K + of brain tissues to varying degrees Content. Moreover, there was a dose-response relationship; the effects of the high-dose and high-dose M 1 and M II groups were more obvious, compared with the blank control group <0.001.

The above results indicate that: M l and M il can significantly reduce cerebral edema caused by ischemia.

Example 4. Effects of MI and Mil on stroke in spontaneously hypertensive rats Experimental animals: 6-week-old spontaneously hypertensive rats

Experimental method: 6-week-old spontaneously hypertensive rats were divided into 7 groups of 10 rats each, and fed daily with 0.8-0.9 g of salt daily from the 6th week of age, and gradually increased to 1.2-1.3 g per day thereafter, starting from the 8th week Oral administration of Ml and Mil (12.5, 25, 50mg / kg / d) and nimodipine (37mg / kg / d) were started, and 3 weeks after the onset of stroke, the nerve loss score and the time of death were recorded.

The experimental group was divided into 8 groups: model group, positive drug group (nimodipine 37mg / kg / d), group A (MI 12.5mg / kg / d), group B (MI 25mg / kg / d), and group C (MI 50mg / kg / d), group D (MII 12.5mg / kg / d), group E (MII 25mg / kg / d), and group F (MII 50mg / kg / d).

Experimental results: Compared with the model group, compared with the positive drug group, group A, group B, group C, group D, group E, and group F, the time of stroke onset can be delayed to a different extent or significantly, indicating that it can prevent stroke onset At the same time, MI can significantly increase the survival rate and improve the neurological deficit after stroke in a dose-dependent manner, indicating that it has a therapeutic effect on stroke caused by spontaneous hypertension. It is suggested that M I has preventive and therapeutic effects on patients with stroke caused by severe hypertension, and M II has preventive effects on patients with stroke caused by severe hypertension, and the therapeutic effect is not obvious.

Example 5.Effects of MI and Mil on brain energy metabolism of global cerebral ischemia caused by decapitation in mice

Experimental animal: same as in Example 1

Experimental drugs: MI and M II injections formulated with Tween 80 emulsion Experimental method: 10-22 g of Kunming mice were divided into 8 groups, 10 mice in each group, and Ml and MΠ (25, 50, 100 mg / kg / d) were administered orally as a blank control group, and phenobarbital sodium (225 mg / kg ip) 30 minutes after administration, decapitation to homogenization time of 15 seconds was used as cerebral ischemic time. Centrifugal extraction was performed according to the Folbergrova method and ATP, creatine phosphate (PCr) and lactic acid (LA) contents were measured by enzymatic method.

Experimental grouping: divided into 8 groups, blank control group, positive drug group (phenobarbital sodium 225mg / kg, ip), group A (25mg / kg / d), group B (50mg / kg / d), C Group (100 mg / kg / d D 25 mg / kg / d :), £ group (50 mg / kg / d), F 100 mg / kg / d) Experimental results: See Table 3. Compared with the blank control group, the ip-positive control group showed an increase in ATP and PCr and a decrease in lactic acid content, indicating that this method is reliable. After oral administration of Ml, ATP and PCr increased, and the content of AL decreased in the middle and high-dose groups, which was very different from that in the blank control group; the ΜΠ group had this effect only in the high-dose group. The results suggest that both M I and ΜΠ can improve brain energy metabolism, and Ml has a better effect.

Table 3: Effects of Ml and MΠ on brain energy metabolism of global cerebral ischemia induced by decapitation in mice (± _s , μηιΐ / g)

Group ATP AL PCr

Blank control 0.85 + 0.22 10.5 ± 1.62 2.15 ± 0.44 Positive drug group 2.69 ± 0.41 5.39 ± 1.6 3.69 ± 0.51

Group A 1.14 + 0.55 8.34 + 1.33 2.64 ± 0.35

Group B 1.58 ± 0.36 6.35 ± 1.63 ^x 2.98 ± 0.43 ^s *

Group C 2.45 ± 0.54 5.58 ± 1.84 3.85 ± 0.64

Group D 0.95 ± 0.69 9.95 + 1.69 2.22 ± 0.58

Group E 1.44 ± 0.6 ^ 8.46 + 1.88 ^ 2.44 soil 0.47

Group F 1.69 ± 0.55 6 · 53 ± 1.75 · 2.99 ± 0 · 65 Compared with blank control ^¾ cadaver <0.05, ^M '<0.01

Example 6, Effects of MI and Mil on Local Cerebral Blood Flow

Experimental equipment: SXP-operative microscope, 62TZ-V high-frequency electric knife

Real Face Animal: Male Wistar Rat

Stereotactic instrument: Shanghai Jiangwan Type I C

Tissue gas analyzer (Diamond Electro-Tech chemical microsensor 1231): manufactured by Diamond General, USA

Two-channel physiological recorder: produced by Chengdu Instrument Factory

experimental method

(1) M I and Mil (15, 30, 60 mg / kg) and a solvent were orally administered to normal rats, and changes in cerebral blood flow at different times after the administration were measured.

