WO2009100587A1

WO2009100587A1 - A drug composition for treatment and prevention of ischemic stroke and its preparation methods

Info

Publication number: WO2009100587A1
Application number: PCT/CN2008/000349
Authority: WO
Inventors: Huiwan Han; Liang JI; Yanda Li; Mingyu Ding
Original assignee: Tsinghua University
Priority date: 2008-02-14
Filing date: 2008-02-14
Publication date: 2009-08-20
Also published as: CN101677989A; CN101677989B

Abstract

A drug composition for treatment and prevention of ischemic stroke and its preparation methods. The drug composition consists of Sedanolide 19-72%, Ligustilide 17-67%, Gastrodin 2-29% and solvent. It has a high purity of more than 98%. The drug composition can be prepared into different forms of drugs, including injection, and it can effectively alleviate symptoms of the disease.

Description

Medicine composition for preventing and treating ischemic stroke and preparation method thereof

The invention belongs to a traditional Chinese medicine monomer composition, in particular to a pharmaceutical composition for preventing and treating ischemic stroke and a preparation method thereof.

Background technique

Stroke is one of the leading causes of death and disability in middle-aged and elderly people. Nearly 80% of cases of ischemic stroke are caused by cerebral arterial embolism or atherothrombosis. According to clinical symptoms and duration, cerebral ischemia can be divided into transient ischemic attack (TIA) with complete remission of symptoms within 24 hours, reversible ischemic neurological deficit (RIND) with symptoms lasting for 6 weeks, and Symptoms persist in stroke. According to data released by the WHO, 40 of the 57 countries have listed the mortality rate of cerebrovascular disease in the top three causes of death, ranking first in Japan and China. However, the existing drug control effect is not good, so it is urgent to develop a clinical drug with better efficacy.

Medicinal Chuanxiong is the rhizome of Ligusticum chuanxiong Hort, a perennial herb of the family Umbelliferae (English name Rhizoma chuanxiong). The main chemical constituents are volatile oil, alkaloids, phenolic components, lactones, ferulic acid, etc. The chemical component of the effective part of Chuanxiong, which improves cerebral ischemia in vivo, is a Chuanxiong lactone compound. The lactone is cetanolactone (4,5-dihydro-3-butylbenzene peptide, 4, 5-dihydro- 3-butyl-phthalide ) (Formula ( I ) and Artemisactone (4, 5-dihydro-3-butylidene phenyl peptide, 4, 5-dihydro-3-buty 1 i dene-phthal ide ) Formula (11).

(I) (II)

Medicinal Gastrodia (Gastrodia elata Blume, English name Rhizoma Gastrodiae) is a perennial orchid family high-grade fungus plant. Gastrodia contains gastrodin, vanillin, succinic acid, β-sitosterol, vitamin-like substances, terpenoids, Alkaloids, mucilage and gastrodia polysaccharides. Among them, gastrodin and gastrodia elata polysaccharide are the main components. It is now possible to artificially synthesize gastrodin (Tianma) (Zhou Shuzhen, etc., Chinese pharmaceutical industry miscellaneous Zhi, 1985, 05)

Gastrodin (English name is Gastrodin), its chemical name is 4-hydroxybenzene-β-D-glucopyranoside. The molecular formula is: C13H1807. Gastrodia has a variety of pharmacological effects. It can restore the imbalance between the excitatory and inhibitory processes in the cerebral cortex, and has central inhibition such as sedation, sleep and analgesia. The acute toxicity of gastrodin (tianzin) is small.

Since ancient times, there have been groups of Chuanxiong and Tianma used to promote blood circulation and remove blood stasis, such as Dachuan Zhiwan. In addition, there are other traditional Chinese medicines consisting of Chuanxiong and Tianma, such as "Tianzhu Injection", which is a traditional Chinese medicine preparation consisting of Chuanxiong and Tianma. The monitoring index of Chuanxiong is Awei acid (Huang Dongping et al. Chinese Herbal Medicine, 2004, 35 (4), 409-411). "Tianyi Head Ning Capsule" is a prescription of Tianma as a drug, consisting of dozens of traditional Chinese medicines (Shen Yuhua et al. New Drugs and Clinical Pharmacology, 1995, 6 (3), 33-35). The main ingredients of Tianzhu Soft Capsule are Tianma and Rhubarb, and the ratio of Tianma and Rhubarb in the place is 3:1 (Guo Zengjun et al. Chinese patent medicine 2005, 27 (4), 467-468). Most of these prescriptions are mainly for the treatment of migraine, and the active ingredients are not used as indicators in the preparation process.

In addition to the above traditional Chinese medicine preparations, there are also some medicines that use Chuanxiong and Tianma as active ingredients. For example, a patent application "pharmaceutical composition and preparation containing active ingredients of gastrodia elata and chuanxiong" (application number: 200610020605. 2) discloses a pharmaceutical composition comprising gastrodia elata and chuanxiong as active ingredients; patent application "Dachuan dry powder preparation and preparation method" And Application (Application No.: 200610046747. 6) discloses a dry powder preparation prepared by mixing Gastrodia elata extract and Ligusticum chuanxiong extract. The patent application "Chloraria lactone for the treatment of ischemic heart disease and its preparation method" (Application No. 03146029. 1) discloses the endogenous lactone, including the two lactone components of cedar lactone and artemisinide. Patent application "A pharmaceutical composition for treating cerebrovascular diseases and a preparation method thereof" (Application No.: 200510101971. 6) discloses a composition composed of artemisinide and sedative lactone and "a kind of chuanxiong lactone extract and Its preparation method and application (Application No.: 200610130680. 4) discloses the application of ligustilide in the treatment of ischemic cardiovascular disease. Patent "A kind of Chuanxiong volatile oil soft capsule and preparation method thereof" (Application No.: 200410037185. X) discloses a method for preparing volatile oil of Ligusticum chuanxiense by supercritical carbon dioxide extraction method.

