CN101677989A - A drug composition for treatment and prevention of ischemic stroke and its preparation methods - Google Patents

A drug composition for treatment and prevention of ischemic stroke and its preparation methods Download PDF

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Publication number
CN101677989A
CN101677989A CN200880001478A CN200880001478A CN101677989A CN 101677989 A CN101677989 A CN 101677989A CN 200880001478 A CN200880001478 A CN 200880001478A CN 200880001478 A CN200880001478 A CN 200880001478A CN 101677989 A CN101677989 A CN 101677989A
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ligustilide
separation
separation reactor
sedanolide
extraction kettle
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CN101677989B (en
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韩慧婉
季梁
李衍达
丁明玉
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Tsinghua University
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Tsinghua University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/236—Ligusticum (licorice-root)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/898—Orchidaceae (Orchid family)
    • A61K36/8988—Gastrodia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin

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. Ithas 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

A drug composition for treatment and prevention of ischemic stroke and its preparation methods
A kind of pharmaceutical composition for preventing and treating cerebral arterial thrombosis and preparation method thereof technical field
The invention belongs to a kind of traditional Chinese medicine monomer composition, a kind of pharmaceutical composition for preventing and treating cerebral arterial thrombosis and preparation method thereof is related in particular to.
Background technology
Cerebral apoplexy be in, the elderly is lethal and the one of the main reasons that disables.Caused by nearly 80% cerebral arterial thrombosis case is due to cerebral embolism or atherosis thrombosis.Cerebral ischemia can be divided into according to clinical symptoms and duration by the transient ischemic attack of symptom complete incidence graph in 24 hours（TIA), symptoms last up at 6 weeks just alleviate reversible ischemic neurologic afunction（) and symptomatic persistent palsy RIND.The data announced according to WHO, has 40 countries the death rate of cerebrovascular disease to be included in the front three of the cause of death in 57 countries, and first place is then accounted in Japan and China.But existing medical treatment effect is bad, therefore it is badly in need of the more preferable clinical application of development efficacy.
Medicinal Ligusticum wallichii is Umbelliferae herbaceos perennial Ligusticum wallichii（Ligusticum chuanxiong Hort) rhizome（The main chemical compositions of the entitled Rhizoma chuanxiong Ligusticum wallichiis of English are volatile oil, alkaloid, phenolic constituent, lactone, forulic acid etc..The chemical composition for wherein entering in vivo and improving the extract of Ligusticum chuanxiong Hort of cerebral ischemia is chuanxiong lactone kind compound.Lactone therein is sedanolide（4,5 dihydro-3- butyl benzene peptides, 4,5-dihydro-3-butyl-phthalide) (logical formula (I) and ligustilide (4,5 dihydro-3-butane benzene peptides, 4,5-dihydro-3-buty 1 i dene-phthal ide) (formula（11).
(I) (II)
Medicinal rhizoma Gastrodiae (Gastrodia elata Blume, the entitled Rhizoma Gastrodiae of English) it is the fungivorous plants such as perennial orchid family height, rhizoma Gastrodiae includes Gastrodin, vanillin, butanedioic acid, β-paddy alcohol, vitamin Α samples material, glucoside, alkaloid, mucilaginous substance and gastrodia elata polysaccharide etc..Wherein Gastrodin and gastrodia elata polysaccharide are its main components.Can artificial synthetic gastrodin（Gastrodin）（Zhou Shushun etc., Chinese Medicine industry is miscellaneous Will, 05 phase in 1985)
Gastrodin（English name is Gastrodin), its chemical name is 4- hydroxy benzenes-β-D glucopyranosides.Molecular formula is： C13H1807.Rhizoma Gastrodiae has many pharmacological actions.The excited dysequilibrium between process of inhibition of cerebral cortex can be recovered, with central inhibitory actions such as calm, sleeping and analgesias.Gastrodin（Gastrodine）Acute toxicity very little.