(2) Rats were subjected to cerebral arterial ligation according to the Tamura method, and were orally administered at 10 minutes after operation. They were divided into 8 groups, a sham operation group, a solvent control group, and a M1 and MΠ (15, 30, 60 mg / kg) group.

Experimental results: Cerebral blood flow increased significantly after 30 minutes of Ml in normal rats.

Cerebral blood flow remained at a high level for 60 minutes. Later, as time went on, the increase was gradually reduced, and it had no effect on blood pressure. It can be seen that Ml can increase cerebral blood flow and improve brain blood supply without substantially affecting blood pressure. After arterial ligation, the Ml group can significantly increase the local blood flow in the cerebral arteries and block the lateral striatum. Compared with the solvent control group, there is a significant difference, and there is a dose-response relationship. The effect of the ΜΠ (60mg / kg) group was more obvious, compared with the blank control group, PO.05, and there was no significant effect in the other drug groups. It shows that Ml and Mil can not only increase the cerebral blood flow in normal rats, but also increase the cerebral blood flow in the ischemic area. Ml has a better effect.

Example 7, Effects of MI and Mil on Cerebral Infarction Area and Neural Function Loss in Focal Cerebral Ischemia Rats Experimental animal: same as in Example 1

Practical method: Rats were subjected to brain movement and permanent ligation according to the Tamura method, and were orally administered 10 min after surgery. They were divided into 8 groups, a sham operation group, a solvent control group, and MI and M (15, 30, 60 mg / kg) group.

Experimental results: Neurological symptoms caused by cerebral ischemia in rats. After oral administration, compared with the surgical control group, the MI group can significantly improve the neurological symptoms and reduce the infarct area in a dose-dependent manner. Compared with the blank control group, it is <0.05, which indicates that the two metabolites M l and M II of butylphthalide can significantly improve the neurological symptoms caused by ischemia.

Example 8. M I and Mil antiplatelet aggregation and antithrombotic effect

Experimental drugs and instruments: Aspirin (Asp), adenosine diphosphate (ADP), collagen (Col), arachidonic acid (AA).

Platelet Aggregation Instrument (PAT-4A MEGURO-KU TOKYO JAPAN) Animals

Male Wistar rats, weighing 280-320g; male Kunming mice, weighing 22-26g.

(1) Rat platelet aggregation in vitro

Rats were anesthetized with 10% chloral hydrate (30mg / kg), blood was collected from the carotid arteries, and platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared according to conventional methods. Refer to the Bom's method, and take PKK 200 μ1, and add the drugs separately. The two concentration curves for M l and MΠ range from 1, 3, 10, 30, and 100 μM. Place it on a platelet aggregator and pre-warm it at 37 ° C for 5 minutes. Then add ADP as the final concentration to 5 μM, and determine the maximum aggregation rate of the platelet after 5 minutes.

(2) Time-dependent relationship between Μ I and Μ II on platelet aggregation in vivo in rats

The rats were randomly divided into 15 groups of 3 animals each, which were the solvent control group and M l And Mll (50mg / kgp.o.) Blood was taken at different times (30, 45, 60, 90, 120, 180, 240min). Animals in each group were anesthetized with chloral hydrate (30mg / kg) at the corresponding time points after administration, and blood was collected from the carotid artery. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared according to conventional methods, with reference to Bom's Method: Take 200μ1 of PKP, place it on a platelet aggregation meter, and pre-incubate at 37 ° C for 5 minutes, then add the inducer ADP (5 mol / l), and determine the maximum platelet aggregation rate after adding the attractant for 5 minutes.

(3) In vivo platelet aggregation test in rats

The rats were divided into 17 groups of 10 animals each, which were the control group, the aspirin group, the M1 and MΠ (3, 10, 30, 100mg / kg po single administration) group, and the celopidine (100mg / kg continuous administration for 3 days) group. Blood was taken from the carotid artery after anesthesia with chloral hydrate (30mg / kg) 2h after the last administration, and platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared according to the conventional method. Refer to Born's method, and take PKP 200 μ1, Place it on a platelet aggregator and pre-warm it at 37 ° C for 5 minutes, then add the attractant ADP, collagen and AA to make the final concentration of 5μιηο1 / 1, 100μΙ / mL 0.5mmol / L, determine Maximum platelet aggregation rate after minutes.