The above prior art has the following disadvantages: (1) The purity of the drug component is not high, and it cannot be made into a traditional Chinese medicine preparation with a clear chemical composition, in particular, it cannot be prepared into an injection having a clear chemical composition and a high purity; (2) The traditional Chinese medicine preparations for the prevention and treatment of stroke, which are mainly based on extracts of Chuanxiong, are all based on the chemically stable and stable artemisinone as the monitoring index, which affects the accuracy of the detection.

A pharmaceutical composition consisting of seledanolactone, artemisinide and gastrodin was not found by searching. Summary of the invention One of the objects of the present invention is to provide a pharmaceutical composition for preventing and treating ischemic stroke.

A second object of the present invention is to provide a process for the preparation of the above pharmaceutical composition.

Another object of the present invention is to provide the use of the above pharmaceutical composition for the preparation of a medicament for treating ischemic stroke.

In order to achieve the above object, the technical solution adopted by the present invention is:

A pharmaceutical composition for preventing and treating ischemic stroke, the composition thereof and the weight percentage thereof are: Cetanolactone 19~72%

Artemispermide 17~67%

Gastrodin 2~29%

Solvent balance

The solvent described in the above pharmaceutical composition means sodium carboxymethylcellulose, Tween-80 or vegetable oil and the like. The vegetable oil refers to one or more of vegetable oils such as olive oil, peanut oil, corn oil, soybean oil, sunflower oil, sesame oil, castor oil, almond oil or peach kernel oil.

The dosage form prepared by the above pharmaceutical composition may be a pharmaceutically acceptable dosage form according to a usual method, and may be, for example, an injection, a spray, a capsule, a dropping tablet, a tablet, a granule, an emulsion, a dispersing agent, and a sustained release. Agents, etc.

The use of the above pharmaceutical composition for the preparation of a medicament for treating ischemic stroke.

The above-mentioned pharmaceutical compositions are all pure compounds of serranolol, artemisinide and gastrodin, all of which are more than 98% pure, and gastrodin is commercially available.

The preparation method of the above-mentioned pharmaceutical composition for preventing and treating ischemic stroke, according to the ratio of cetanolactone, artemisinide, gastrodin and cosolvent, and then uniformly mixed.

The preparation method of the above-described seletanolactone comprises the following steps:

(1) pulverizing the dried roots of Chuanxiong to particles having a diameter of l~3 mm, and then placing them in an extraction vessel;

(2) set the temperature knob of the extraction kettle to 42~58 °C, and heat; when the temperature of the extraction kettle is 42~58 ^D C, turn on the C0 ₂ air intake pump;

(3) Extraction and separation, after passing through the extraction vessel, the C0 ₂ gas stream enters the separation reactor I and enters the separation reactor II; the pressure at the extraction reactor reaches 18 to 32 MPa; wherein the pressure of the separation reactor I is 8 to 14 MPa, and the temperature is 45 to 55 ^Q. C, extraction time is 60~120 min, C0 ₂ flow rate is 220~350kg/h; separation kettle II pressure is 4~8Mpa, temperature is 35~45 ⁰ C, extraction time is 60~120 min;

(4) After the first extraction and separation, the same size of the Chuanxiong root granules are again fed into the extraction vessel, and steps (2) to (3) are repeated to carry out the second extraction and separation;

) £正页(fine 91) (5) After the second extraction and separation, the entire contents are discharged from the discharge ports of the separation kettle I and the separation kettle II at a time;

(6) The above two contents were separately placed in a separatory funnel until stratification, and the upper layer liquid was collected after stratification, respectively, to obtain a grade I separation product of chuanxiong lactone (from the separation kettle) and a grade II separation product (from Separation kettle II);

(7) A step-by-step separation product of the Chuanxi lactone of the step (6) or a second-stage separation product of the Chuanxiong lactone or a mixture of the two products is added in an amount of 3 to 7 ml of petroleum ether: ethyl acetate (95: 5). Mix the solution to 8~12ml, shake the hook; inject into Biotage 40+M normal phase liquid chromatography silica gel column; its mobile phase is A petroleum ether, B ethyl acetate; flow rate 50ml/min; detection wavelength 280nm; gradient elution , 0.01~12minA: B= 95: 5, 15~30minA: B = 85: 15; collect the eluent between 18~25min;

(8) Repeat step (7) to sequentially elute the grade I and / or II separation products.

, --,

Responsible

(9) The eluate of step (8) is rotary evaporated in a water bath at 30 to 40 ° C to remove the solvent, thereby obtaining a primary purified product of seletanolactone;

(10) 3~7ml of the primary purification product in the step (9) is added to methanol and dissolved in Shimadzu LC-8A reverse phase preparative liquid phase system; wherein the column is Phenomenex Luna ΙΟμπι, 250mm×50mm ID _{; the} mobile phase is water (A) And methanol (B), flow rate of 80 ml / min, isocratic elution, A: B = 40: 60; spectroscopic wavelength of 280 nm; collecting eluate between 18~26 min;

(11) The eluate obtained in the step (10) is subjected to rotary evaporation in a water bath at 30 to 40 ° C to remove methanol; and the residue is extracted with ethyl acetate for 3 to 5 times to obtain an extract;

(12) The above extract was rotary evaporated in a water bath at 30 to 40 ° C to remove the solvent to obtain a cearyl lactone. The preparation method of the artemisinone comprises the following steps:

Step (1) ~ (6) The same as the preparation method of the synthion lactone (1) ~ (6);

(7) the threonine lactone step (7), collecting the eluent between 6 and 12 min;

(8) The solvent is removed by rotary evaporation in a water bath of 30 to 40 ° C, and the obtained product is artemisinide. The supercritical carbon dioxide extraction tank described in the above preparation method step (1) may be

HA221-40-48 or HA421- 40- 963. 13-3.