Just there is the prescription for the promoting blood circulation and removing blood stasis, Ligusticum wallichii of ceases wind analgesic and rhizoma Gastrodiae in China from ancient times, such as DCXW.In addition, there are the other Chinese medicinal formulaes being made up of Ligusticum wallichii and rhizoma Gastrodiae, such as " Tianxiong injection ", the medicine belongs to traditional Chinese medicine compound preparation, is made up of Ligusticum wallichii, the taste Chinese medicine of rhizoma Gastrodiae two.The monitoring index of its Ligusticum wallichii is forulic acid（The Chinese herbal medicines such as Huang Dongping, 2004,35 (4), 409-411)." day rhizome of chuanxiong Toutongning capsule for headache " is the prescription being made up of ten a few herbs using rhizoma Gastrodiae as monarch drug in a prescription（The such as Shen Yuhua new Chinese medicines and clinical pharmacology, 1995,6 (3), 33-35)." day rhizome of chuanxiong soft capsule " Main Ingredients and Appearance is rhizoma Gastrodiae and rheum officinale, and the wherein ratio of rhizoma Gastrodiae and rheum officinale in prescription is 3：1 (such as Guo Zengjun Chinese patent drugs 2005,27 (4), 467-468).These prescriptions are more to treat based on antimigraine, and not using active ingredient as index in preparation process.
In addition to above-mentioned traditional Chinese medicine compound preparation, the modern times also have some using Ligusticum wallichii and rhizoma Gastrodiae as the medicine of active ingredient.Such as patent application《Pharmaceutical composition and preparation comprising rhizoma Gastrodiae and effective Chuanxiong component》（Application number：2) 200610020605. disclose the pharmaceutical composition constituted using rhizoma Gastrodiae and Ligusticum wallichii as effective ingredient；Patent application《Dry powder preparation of chuanxiong rhizome and preparation method and application》（Application number：200610046747. 6) disclose the dry powder formulations being mixed by Rhizoma Gastrodiae extract and Rhizoma Chuanxiong extract.Patent application《Treat ligustilide of ischemic heart disease and preparation method thereof》（Application number：1) 03146029. disclose ligustilide, including two kinds of Ginkgolide Components of sedanolide and ligustilide.Patent application《It is a kind of to treat pharmaceutical composition of cranial vascular disease and preparation method thereof》（Application number：200510101971. 6) disclose ligustilide and senkyunolide composition composition and《A kind of extract of cnidium lactone and its preparation method and application》（Application number：4) 200610130680. disclose the application for being related to ligustilide in treatment ischemic cardiovascular disease.Patent《A kind of Chuanxiong rhizome volatile oil soft capsule and preparation method thereof》（Application number：200410037185. X) disclose a kind of method that use carbon dioxide supercritical extraction method prepares rhizoma chuanxiong volatile oil.
Above-mentioned prior art has the following disadvantages：（1) drug ingredient purity is not high, it is impossible to the Chinese medicine preparation of specific chemical components is made, and specific chemical components particularly can not be made and purity very high injection；（2) ligustilide being not sufficiently stable in the Chinese medicine preparation of the existing preventing and treating cerebral apoplexy based on Rhizoma Chuanxiong extract using chemical property influences the accuracy of detection as monitoring index.
Through retrieval, the Pharmaceutical composition being made up of sedanolide, ligustilide and Gastrodin is not found.The content of the invention One of the object of the invention is to provide a kind of Pharmaceutical composition for preventing and treating cerebral arterial thrombosis.
Second object of the present invention is to provide the method for preparing aforementioned pharmaceutical compositions.
It is a further object of the present invention to provide application of the aforementioned pharmaceutical compositions in treatment cerebral arterial thrombosis medicine is prepared.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of pharmaceutical composition for preventing and treating cerebral arterial thrombosis, its constituent and its percentage by weight are：Sedanolide 19 72%
Ligustilide 17 67%
Gastrodin 2 29%
Balance of solvent
Solvent described in aforementioned pharmaceutical compositions refers to sodium carboxymethylcellulose, tween -80 or vegetable oil etc..Described vegetable oil refers to the one or more in the vegetable oil such as olive oil, peanut oil, corn oil, soybean oil, sunflower oil, sesame oil, castor oil, apricot kernel oil or persic oil.
According to usual way, the formulation prepared with aforementioned pharmaceutical compositions can be a kind of pharmaceutically acceptable formulation, such as can be injection, spray, capsule, dripping pill, tablet, granule, emulsion, dispersant, sustained release agent.
Application of the aforementioned pharmaceutical compositions in treatment cerebral arterial thrombosis medicine is prepared.
Sedanolide, ligustilide and Gastrodin are sterling compound in aforementioned pharmaceutical compositions, and purity is all more than 98%, and Gastrodin is commercially available.