(4) Experimental model of arteriovenous loop in rats

Rats were randomly divided into 10 groups with 10 animals in each group. Fasting the night before the experiment. Oral Asp, MI and ΜΠ (dose of 3, 10, 30, 100 mg / kg for each drug) and the same volume of vegetable oil. Two hours after the administration, the rats were anesthetized with 30 mg / kgip sodium pentobarbital, fixed in the supine position, and the right common carotid artery and the left external jugular vein were separated. Take a three-section polyethylene tube with an inner diameter of 0.9 mm and a length of about 12 cm (built-in 6 cm long silk thread, and the silk thread is weighed). The tube is filled with normal saline and connects the right common carotid artery and the left external jugular vein. Open the arterial clamp, blood flows from the right common carotid artery into the polyethylene tube, returns to the left external jugular vein, and the blood flow is interrupted 15 minutes after the blood flow is opened. Quickly remove the silk thread and weigh it. The wet weight of the silk minus the dry weight of the original silk thread is the wet weight of the formed thrombus. (5) Male Kunming mice, a model of pulmonary thrombosis induced by collagen and epinephrine, were fasted on the eve of the experiment. M1 and MΠ, Asp (30, 100 mg / kg of each drug) and the same volume of vegetable oil were administered to mice by gavage. After 2 hours of administration, collagen 1000 g / kg (including epinephrine 8) was injected into the tail vein. (^ g / kg), the injection was completed within 20 seconds, and the mortality of the animals was observed within 5 minutes. The protective effect of the drug was represented by the decrease in the ratio of the number of surviving animals to the number of experimental animals.

(6) Bleeding time experiment in mice

Male Kunming mice were fasted before the experiment. The mice were divided into 8 groups, a solvent control group, an Aspl00 mg / kg, MI and Mil (10, 30, 100 mg / kg) group. 2 hours after gavage, cut off the rat tail 5iran, start timing, and gently touch the sore surface of rat tail bleeding with qualitative filter paper every 30 sec, so that blood is printed on the filter paper, and printed once every 30 sec until there is no blood on the filter paper. Time to start bleeding to no blood.

Experimental results:

1. Effects of M I and M II on platelet aggregation in vitro of rats

M I and Mil (1 ~ 100 μM) significantly inhibit platelet aggregation.

2. The time-dependent relationship of the effects of L-butylphthalide on platelet aggregation in vivo in rats showed that the inhibitory effects of M I and M II at 50 mg / kg orally increased gradually over time, and reached a peak in about 2 hours. The inhibition rate reached about 50%, and then gradually recovered. Therefore, in vivo experiments for measuring M1 and MΠ to inhibit platelet aggregation were selected to take blood at 2 hours after administration.

3. Effects of M I and Mil on platelet aggregation in vivo in rats

The same concentrations of Ml and MII (3, 10, 30, 100 mg / kg) all inhibited collagen, ADP, and AA-induced platelet aggregation in a dose-dependent manner. Ml had a stronger effect, while Mil had a weaker effect. Inhibitory effect of Ml on platelet aggregation induced by collagen and ADP Stronger than aspirin at the same dose, but weaker on AA-induced platelet aggregation than Asp. The effect on ADP-induced platelet aggregation was similar to that of seclopidine for three days.

4. Effects of M I and Μ Π on experimental thrombosis of arteriovenous loop in rats

M l and MII (3, 10, 30, 100 mg / kg p.o) can inhibit experimental thrombosis in a dose-dependent manner, all in a dose-effect relationship. The inhibitory rates of M l (3, 10, 30, 100 mg / kg p.o) on thrombosis were 13%, 25%, 44%, and 45%, respectively. The inhibition rates of Asp (3, 10, 30, 100 mg / kg p.o) were 4%, 27%, 44%, and 48%, respectively. The results showed that the antithrombotic effect of M I was slightly weaker than that of aspirin at the same dose, and Mil had antithrombotic effect only at high doses (30, 100 mg / kg p.o).

5. Effects of M l and Mil on collagen and epinephrine-induced pulmonary thrombosis in mice In the tail vein injection of collagen and epinephrine-induced pulmonary embolism in mice, the survival rate of the control group was 31%, and M l And Mil (100mg / kg, po) significantly increased the survival rate of pulmonary embolism mice, and the survival rates of the mice were 58% and 66%, respectively. , The intensity of action is comparable to aspirin (65%) and eclopridine (70%).

6. Effects of L-butylphthalide on bleeding time in mice

M l (10, 30, 100 mg / kg) dose-dependently increased bleeding time in mice. M l had a stronger effect than aspirin at the same dose, and ML only had this effect at large doses (100 mg / kg p.o).

Example 9. Preventive and therapeutic effects of M I and ΜΠ on dementia

Instrument: Morris Water Maze Automatic Monitor

2-VO model establishment: Male Wistar rats, 10 weeks old, weighing about 280 grams, anesthetized with sodium pentobarbital, and bilateral common carotid artery ligation. The sham operation group underwent the same operation except that the common carotid artery was not ligated.