The gastrodin can be purchased directly from the market, such as gastrodin or acetyl gastrodin produced by Kunming Pharmaceutical Group Co., Ltd.

The active constituents extracted and purified by the method of the present invention have a purity of 98% or more. The commercially available gastrodin (or acetyl gastrodin) has a purity of 98% or more. Correction page (fine ⁴ , rule 91) The invention has the advantages and beneficial effects: (1) high drug efficacy, can effectively alleviate the pathological changes and behavioral disorders of ischemic stroke, and the drug effect is obviously better than the existing clinical drugs; (2) the pharmaceutical composition of the invention has exact efficacy , the dosage is small, the action is strong; (3) the purity is high, the components of the pharmaceutical composition of the invention are all pure, the purity is above 98%, and the preparation can be prepared including the injection; (4) the stability is good, The process of the invention reaches the level of the preparation standard; (5) The invention indicates that the chemical property of the cyanuric acid lactone is more stable than that of the artemisinol, and is more suitable as a monitoring index for the quality of the Chuanxiong oil product.

DRAWINGS

Figure 1 is a preparative spectrum of the artemispermide and the selenate lactone product isolated from the supercritical extract (forward HPLC method) (where QHNP-1 is artemisinide; QHNP-2 is in the seletanic acid) Ester)

Figure 2 shows the detection spectrum of artemisinide (reverse HPLC detection)

Figure 3 shows the preparative spectrum of the primary purified product of seledanolactone (reverse HPLC method)

Figure 4 shows the detection spectrum of the final purified product of the seledanolactone (reverse HPLC detection). Figure 5 shows the infrared identification spectrum of the seledanolide.

Figure 6 shows the infrared identification spectrum of artemisinide

Figure 7 shows the UV identification spectrum of the seletanide.

Figure 8 shows the UV identification spectrum of artemisinide

Figure 9 shows the mass spectrometric spectrum of the seletanide

Figure 10 shows the mass spectrometric spectrum of artemis lactone

Figure 11 shows the nuclear magnetic resonance spectrum of threonolactone (hydrogen spectrum)

Figure 12 shows the nuclear magnetic resonance spectrum of artemisinide (hydrogen spectrum)

Figure 13 shows the nuclear magnetic resonance spectrum of threonolactone (carbon spectrum)

Figure 14 shows the nuclear magnetic resonance spectrum of artemisinide (carbon spectrum)

detailed description

Example 1

Preparation of seletanolactone

(1) Weigh 30kg of the dried root block of Ligusticum chuanxiong and pulverize it to a diameter of 2.5 to be placed in the HA221-40-48 extraction kettle;

(2) Rotate the extraction kettle temperature setting knob to 50° (:, when the temperature reaches 50 ^Q C, turn on the C0 ₂ intake pump;

(3) Start the equipment for the first extraction and separation; C0 ₂ flow through the extraction tank and enter the points

article) From the kettle I, and then into the separation kettle II; the pressure of the extraction kettle is 25Mpa, the pressure of the separation kettle I is llMpa, the temperature of the separation kettle I is 50 ° C, the extraction time is 90 min, the flow rate of C0 ₂ is 250 kg / h, the pressure of the separation kettle II is 6 Mpa, the separation kettle II Temperature 40 ° C;

(4) Re-feed 30kg, carry out the second extraction and separation according to steps (1) ~ (3);

(5) releasing the entire contents from the discharge ports of the separation reactor I and the separation reactor II at a time;

(6) The above two contents were separately placed in a separatory funnel until stratification. After the stratification, the upper liquid was collected to obtain a first-stage separation product of Ligustilide (from Separation Tank I) and a second-stage separation product (from Separation Tank 11).

(7) Aspirating 5 ml of the Chuanxi lactone Grade II product in the step (6), adding a mixed solution of petroleum ether and ethyl acetate (petroleum ether: ethyl acetate: 95:5) to 10 ml, and shaking; Biotage 40+M normal phase liquid chromatography silica gel column; its mobile phase is A petroleum ether, B ethyl acetate; flow rate 50ml/min; detection wavelength 280nm; gradient elution, 0.01~12min A: B= 95: 5, 15 ~30min A: B =85

: 15; Collect the eluent between 19.6 and 23.2 min (see Figure 1QH P-2);

(8) repeating step (7), the injection of the Chuanxi lactone class II separation product is completed, and an eluent is obtained;

(9) The elastate collected in the step (8) is subjected to rotary evaporation in a water bath at 35 ° C to remove the solvent, thereby obtaining a primary purified product of the seletanolactone;

(10) Aspirate 5ml of methanol from the product of step (9) and dissolve it into Shimadzu LC-8A reverse phase preparative liquid phase system; the column is Phenomenex Luna ΙΟμηι, 250mmx50mm ID, mobile phase is A water B methanol, flow rate 80ml/min, isocratic elution, A: B = 40: 60, detection wavelength is 280nm; collect eluent between 20.3~24.5min (see Figure 3);

(11) The eluate obtained in the step (10) is subjected to rotary evaporation in a water bath at 35 ° C to remove methanol, and the residue is extracted with ethyl acetate three times to obtain an extract;

(12) The extract obtained in the step (11) was rotary evaporated in a 35 hr water bath to remove the solvent to obtain a final product of 347.3 g.

(13) The final product obtained was tested according to the test method described in Appendix VI D "High Performance Liquid Chromatography" of the Chinese Pharmacopoeia, 2005, and its purity was 99.17% (see Figure 4). Infrared, ultraviolet, mass spectrometry, and nuclear magnetic resonance (including hydrogen and carbon spectra). The four major spectroscopy certifications indicate that the product is cedar lactone (see Figure 5, Figure 7, Figure 9, Figure 11 and Figure). 13).