The preparation method of the pharmaceutical composition of above-mentioned preventing and treating cerebral arterial thrombosis, proportionally takes sedanolide, ligustilide, Gastrodin and cosolvent, is then well mixed.
The preparation method of above-mentioned sedanolide, comprises the following steps：
(1) dry Ligusticum wallichii root block is crushed to diameter l 3mm particle, is subsequently placed in extraction kettle；
(2) temperature adjustment knob of extraction kettle is set to 42 58 °C, heated；When extraction kettle temperature is 42 58DDuring C, C0 is opened2Air intake pump； '
(3) extract and separate, C02Air-flow enters separation reactor I after extraction kettle, enters back into separation reactor I I；It is 18 32Mpa to reach extraction kettle pressure;Wherein separation reactor I pressure is 8 14Mpa, and temperature is 45 55QC, extraction time is 60 120 min, C02Flow is 220 350kg/h;Separation reactor I I pressure is 4 8Mpa, and temperature is 35 450C, extraction time is 60 120 min;
(4) first time extracts and fed intake again into extraction kettle after the completion of separating the Ligusticum wallichii root block particle of same particle diameter, repeat step（2 ) 〜 （3) second, is carried out to extract and separate；
) the positive pages of ￡ are (thin 91st article） (5) after extracting and separate for the second time, the discharging opening from separation reactor I and separation reactor I I disposably releases entire contents respectively；
(6) above two content is respectively placed in separatory funnel until layering, supernatant liquid is collected after layering, the I levels for respectively obtaining ligustilide separate product（Come from separating still work）With II grades of separation products（Come from separation reactor I I);
(7) aspiration step（6) 7ml of mixture 3 of the I levels separation product of ligustilide or II levels separation product either both products of ligustilide adds petroleum ether：Ethyl acetate（95：5) mixed solution shakes hook to 8 12ml；Sample introduction is to Biotage 40+M positive liquid chromatogram silicagel columns；Its mobile phase is A petroleum ethers, B ethyl acetate；Flow velocity 50ml/min;Detection wavelength 280nm;Gradient elution, 0.01 12minA： B= 95：5,15 30minA： B =85： 15;Eluent is collected between 18 25min；
(8) repeat step（7), I grades and/or II grades separation product sample introductions are finished successively, must be eluted
、―、
Hand；
(9) by step（8) eluent rotary evaporation in 30 40 °C of water-baths removes solvent, produces sedanolide Primary purification product；
(10) aspiration step（9) 7ml of Primary purification product 3 in adds methanol and is dissolved to the anti-phase preparation liquid phase systems sample introductions of Shimadzu LC-8A；Wherein chromatographic column is Phenomenex Luna Ι Ο μ π ι, 250mmx50mm I.D.;Mobile phase is water（) and methanol A（B), flow velocity is 80ml/min, isocratic elution, A： B =40： 60；It is 280nm to pick up survey wavelength;Eluent is collected between 18 26min；
(11) by step（10) gained eluent rotary evaporation in 30 40 °C of water-baths removes methanol；Residue is extracted with ethyl acetate 35 times, extract is obtained；
(12) by above-mentioned extract, rotary evaporation removes solvent in 30 40 °C of water-baths, obtains sedanolide.The preparation method of described ligustilide, comprises the following steps：
Step（1 ) 〜 （6) with sedanolide preparation method step（1 ) 〜 （6) ；
(7) with sedanolide step（7) eluent between 6 12min, is collected；
(8) rotary evaporation removes solvent in 30 40 °C of water-baths, and products obtained therefrom is ligustilide.Above-mentioned preparation method step（1) the supercritical carbon dioxide extracting kettle described in can be
The 13-3. 43 of HA221-40-48 or HA421- 40- 963..
Described Gastrodin can directly commercially, Gastrodin or Acegastrodine that such as Kunming Medicine Group Stock Co., Ltd produces.
The active ingredient sedanolide and ligustilide extracted with the inventive method, purified, purity is more than 98%.Commercially available Gastrodin（Or Acegastrodine）Purity is more than 98%.Correct page (thin4Then the 91st article） Advantage and beneficial effect of the present invention：（1) drug effect is high, can effectively mitigate pathological change and the behavior disorder of cerebral arterial thrombosis, and drug effect is significantly better than existing clinical application；（2) pharmaceutical composition drug effect of the present invention is definite, and consumption is small, and effect is strong；（3) purity is high, and pharmaceutical composition each component of the present invention is sterling, and the preparation including injection can be made all more than 98% in purity；（4) stability is good, and present invention process, which reaches, prepares standard items level；（5) present invention points out that the chemical property of sedanolide is more stable than ligustilide, is more suitable for the monitoring index of cnidium oil product quality.