Experimental grouping and design: There are 8 groups in total, the same as in Example 2. Dosing started 10 days after surgery, A water maze experiment was performed on days 29-33, and a dark avoidance experiment was performed on days 34-35. Animals were sacrificed on day 36. In behavioral experiments, they were administered 40 minutes before the experiment.

Experimental results:

①Effects on water maze experiments: See Table 4, compared with the sham operation group, the number of active avoidances in the model group was significantly less, and the active avoidance latency and passive escape latency were also significantly longer, suggesting that their conditional learning and memory ability was significantly reduced. Compared with the model group, the number of active avoidances in groups B, C, E, and F increased significantly, indicating that M1 and ΜΠ can improve the memory impairment caused by dementia. Ml and ΜΠ can significantly reduce the latency of their active avoidance and passive escape responses, suggesting that butylphthalide can significantly improve the conditional learning and memory dysfunction in rats caused by cerebral artery ligation.

Table 4: Effects of Ml and Mil on water maze in dementia rats

Number of active avoidance times Active avoidance latency Passive avoidance latency Model group 6.5 ± 4.1 4.25 ± 1.43 4.05 ± 3.10 Sham operation group 18 · 5 ± 3 · 1 ^ΔΔ 0.15 ± 0 · 31 ^ΔΔ 0.05 ± 0.41 ^ΔΔ

Group A 8.5 + 5.3 3.25 + 0.43 3.12 ± 1.35

Group B 12.5 tG ^ ⁵⁸ 1.85 ± 1.53 0.59 ± 0.4

Group C 15.2 ± 4.1 0.58 ± 1.49 ^ 0.05 ± 0.50

Group D 9.1 ± 4.7 2.95 ± 1.23 3.25 ± 1.41

Group E 13.8 ± 3.9 1.64 ± 1.38 '0.46 soil 0.56

F group 15.09 ± 5.5 ^{5K 25} 0.63 ± 1.55 0.07 ± 0.47 compared with sham operation group <0.05 ^ΔΔ <ο.οΐ; compared with model group ^^ o.os, cadaver <0.01

②Effects on avoidance experiment of dementia rats: See Table 5. The dark avoidance latency of dementia rats was significantly shorter than that of the sham operation group. The trend of the dark incubation period significantly reduced the number of times of avoiding dark errors in dementia rats.

Table 5: Effects of M I and ΜΠ on dark avoidance experiments in dementia rats (± s)

Dark avoidance latency (s) Dark avoidance errors

Model group 50.5 ± 24.1 5.5 ± 1.2

Sham operation group 28.5 ± 31.3 ^ΔΔ 2.1 ± 1.3 ^ΔΔ

Group A 48.5 + 33.3 4.2 ± 1.1

Group B ΐZό ^ 3.5 + 1.5 ^

Group C 31.2 ± 34.1 2.5 ± 1.4 ⁵⁸

Group D 49.1 ± 24.7 4.9 ± 1.3

Group E 43.8 iSS ⁵⁸ 3.6 ± 1.6 ^s5

Group F 35.09 ± 45.5 ' ^χ ' 2.6 ± 1.5

Compared with the sham ^{^{group, Δ Ρ <0.05, ΔΔ Ρ}} <0.01; compared with model group ※ after dead <0.05, ※※ P <0.01 The results show that oral administration, Μ I and Mil near to improve cerebral insufficiency rats Memory impairment and spatial location impairment have significant effects. It is suggested that MI and MΠ may have obvious therapeutic and preventive effects on vascular dementia.

Claims

Rights request

1. Use of butylphthalide homologs 3- (3'-hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide in the preparation of a medicament for the prevention and treatment of diseases caused by cerebral ischemia .

2. The use according to claim 1, wherein the disease caused by cerebral ischemia is selected from the group consisting of neurological symptoms caused by cerebral ischemia, memory disorders caused by cerebral ischemia, cerebral edema caused by cerebral ischemia, and cerebral ischemia. Stroke, impaired energy metabolism due to cerebral ischemia, cerebral infarction caused by cerebral ischemia, and loss of nerve function.

3. Use of butylphthalide homologues 3- (3'-hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide in the preparation of a medicament for improving cerebral blood flow in a cerebral ischemic region.

4. Use of butylphthalide homologues 3- (3'-hydroxy) butylbenzylphthalate and 3-hydroxy-3-butylphthalide in the preparation of antiplatelet aggregation and antithrombotic drugs.

5. Use of butylphthalide homologues 3- (3'-hydroxy) butylphthalide and 3-hydroxy-3-butylphthalide in the preparation of a medicament for the treatment and prevention of dementia.