Example 2

Preparation of seletanolactone

(1) Weigh 30kg of the dried root block of Ligusticum chuanxiong. In the extraction tank of HA221-40-48;

(2) Rotate the extraction kettle temperature setting knob to 58 ° C, when the temperature reaches 58 ° C, turn on the C0 ₂ air intake pump;

(3) Start the equipment, carry out the first extraction and separation, and the C0 ₂ gas stream enters the separation tank I through the extraction tank, and then enters the separation tank II. The pressure of the extraction tank is 32Mpa, the pressure of the separation tank I is 14Mpa, and the temperature of the separation tank I is 55°C. , extraction time 120min, C0 ₂ flow 280kg / h; separation kettle II pressure 8Mpa, separation kettle II temperature 45 ° C;

(4) Re-feeding Chuanxiong Granules 30kg in HA221- 40-48 extraction kettle, repeat steps (2)~(3) for the second extraction and separation;

(6) The above two contents were separately placed in a separatory funnel until stratification; after stratification, the upper liquid was collected to obtain a grade I separation product of Ligustilide (from Separation Tank I) and a Grade II separation product (from Separation kettle II);

(7) thoroughly mixing the above two kinds of ligustilide products;

(8) Aspirate 5 ml of a mixed solution of petroleum ether and ethyl acetate (petroleum ether: ethyl acetate: 95:5) to 10 ml from the product of step (7), shake well; and inject into Biotage 40+M normal phase Liquid chromatography silica gel column; its mobile phase is petroleum ether (A) and ethyl acetate (B); flow rate 50 ml / min _; detection wavelength 280 nm; gradient elution, from 0.01 to 12 min A: B = 95: 5, 15~ 30 min A: B = 85: 15; The eluate was collected between 18 and 25 min;

(9) The collected eluate is subjected to rotary evaporation in a water bath at 30 ° C to remove the solvent to obtain a primary purified product of the seledanolactone;

(10) Aspirate 5 ml of methanol from the product of step (9) and dissolve it into Shimadzu LC-8A reverse phase preparative liquid phase system; the column is Phenomenex Luna ΙΟμηι, 250 mm 50 mm ID, and the mobile phase is A water B methanol. The flow rate was 80 ml/min, isocratic elution, A: B = 40: 60, the detection wavelength was 280 nm; the eluate was collected between 18 and 26 min;

(11) removing methanol by rotary evaporation in a 30 ° C water bath, and then extracting the residue with ethyl acetate for 5 times to obtain an extract;

(12) The extract was rotary evaporated in a water bath at 30 ° C to remove the solvent to give cerdactone 309 g. Example 3

The preparation of artemisinone comprises the following steps - (1) Weighing 30 kg of dried roots of Chuanxiong, pulverizing to particles of diameter 3, placed In the extraction tank of HA221-40-48;

(2) The temperature of the extraction kettle is 42 ° C. When the temperature reaches the set value, the C0 ₂ intake pump is turned on;

(3) The pressure of the extraction kettle is 18Mpa, the pressure of the separation kettle I is 8Mpa, the temperature of the separation kettle I is 45°C, the extraction time is 60min, the flow rate of C02 is 220kg/h, the pressure of the separation kettle II is 5Mpa, and the temperature of the separation kettle II is 40°C. Secondary extraction and separation;

Step (4) ~ (7) Same as step (4) of Example 2 ~ (7);

(8) Aspirate 5 ml from the product of step (7) onto a Biotage 40+M normal phase liquid chromatography silica gel column; chromatographic conditions are the same as in Example 2; the eluate is collected between 7 and 10.6 min to obtain a halo product. 498.2g (see QH P-1 in Figure 1).

(9) The final product obtained was tested according to the detection method described in the Chinese Pharmacopoeia 2005 Edition, Appendix VI D "High Performance Liquid Chromatography", and the purity was 98.32%. Infrared, ultraviolet, mass spectrometry and nuclear magnetic resonance (including hydrogen and carbon spectra) four spectroscopy certifications that the product is ligustilide (see Figure 6, Figure 8, Figure 10, Figure 12 and Figure 14) ).

Example 4 '

The cetyl lactones prepared in Examples 1 to 3 (specific gravity: 0.95, the same hereinafter) were collected in an amount of 4.2 g, ligustilide 1. Og (specific gravity 0.95, the same hereinafter), and gastrodin 0.6 g, To 20 g of pure olive oil, ultrasonically mixed uniformly to obtain a composition A 25.8 _{g of the} present invention.

Example 5 ' 0.6 g of cearyl lactone, 1.8 g of ligustilide, and 0.75 g of gastrodin were respectively added to 20 g of soybean oil, and ultrasonically mixed uniformly to obtain 23.15 g of the composition B of the present invention.

Example 6

Sesame lactone 3.6 g, ligustilide 3.3 g, and gastrodin 2.9 g, respectively, were added to pure peanut oil to 20 ml, and ultrasonically mixed uniformly to obtain a composition C 29.8 g of the present invention.

Example 7 - 2.8 g of cearyl lactone, 1.2 g of artemisinide, 0.08 g of gastrodin, respectively, and 0.5% sodium carboxymethylcellulose to 21.6 ml were added to obtain the composition D 25.68 of the present invention. g. Taking propanol lactone 1.4g, ligustilide 3.0g, and gastrodin 0. l _g , added to 2% sodium carboxymethylcellulose to 20ml, and ultrasonically mixed uniformly, the composition E 25.5g of the present invention was obtained. . Determined by high performance liquid chromatography (see Appendix VI of the Chinese Pharmacopoeia 2005 edition).