Brief description of the drawings
Fig. 1 is that the ligustilide and sedanolide product separated from supercritical extract liquid prepares spectrogram（Positive HPLC methods）（Wherein QHNP- 1 is ligustilide；QHNP- 2 is sedanolide）
Fig. 2 is the detection spectrogram of ligustilide（Reverse hplc is detected）
Fig. 3 prepares spectrogram for sedanolide Primary purification product（Reverse hplc method）
Fig. 4 is the detection spectrogram of the final purified product of sedanolide（Reverse hplc is detected）Fig. 5 is the infrared identification spectrogram of sedanolide
Fig. 6 is the infrared identification spectrogram of ligustilide
Fig. 7 is the ultraviolet identification spectrogram of sedanolide
Fig. 8 is the ultraviolet identification spectrogram of ligustilide
Fig. 9 is the Mass Spectrometric Identification spectrogram of sedanolide
Figure 10 is the Mass Spectrometric Identification spectrogram of ligustilide
Figure 11 identifies spectrogram for the nuclear magnetic resonance of sedanolide（Hydrogen is composed）
Figure 12 identifies spectrogram for the nuclear magnetic resonance of ligustilide（Hydrogen is composed）
Figure 13 identifies spectrogram for the nuclear magnetic resonance of sedanolide（Carbon is composed）
Figure 14 identifies spectrogram for the nuclear magnetic resonance of ligustilide（Carbon is composed）
Embodiment
Embodiment 1
The preparation of sedanolide
(1) the dry root block 30kg for weighing Ligusticum wallichii is crushed to the Let of diameter 2. 5 particle, is placed in HA221-40-48 extraction kettles；
(2) rotation extraction kettle temperature setting knobs to 50 ° (：, when temperature reaches 50QDuring C, C0 is opened2Air intake pump；
(3) starting device, carries out first time extraction and separation； C02Air-flow enters after extraction kettle and divided Bar） From kettle I, enter back into separation reactor I I;Extraction kettle pressure 25Mpa, separation reactor I pressure llMpa, 50 °C of separation reactor I temperature, extraction time 90 min, C0240 °C of flow 250kg/h, separation reactor I I pressure 6Mpa, separation reactor I I temperature;
(4) feed intake 30kg again, by step（1 ) 〜 （3) second is carried out to extract and separate；
(5) discharging opening respectively from separation reactor I and separation reactor I I disposably releases entire contents；
(6) above two content is respectively placed in separatory funnel until being layered.Supernatant liquid is collected after layering, the I levels separation product of ligustilide is respectively obtained（Come from separation reactor I) and II grades of separation products（Come from separating still 11).
(7) II grades of separation product 5ml of the ligustilide in aspiration step (6), add the mixed solution of petroleum ether and ethyl acetate（Petroleum ether：Ethyl acetate is 95:5) to 10ml, shake up；Sample introduction is to Biotage 40+M positive liquid chromatogram silicagel columns；Its mobile phase is A petroleum ethers, B ethyl acetate；Flow velocity 50ml/min;Detection wavelength 280nm;Gradient elution, 0.01 12min A： B= 95：5,15 30min A： B =85
： 15；Eluent is collected between 19.6 23.2min (see Fig. 1 QH P-2);
(8) repeat step (7), II grades of separation product sample introductions of ligustilide are finished, eluent is obtained；
(9) by step（8) eluent collected by obtains sedanolide Primary purification product in 35 °C of water-baths after rotary evaporation removing solvent；
(10) from step（9) 5ml addition methanol is drawn in product and is dissolved to the anti-phase preparation liquid phase systems sample introductions of Shimadzu LC-8A；Wherein chromatographic column is Phenomenex Luna Ι Ο μ η ι, 250mmx50mm I.D., and mobile phase is A water B methanol, and flow velocity is 80ml/min, isocratic elution, A： B =40：60, Detection wavelength is 280nm;Eluent is collected between 20.3 24.5min (see Fig. 3);
(11) in step（10) gained eluent rotary evaporation in 35 °C of water-baths removes methanol, and residue is extracted with ethyl acetate 3 times, extract is obtained；
(12) by step（11) gained extract rotary evaporation in 35 water-baths removes solvent, obtains final products 347.3g.