Chromatographic conditions and system suitability test: Shimadzu LC-2010 high performance liquid chromatography column was selected. Using octadecylsilicone bonded silica as a filler (Diamonsil C18, 4.6x150mm, 5 um) _; acetonitrile monohydrate (42: 58) as mobile phase; column temperature 35 ° C; detection wavelength 280 nm, 230 nm _; flow rate Lml/min. The number of theoretical plates is not less than 16,000 based on the peak of the seletanide.

Preparation of standard solution: The cetanolactone prepared in Example 1 was accurately weighed, and methanol was added to make a solution containing 0.08 mg of seletanolactone per 1 ml, respectively, to obtain a standard having a purity greater than 99%. Solution.

Preparation of the test solution: Take the appropriate amount of the cetanolactone obtained in Example 1, accurately weighed, dissolved in methanol and quantitatively diluted to prepare a solution containing 0.08 mg per 1 ml, as a testosterone lactone for the test sample. Solution.

The measurement method is as follows: 10 ul of each of the standard sample of the seletanolactone and the test solution is accurately extracted, and injected into a liquid chromatograph, and the method can be determined according to the method described in the step (13) of the first embodiment.

Table 1 Results of stability test of seletanolactone (% normalized purity, 280 nm)

Results (See Table 1) The stability of the curtanolactone of the present invention is mainly affected by high temperature, light and oxygen. Under the storage conditions of nitrogen and darkness for 30 days, no matter whether it is room temperature, refrigerated or frozen, there is basically no effect on the stability of the sirolimus (the relative standard deviation of the content is <2%). It shows that the properties of seletanol are relatively stable. As long as the nitrogen is stored in the dark and protected from light, the purity will not decrease within 30 days and can be used as a standard.

Example 10

Ligustilide stability test

It was determined by high performance liquid chromatography as shown in Appendix VI D of the 2005 edition of the Chinese Pharmacopoeia. Chromatographic conditions and system suitability test: Shimadzu LC-2010 high performance liquid chromatography column was selected. Using octadecylsilane bonded silica as a filler (Diamonsil C18, 4.6 <150mm, 5 nm); Water (42: 58) was the mobile phase; column temperature was 35 ° C; detection wavelength was 280 nm, 230 nm _; flow rate was 1 ml/min. Preparation of the standard solution: The ligustilide prepared in Example 3 was accurately weighed, and a solution of 0.4 mg of ligustilide per 1 ml of methanol was added to obtain a standard solution having a purity of more than 99%.

Preparation of test solution: The appropriate amount of ligustilide obtained in Example 3 was accurately weighed, dissolved in methanol and quantitatively diluted to prepare a solution containing 0.4 mg per 1 ml, which was used as a sample solution for ligustilide.

Measurement method: Each of 10 ul of the standard sample and the test solution was aspirated, and injected into a liquid chromatograph to carry out the measurement according to the method described in the step (9) of Example 3.

Results (see Table 2). The stability of the ligustilide of the present invention is mainly affected by light, temperature and oxidation; the purity falls from 98% to less than 95% after being placed at room temperature for 10 days. It is indicated that the ligustilide standard must be stored under nitrogen, light, refrigerated or frozen conditions. Must be freshly prepared. The amount of preparation per time does not exceed 5 days.

Table 2 Experimental results of the stability of ligustilide (% of normalized purity, 280 nm)

Example 11

Effect of pharmaceutical composition of the invention on cerebral ischemia model of suture method

(1) The test method is carried out as follows:

(1). Grouping and administration: 150 healthy male SPF/VAF grade Wistar rats weighing 240 to 250 g were used for the test. (Provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., Certificate No.: SCXK Jing 2002-0003). They were randomly divided into 9 groups, namely, blank control group, model control group, Mailuoning positive control group 6 ml/kg, and composition A, B, and C two doses (6 mg/kg and 12/kg). For the preparation method of the compositions A, B, and C, see Examples 4 to 6. A composition or saline or positive drug was intraperitoneally injected 1 hour before surgery and 2 hours after reperfusion. The positive drug was selected from the traditional western medicine Mailuoning injection (lOrnl/branch, Jinling Pharmaceutical Co., Ltd. Nanjing Jinling Pharmaceutical Factory, batch number: 200503011) for clinical treatment of ischemic stroke. The blank control group and the model control group were intraperitoneally injected with an equal volume of normal saline.

(2). The preparation of the wire plug: a nylon wire having a diameter of 0.23 mm made in Japan is formed into a straight line and cut into a 5 cm line segment, the front end is smoothed with a fine sandpaper, and the rubber is smoothed, at a distance of 1. 6 cin from the front end. 1. 8cm, 2. 0cm for labeling and spare, the helixin is taken from the front end of the nylon wire.

(3). Model replication: 400mg/kg intraperitoneal injection of 10% chloral hydrate anesthesia; neck median incision, exposure of the left common carotid artery (referred to as CCA), external carotid artery (ECA), internal carotid artery (referred to as ICA Ligation of the proximal end of the CCA, ECA root; at the end of the CCA ligation, cut a slant, insert a nylon thread, through the CCA bifurcation through the ICA into the cerebral artery (ACA), block the blood flow of MCA; The average insertion depth of the silk is 18. 5 ± 0. 5 legs, ligation of ICA. The skin was sutured and the ends of the nylon filaments were exposed. After 2 hours of ischemia, the nylon filaments were pulled out to observe changes in different time courses after reperfusion; the sham operation group was not inserted with nylon filaments.

(II) Observation indicators:

(1). Neurobehavioral score: A five-point scale (0-4 points) was used. Behavioral observations of animals were performed at the end of reperfusion. The rat tail was about 1 foot away from the ground, and the condition of the two forelimbs was observed. The rats were placed on the horizontal ground, and their shoulders were pushed to observe whether there was any difference in resistance between the two sides; the rats were placed on the ground to observe the walking. The higher the score, the more severe the neurobehavioral damage. (by the highest score)

a. The behavior is completely normal for 0 points;

b. Lift the rat tail off the ground, and the contralateral forelimb of the operation will be rotated and the recipient will be 1 point;

c. Rats to the ground, squeeze the sides to check their resistance, and the contralateral resistance of the surgery is reduced, 2 points;

d. Rats to the ground, observe their walking, and circle around the opposite side of the operation, 3 points;

e. The damage is extremely serious, and those who are unable to move independently are 4 points.

(2). Cerebral infarction area test: After 24 hours, the brain was decapitated, and the left brain was cut into 2 slices and a total of 5 slices and 4 mm thick. It was stained with 4% red tetrazolium (TTC) at 37 ° C for 30 minutes in the dark; it was flipped every 5 minutes. After TTC staining, the normal tissue was stained red and the infarcted tissue was white. Each group of brain slices is arranged neatly and photographed and saved. Processing and statistics were performed using image analysis system software (SPOT 3.5 software). The infarct size of each piece was calculated and finally superimposed into the infarct volume. The infarct volume is expressed as a percentage of the cerebral hemisphere. Calculated as follows:

Cerebral infarction volume (%) = (the volume of the contralateral hemisphere of the operation - the uninfarcted part of the surgical side hemisphere Volume) I volume of the contralateral hemisphere X 100%

(3). Determination of brain water content:

After 24 hours of reperfusion, the brain was immediately decapitated, the olfactory bundle, cerebellum and lower brain stem were removed, and the surgical side and the normal side hemisphere were separated and weighed separately (wet weight). After being baked in the oven at 95 ° C for 24 hours to constant weight, weigh the brain again (dry weight). Brain water content is calculated according to the following formula:

Brain water content (%) = (wet weight to dry weight) / wet weight X 100 %

The experimental data were expressed as ± SZ), and the differences between the groups were compared by t test.

Table 3. Results of the therapeutic test of the medicinal composition of the present invention on the model of cerebral ischemia

Note: Compared with the blank control group ΔΔΡ<0. 01 ΔΔΡ<0. 05;

Compared with the model control group **Ρ<0· 01 *Ρ<0. 05;

Results (See Table 3) The three groups of pharmaceutical compositions A and B, and the positive drug group of the present invention all improved the area of focal cerebral ischemia and infarction in the middle cerebral artery of rats. Among them, the large dose of composition B (12mg/kg body weight) is the best, and the drug effect is better than the commonly used clinical drug Mailuoning injection. It is indicated that the pharmaceutical composition of the present invention can be used for treating ischemic stroke and reducing the area of cerebral infarction.

Example 12

(1) The test is carried out as follows:

(1) Grouping and administration: 78 healthy male SD rats of 20 0 to 220 _g were randomly divided into 6 groups, which were a normal control group, a model group, a positive control drug ligustrazine group, and Examples 4 to 6 of the present invention. Group Compositions A, B, and C. The positive control drug ligustrazine group is selected from the group consisting of ligustrazine phosphate tablets, 50 mg/tablet, 100 tablets/bottle, produced by Beijing Yanjing Pharmaceutical Factory, batch number: 040603. Pre-administration for 7 days, once a day, each dose of 50 mg / kg. The decapitation was taken 2 hours before the brain. The normal control group and the model group were given the same amount of normal saline.

(2) Experimental method: 4% chloral hydrate (350 mg/kg, ip) was anesthetized. The rat was fixed in the supine position, and the medial incision was made along the neck. The right common carotid artery was separated and two sutures were used. The external carotid artery was separated and the proximal end was ligated. The internal carotid artery and a branch (winged iliac artery) are separated under the external carotid artery. A suture is placed under the branch and ligated at the proximal end bifurcation. At the proximal end of the isolated common carotid artery, the suture is ligated, and the distal end is blocked by an arterial clamp. During this time, a small opening is made and one end is heated into a bead-shaped (diameter <0.3 mm) nylon. Insert the line (4-10) into the small mouth, remove the arterial clip, push the nylon thread slowly into the forebrain artery (about 20mm), and then pull back about 2mm to the middle cerebral artery, about 17mm long (from the internal and external carotid artery) The bifurcation is used to fix the nylon bar with a suture. Stitch muscles and skin. The field of view temperature was maintained at 37 °C during the procedure. The behavioral score was scored 24 hours after the operation. After the score, the rats were sacrificed and the brain was decapitated. The control group was not inserted, and the behavioral score was sacrificed 24 hours after the operation, and the brain was decapitated. The following operations and calculation methods are equivalent to the relevant parts of Embodiment 11.

(II) Observation indicators: The same as the example 11 observation indicators (2)

Results (see Table 4) The oral administration of the three groups of pharmaceutical compositions A, B and C of the present invention and the positive drug group can improve the area of focal cerebral ischemia infarction of the middle cerebral artery in rats for 7 days; The pharmacological effects of the three groups of B, C and C were significantly better than those of the commonly used clinical drug, Ligustrazine. It is indicated that the pharmaceutical composition of the invention can prevent and treat ischemic stroke and reduce the area of cerebral infarction. The drug effect was significantly better than that of the commonly used clinical drug, Ligustic Acid Pyridazine (P<0.01).

Table 4 Results of the model test of the medicinal composition of the present invention against the cerebral ischemia model

Note: *Compared with the model control group P<0.05, ** compared with the model control group P<0.01. Example 13

Effect of pharmaceutical composition of the invention on cerebral ischemia model of ferric chloride

(1) The experiment is carried out as follows:

(1) Grouping and administration: 110 healthy male SD rats weighing 180 to 200 g were randomly divided into 9 groups according to body weight. The blank control group, the model control group, the ligustrazine phosphate 120 mg/ml group (the drug source was the same as in Example 12), and the components of each composition were 80 mg/ml and 40 mg/ml in two dose groups, 12 in each group. Oral administration once a day for 30 consecutive days, the blank group and the model control group were given an equal volume of edible oil.