(13) according to《Chinese Pharmacopoeia》The described detection method of the one annex VI D " high performance liquid chromatography " of version in 2005 is detected to resulting final products, obtains its purity for 99.17% (see Fig. 4).Through infrared, ultraviolet, mass spectrum and nuclear magnetic resonance（Two methods are composed including hydrogen spectrum and carbon）Four big spectroscopy certifications learn that the product is sedanolide（See Fig. 5, Fig. 7, Fig. 9, Figure 11 and Figure 13).
Embodiment 2
The preparation of sedanolide
(1) the dry root block 30kg for weighing Ligusticum wallichii is crushed to the awake particle of diameter 1, is placed in In HA221-40-48 extraction kettles；
(2) rotation extraction kettle temperature setting knobs are to 58 °C, when temperature reaches 58 °C, open C02Air intake pump；
(3) starting device, carries out first time extraction and separation, C02Air-flow enters separation reactor I after extraction kettle, enters back into separation reactor I I, extraction kettle pressure 32Mpa, separation reactor I pressure 14Mpa, 55 °C of separation reactor I temperature, extraction time 120min, C02Flow 280kg/h;45 °C of separation reactor I I pressure 8Mpa, separation reactor I I temperature;
(4) Ligusticum wallichii particle 30kg is fed intake again in the extraction kettles of HA221- 40- 48, repeat step（ 2 )〜（3) second is carried out to extract and separate；
(5) discharging opening respectively from separation reactor I and separation reactor I I disposably releases entire contents；
(6) above two content is respectively placed in separatory funnel until being layered；Supernatant liquid is collected after layering, the I levels separation product of ligustilide is respectively obtained（Come from separation reactor I) and II grades of separation products（Come from separation reactor I I)；
(7) above two ligustilide product is sufficiently mixed；
(8) from step（7) mixed solution that 5ml adds petroleum ether and ethyl acetate is drawn in product（Petroleum ether：Ethyl acetate is 95:5) to 10ml, shake up；Sample introduction is to Biotage 40+M positive liquid chromatogram silicagel columns；Its mobile phase is petroleum ether (A) and ethyl acetate（B);Flow velocity 50ml/min;Detection wavelength 280nm;Gradient elution, from 0.01 12min A: B= 95：5,15 30minA: B =85: 15；Eluent is collected between 18 25min；
(9) it is to obtain sedanolide Primary purification product that by collected eluent, rotary evaporation, which is removed after solvent, in 30 °C of water-baths；
(10) from step（9) 5ml addition methanol is drawn in product and is dissolved to the anti-phase preparation liquid phase systems sample introductions of Shimadzu LC-8A；Wherein chromatographic column is Phenomenex Luna Ι Ο μ η ι, 250mm 50mm I.D., and mobile phase is A water B methanol, and flow velocity is 80ml/min, isocratic elution, A： B =40：60, Detection wavelength is 280nm;Eluent is collected between 18 26min；
Rotary evaporation removes methanol in (11) 30 °C of water-baths, and then residue is extracted with ethyl acetate 5 times, extract is obtained；
(12) by extract, rotary evaporation removes solvent in 30 °C of water-baths, obtains sedanolide 309g.Embodiment 3
The preparation of ligustilide, comprise the following steps-(1) weigh dry Ligusticum wallichii root block 30kg, the awake particle of diameter 3 is crushed to, is placed in In HA221-40-48 extraction kettles；
(2) 42 °C of extraction kettle temperature, when temperature reaches setting value, open C02Air intake pump；
(3) extraction kettle pressure 18Mpa, separation reactor I pressure 8Mpa, 45 °C of separation reactor I temperature, extraction time 60 min, C02 flow 220kg/h, separation reactor I I pressure 5Mpa, 40 °C of separation reactor I I temperature, carries out first time extraction and separation；
Step（4) 〜 （7) the step of be the same as Example 2（4) 〜 （7) ；
(8) from step（7) 5ml sample introductions are drawn in product to Biotage40+M positive liquid chromatogram silicagel columns；Chromatographic condition be the same as Example 2;Eluent is collected between 7 10.6min, obtain dizzy finished product 498.2g (see the QH P- 1 in Fig. 1).