(2) Experimental method: One hour after the last administration, the rats were anesthetized by intraperitoneal injection of 12% chloral hydrate solution at 350 mg/ml, fixed in the lateral position, and operated under a surgical microscope. A vertical incision of about 1 cm was made at the midpoint of the eyelid and the external auditory canal. The diaphragm was exposed and peeled off. A small hole with a diameter of about 2 mm was drilled with a dental drill at the upper front of the foramen oval hole, and the needle was pierced with a fine needle. The meninges, exposed to the middle cerebral artery (MCA), were applied to the middle cerebral artery with a filter paper containing 50% ferric chloride solution. After 30 minutes, the middle cerebral artery became black or white. Suture the incision. After 24 hours, the brain was decapitated. The following operations and calculation methods are described in the relevant part of the embodiment 11.

Neurobehavioral tests were performed 6 h and 24 h after surgery, respectively. The scoring criteria and methods are the same as those in Example 11 (1).

(II) Observation indicators: The same as the example 11 observation indicators (1), (2)

Results (See Table 5) The oral administration of the oral and positive drug groups of each group was continuously prevented for 30 days. Compared with the model group, the area of focal cerebral ischemic infarction in the middle cerebral artery of rats was significantly improved. Among them, the composition A of the present invention was significantly better than the commonly used clinical drug lithium phosphinizine tablets (P < 0.05). Since the same dose of the positive drug group could not reflect the efficacy, it was necessary to increase the dose. On the other hand, 1/3 to 2/3 of the dose of the composition of the present invention can exhibit similar effects. It is indicated that the long-term administration of the oral composition of the pharmaceutical composition of the invention can prevent and treat ischemic stroke and reduce the area of cerebral infarction. The drug efficacy is better or not lower than the commonly used clinical drug lithium tetramethylpyrazine tablets.

Table 5. Test results of cerebral ischemia model test for 30 days of prophylactic administration of the pharmaceutical composition of the present invention

Blank control 11 1 0.73 ± 0.65 0.45 ± 0.52 0 Model control 10 1 5.00 ± 1.15 Δ 3.80 ± 1.40 Δ Δ 5.17 ± 2.36 Δ Δ Ligustrazine positive drug group 10 120 3.58 ± 1.44 * 3 · 08 ± 1.56 4.41 ± 0.72 Composition A 10 80 3.58 ±1.44* 3.67±1.50 3·83±0·91 Composition A 9 40 4.00±1.58 3.67±1.80 3.23±0.85* Composition D 11 80 3.36±1.57* 3.55±1.92 4.98±1.15 Composition D 11 40 3.92±1.24* 3.83±1.53 3.92±1.14 Composition E 10 80 3.70±1.34* 3.30±1.16 3.99±2.06 Composition E 9 40 4.33±1.61 3.83±1.27 4.34±0.85

Note: Compared with the blank control group, ΔΔ Ρ<0.01

Compared with the model control group * Ρ<0.05

Example 14

Acute toxicity test of mice of the pharmaceutical composition of the present invention

(1) Experimental and calculation methods:

The SPF Kunming mice were used for oral administration once. The pretest result is the test dose, and the agent distance is 1:0.90.

The preparation method of the drug solution is as follows: Accurately weigh 2.601 _{g of} seletanolactone, 2.502 _{g of} artemispermide, and 0.7502 _{g of} gastrodin, mix well and add appropriate amount of 2% sodium carboxymethylcellulose to dissolve the solution. A solution having a concentration of 0.0575 g/g was prepared; each dose was diluted with an aqueous solution of 2% sodium carboxymethylcellulose as shown in Table 7, and 0.8 ml was orally administered per 20 g of body weight; continuous observation was carried out for 48 hours.

The animals were randomly divided into 9 groups according to the body weight after fasting for 16 hours, 20 rats in each group, half male and half female; the distance between the groups was 1:0.9; the above-mentioned different concentrations of the test substances were administered by 0.8ml/20g. The toxic reaction of each group of animals after administration of the test substance was immediately observed for 48 hours. Record the animal's toxicity and death. Oral LD ₅ was calculated in mice using the Bilss method. Dosage and 95% confidence limits.

The calculation formula is as follows: Number of draws:

Half lethal dose: LD ₅ . =antl. g[X +丄(5-?) ] l. gLD ₅ . Standard error: S. =

LD ₅ . 95% average confidence limit: L ₉₅ =LD _5Q ±4.5 LD ₅₀ - J

b ~∑M) LD ₅ . 99% average confidence limit: L ₉₉ =LD ₅ . ±5.9 LD ₅₀ · "

B^n∑w

The results (see Table 6) show oral administration of LD _{5 to} mice of the pharmaceutical composition of the present invention. The value is 0.7276 _g /k _g; LD ₅ . The 95% average confidence limit is 0, 7276 ± 0.0470 _g /k _g (range: 0.6806-0.7746 g/kg); LD ₅ . The 99% average confidence limit is 0.7276 ± 0.0616 g / kg (range: 0.6660-0 · 7892 g / kg). The inter-group distance was 1:0.9.

This test is an acute toxicity test for chemical grade pure products. The results indicate that the pharmaceutical compositions of the present invention are safe and provide valuable reference data for clinical dosages.

Table 6 Mice oral LD ₅ . Dose and 95% confidence limit experimental results

i=log (1/0.9) =0.0458

b = 12.3675

LD ₅₀ =0.6326g · kg" ¹

L ₉₅ =0.6326±0.0274g · kg- ¹ (range: 0.6052-0.6600 g · kg- ¹ )

L ₉₉ =0.6326±0.0359g · kg- ¹ (range: 0.5967-0.6685g - kg" ¹ )

S3⁄4 ₀ = 0.0096

Claims

Claim

A pharmaceutical composition for preventing and treating ischemic stroke, the composition thereof and the weight percentage thereof being: cetanolactone 19 to 72%

Artemispermide 17~67%

Gastrodin 2~29%

Solvent balance

The pharmaceutical composition according to Claim 1, characterized in that the solvent means sodium carboxymethylcellulose, Tween-80 or vegetable oil or the like.

3. A pharmaceutical composition according to claim 2, characterized in that the vegetable oil means vegetable oil such as olive oil, 'peanut oil, corn oil, soybean oil, sunflower oil, sesame oil, castor oil, almond oil or peach kernel oil. One or several of them.

The pharmaceutical composition according to Claim 1 or 2, which is in the form of an injection, a spray, a capsule, a dropping tablet, a tablet, a granule, an emulsion, a dispersing agent, a sustained release agent and the like.

The use of the pharmaceutical composition according to any one of claims 1 to 4 for the preparation of a medicament for treating ischemic stroke.

6. The method for preparing a pharmaceutical composition according to claim 1, wherein the ratio of seletanol, artemisinide, gastrodin and a solvent is taken in proportion, and then uniformly mixed.

7. The preparation method according to claim 6, wherein the preparation method of the cefonic acid lactone comprises the following steps:

(1) Crush the dried roots of Chuanxiong to the diameter:! ~ 3 painted particles, then placed in the extraction kettle;

(2) Set the temperature knob of the extraction kettle to 42~58 ^Q C, and heat; when the temperature of the extraction kettle is 42~58 ^fl C, turn on the C0 ₂ intake pump;

(3) Extraction and separation, after passing through the extraction vessel, the C0 ₂ gas stream enters the separation reactor I, and then enters the separation kettle II; the pressure at the extraction kettle reaches 18 to 32 MPa; wherein the pressure of the separation reactor I is 8 to 14 MPa, and the temperature is 45 to 55 ^D. C, extraction time is 60~120 min, C0 ₂ flow rate is 220~350kg/h; separation kettle pressure is 4~8Mpa, temperature is 35~45°C, extraction time is 60~: 120 min _;

(5) After the second extraction and separation, the entire contents are discharged from the discharge ports of Separation Tank I and Separation Tank II at a time; Correction Page (Article 91) (6) The above two contents were separately placed in a separatory funnel until layering, and the upper layer liquid was collected after layering to obtain a grade I separation product of Ligusticol (from the separation kettle I) and a separation product of the second grade (from Separation kettle II);

(7) A step-by-step separation product of the Chuanxi lactone of the step (6) or a second-stage separation product of the Chuanxiong lactone or a mixture of the two products 3 to 7 ml of petroleum ether: ethyl acetate (95: 5) The solution was shaken to 8~12ml, and shaken; the sample was injected into Biotage 40+M normal phase liquid chromatography silica gel column; the mobile phase was A petroleum ether, B ethyl acetate; the flow rate was 50ml/min; the detection wavelength was 280nm; gradient elution, 0.01~12minA: B= 95: 5, 15~30minA: B = 85: 15; collect the eluent between 18~25min;

, ―, ―

y instrument;

(10) 3~7ml of the primary purification product in the step (9) is added to methanol and dissolved in Shimadzu LC-8A reverse phase preparative liquid phase system; wherein the column is Phenomenex Luna ΙΟμηι, 250mm×50mm ID _{; the} mobile phase is water (A) And methanol (B), flow rate of 80 ml / min, isocratic elution, A: B = 40: 60; detection wavelength is 280 nm _; collect eluate between 18~26 min;

(12) The above extract was rotary evaporated in a water bath at 30 to 40 ° C to remove the solvent to obtain a cearyl lactone. 8. The preparation method according to claim 6, wherein the preparation method of the artemisinide comprises the following steps:

(2) set the temperature knob of the extraction kettle to 42~58 ° C, and heat; when the temperature of the extraction kettle is 42 to 58 ° C, turn on (0 ₂ intake pump;

(3) Extraction and separation, after passing through the extraction vessel, the C0 ₂ gas stream enters the separation reactor I, and then enters the separation reactor II; the pressure at the extraction reactor reaches 18 to 32 MPa; wherein the pressure of the separation reactor I is 8 to 14 M _P a, and the temperature is 45 〜 55 ° C, extraction time is 60~120 min, C0 ₂ flow rate is 220~350kg / h; separation kettle II pressure is 4~8Mpa, temperature is 35~45 ° C, extraction time is 60~120 min;

18

Correction page (rules ⁹ 1) (5) After the second extraction and separation, the entire contents are discharged from the discharge ports of the separation kettle I and the separation kettle respectively;

(6) The above two contents were separately placed in a separatory funnel until layering, and the upper layer liquid was collected after layering to obtain a grade I separation product of Ligusticol (from the separation kettle I) and a separation product of the second grade (from Separation kettle II);

(7) A step-by-step separation product of the Chuanxi lactone of the step (6) or a grade II separation product of the Chuanxi lactone or a mixture of the two products is added to petroleum ether: ethyl acetate (95: 5) Mix the solution to 8~12ml, shake well; inject into Biot _a ge 40+M normal phase liquid chromatography silica gel column; its mobile phase is A petroleum ether, B ethyl acetate; flow rate 50ml/min; detection wavelength 280nm; gradient Elution, 0.01~12minA: B= 95: 5, 15~30minA: B = 85: 15; Collect the eluent between 6~12min;

(8) The solvent is removed by rotary evaporation in a water bath of 30 to 40 ° C, and the obtained product is artemisinide.