(9) according to《Middle i [pharmacopeia》The described detection method of the one annex VI D " high performance liquid chromatography " of version in 2005 detects that it is 98.32%% to obtain its purity to resulting final products.Through infrared, ultraviolet, mass spectrum and nuclear magnetic resonance（Two methods are composed including hydrogen spectrum and carbon）Four big spectroscopy certifications learn that the product is Ligustilide（See Fig. 6, Fig. 8, Figure 10, Figure 12 and Figure 14).
Embodiment 4 '
Respectively prepared by Example 13（It is same afterwards）Sedanolide（Proportion 0.95, it is rear same）4.2g, Ligustilide l.Og (proportion 0.95, it is rear same）, and Gastrodin 0.6g, it is added in 20g Pure Olive Oils, ultrasonic mixing is uniform, obtains present composition A 25.8g。
Embodiment 5 ' takes sedanolide 0.6g, Ligustilide 1.8g, and Gastrodin 0.75g respectively, is added in 20g soybean oils, and ultrasonic mixing is uniform, obtains present composition B 23.15g.
Embodiment 6
Sedanolide 3.6g, Ligustilide 3.3g, and Gastrodin 2.9g are taken respectively, add pure peanut oil to 20ml, ultrasonic mixing is uniform, obtains present composition C 29.8g.
Embodiment 7-weigh sedanolide 2.8g, ligustilide 1.2g, the g of Gastrodin 0.08 respectively, grinds well, plus 0.5% sodium carboxymethylcellulose is to 21.6ml, obtains present composition D 25.68g.Sedanolide 1.4g, Ligustilide 3.0g, and the l of Gastrodin 0. are taken respectivelyg, 2% sodium carboxymethylcellulose is added to 20ml, and ultrasonic mixing is uniform, obtains present composition E 25.5g. According to high effective liquid chromatography for measuring（See one annex VI D of China's coastal port).
Chromatographic condition and system suitability test：From Shimadzu LC-2010 performance liquid chromatographic columns.Using octadecyl silicon protective embankment bonded silica gel as filler (Diamonsil C18,4.6x150mm, 5 u m);With the water of acetonitrile one（42:58) it is mobile phase；35 °C of column temperature;Detection wavelength 280nm, 230nm;Flow velocity lml/min.Number of theoretical plate is calculated by sedanolide peak is not less than 16000.
The preparation of standard solution：Sedanolide prepared by Example 1, it is accurately weighed, plus every 1ml 0.08mg containing sedanolide respectively solution is made in methanol, produces the standard solution that purity is more than 99%.
The preparation of need testing solution：Sedanolide that Example 1 is obtained is appropriate, accurately weighed, dissolve with methanol and quantifies to dilute the solution containing 0.08mg in every 1ml is made, and is used as sedanolide need testing solution.
Assay method：Precision draws sedanolide standard items and each 10ul of need testing solution, liquid chromatograph is injected, according to the step of embodiment 1（13) methods described is determined.
The sedanolide stability experiment result of table 1（Normalize purity testing %, 280nm)
As a result（Be shown in Table the stability of 1) sedanolide of the present invention is mainly influenceed by high temperature, illumination and oxygen.In 30 days under the condition of storage of nitrogen charging lucifuge, either room temperature, refrigeration or freeze, on the stability of sedanolide substantially without influence（The relative standard deviation of content<2%).Illustrate that sedanolide property is relatively stablized, as long as nitrogen charging is kept in dark place, purity will not decline in 30 days, can be used as standard items.
Embodiment 10
Ligustilide stability experiment
According to《Chinese Pharmacopoeia》High effective liquid chromatography for measuring shown in the one annex VI D of version in 2005.Chromatographic condition and system suitability test：From Shimadzu LC-2010 performance liquid chromatographic columns.Using octadecylsilane chemically bonded silica as filler（Diamonsil C18, 4.6 <150mm, 5 n m);With acetonitrile one Water（42:58) it is mobile phase；35 °C of column temperature;Detection wavelength 280nm, 230nm;Flow velocity lml/min.The preparation of standard solution：Ligustilide prepared by Example 3, it is accurately weighed, plus every 1ml 0.4mg containing Ligustilide solution is made in methanol, produces the standard solution that purity is more than 99%.
The preparation of need testing solution：The gained Ligustilide of Example 3 is appropriate, accurately weighed, dissolve with methanol and quantifies to dilute the solution containing 0.4mg in every 1ml is made, and is used as Ligustilide need testing solution.
Assay method：Standard items and each 10ul of need testing solution are drawn, injection liquid chromatograph is according to the step of embodiment 3（9) methods described is measured.
As a result（It is shown in Table 2).Ligustilide stability of the present invention is mainly influenceed by illumination, temperature and oxidation；Purity drops to less than 95% from 98% after being placed 10 days under room temperature condition.Show that Ligustilide standard items must be stored under nitrogen charging, lucifuge, refrigeration or freezing conditions.Must Fresh.No more than 5 days usage amounts of each preparation amount.
The Ligustilide stability experiment result of table 2（Normalize purity testing %, 280nm)
Embodiment 11
Influence of the Pharmaceutical composition of the present invention to line brush cerebral ischemic model
(1), test method is carried out as follows：
(1) packets and administration：Healthy SPF/VAF grades of Wistar rats of male from 150 body weight in 240 250g are tested.（Beijing Vital River Experimental Animals Technology Co., Ltd. provides, quality certification number:SCXK capital 2002-0003).It is randomly divided into 9 groups, i.e. blank control group, model control group, Mailuoning positive controls 6ml/kg, each two dosage of composition A, B, C（6mg/kg and 12/kg) group.Composition A, B, C compound method are shown in embodiment 46 A kind of composition or physiological saline or positive drug is injected intraperitoneally within 2 hours after operation consent 1 hour and Reperfu- sion.Positive drug chooses the conventional Western medicine MAILUONING ZHUSHEYE of clinical treatment cerebral arterial thrombosis（LOrnl/ branch, Jinling Pharmaceutical Co., Ltd., Jinling Pharmaceutical Factory's production, lot number： 200503011).Isometric physiological saline is injected intraperitoneally in blank control group and model control group.
(2) prepared by lines bolt：Linear is moulded into from a diameter of 0. 23mm of Japan's production nylon yarn and is cut into 5cm line segment, front end polished with fine sandpaper, dip in glue is allowed to smooth, standby being marked at the 6cin of front end 1., 1. 8cm, 2. 0cm, face the used time dips heparin by nylon yarn front end.
(3) model copies：10% chloral hydrate anesthesia is injected intraperitoneally in 400mg/kg；Neck midsection, the left sorrowful arteria carotis communis of exposure（Abbreviation CCA), external carotid artery（Abbreviation ECA), internal carotid（Abbreviation ICA);Ligature CCA near-ends, ECA roots；An angle is cut in distal end at CCA ligation, inserts nylon yarn, cranium is entered to arteria cerebri anterior by ICA through CCA crotches（ACA), MCA blood flow is blocked；The average insertion depth of nylon yarn is 18. 5 ± 0. 5 legs, ligatures ICA.Skin suture, exposure nylon yarn end；Nylon yarn is extracted after 2 hours in ischemic, the change of different time course after observation Reperfu- sion；Sham-operation group does not insert nylon yarn.
(2), observation index：
(1) Neurological deficits：Using Pyatyi point system（0-4 points）.The animal behavioral study of animal is carried out in Reperfu- sion whole latter stage.Rat-tail is put forward from the chi of Jian ground about 1, two forelimb situations are observed；Rat is placed in level ground, its both shoulders is promoted, observation both sides resistance has indifference；Rat is placed in ground, observes its walking situation.Fraction is higher, shows that the damage of its neurobehavioral is more serious.（Based on best result）
A. behavior is normally 0 point completely；
B. lift rat-tail and leave ground, operation offside forelimb inward turning, interior receipts person, are 1 point；
C. rat is to ground, and with its drag of hand extruding both sides inspection Check, perform the operation offside drag descender, is 2 points；
D. rat observes its walking to ground, is 3 points around the operation offside person of turn-taking；
E. damage extremely serious, can not autonomic activities person, be 4 points.
(2) cerebral infarct sizes are tested：Broken end takes brain after 24 hours, and it is thick totally 1 that left side brain is cut into 2 pictures thickness totally 5,4mm.Through 4% red tetrazolium（TTC) 37 °C of lucifuges are dyed 30 minutes；Stirred once every 5 minutes therebetween.After being dyed through TTC, normal structure dye deeply takes on a red color, and infarction tissue is white.By every group of brain piece marshalling, preservation of taking pictures.Application image analysis system software（The softwares of SPOT 3.5）Handle and count.Calculate the infarct size of every and be finally superimposed and be converted into infarct volume.Infarct volume is represented with the percentage of shared cerebral hemisphere.Calculation formula is as follows：
Cerebral infarct volume（%)=(the operation non-infarcted portion of side hemisphere of volume one of operation contralateral hemisphere Volume）The volume X 100% of I operation contralateral hemispheres
(3) brain water contents are determined：
After Reperfu- sion 24 hours, broken end takes brain at once, removes tractus olfactorius, cerebellum and low brain stem, separately perform the operation side and normal side hemisphere, weighs respectively（Weight in wet base）.It is placed in baking box after 95 °C of bakings 24 hours to constant weight, brain weight is weighed again（Dry weight）.Brain water content is according to the following formula：
Brain water content（%)=(dry weight of weight in wet base one）The % of/weight in wet base X 100
Experimental data use ± SZ) represent, examined through t and compare group difference.
The of table 3 Pharmaceutical compositions of the present invention are to line brush cerebral ischemic model therapeutic test result
Note：The Δ Δ Ρ compared with blank control group<0. 01 ΔΔΡ<0. 05；
The * Ρ of * * Ρ ＜ 0 01 compared with model control group<0. 05；
As a result（Intraluminal middle cerebral artery occlusion in rats focal cerebral ischemia infarct size can be improved by being shown in Table 3) tri- groups of Pharmaceutical compositions of A, B, C of the present invention and positive drug group.It is wherein heavy dose of with composition B（12mg/kg body weight）Preferably, drug effect is better than conventional clinical application MAILUONING ZHUSHEYE to effect.Illustrate that Pharmaceutical composition of the present invention can be used for treatment cerebral arterial thrombosis, mitigate cerebral infarct size.
Embodiment 12
Influence of the Pharmaceutical composition of the present invention to line brush cerebral ischemic model
(one）, experiment carry out as follows：
(1) packet and administration： 20 0〜220gHealthy male SD rat 78, be randomly divided into 6 groups, the respectively group of Normal group, model group, positive control drug ligustrazine group and the embodiment of the present invention 46 Compound A, B, C group.Positive control drug ligustrazine group is selected from Ligustrazine Phosphate pill, 50mg/ pieces, 100 pieces/bottle, the production of Beijing Beijing pharmaceutical factory, lot number： 040603.Advance gastric infusion 7 days, once a day, each dosage 50mg/kg.Broken end takes 2 h before brain to be administered once again.Normal group and model group gavage normal saline.
(2) experimental method：4% chloraldurate（350mg/kg, ip) anesthesia.Rat dorsal position is fixed, along neck midsection, right carotid is separated and to wear 2 sutures standby, then separate external carotid artery, proximal part ligation.Internal carotid and its one, side branch are isolated below external carotid artery（Pterygoid process arteria palatina）, a suture is worn under branch, in the ligation of proximal part crotch.In separated arteria carotis communis proximal part suture ligature, distal end artery clamp blocking blood flow between, cuts an osculum, by heating one end into round bead shape（Diameter<Nylon wire 0.3mm)（0) 4 one insert osculum, removes artery clamp, nylon wire is slowly pushed into anterior cerebral artery（About 20mm), draw about 2mm to arteria cerebri media mouthful, to be about 17mm and (counted from inside and outside neck at aortic bifurcation further back）, nylon bar is fixed with suture ligature.Suture muscle and skin.In surgical procedure, visual field temperature is kept for 37 °C.Postoperative 24h carries out neurological deficit score, after scoring, puts to death rat, and broken end takes brain.Control group not plug wire, postoperative 24h put to death after neurological deficit score, and broken end takes brain.Relevant partial content in operation and computational methods equivalent integers 11 below.
(two）, observation index：The observation index of be the same as Example 11（2)
As a result（Intraluminal middle cerebral artery occlusion in rats focal cerebral ischemia infarct size can be improved in 7 days by being shown in Table 4) tri- groups of Pharmaceutical composition oral agents of A, B, C and positive drug group prevention administration of the present invention；Tri- groups of Pharmaceutical composition drug effects of A, B, C of the present invention are significantly better than conventional clinical application Ligustrazine Phosphate pill.Illustrate that Pharmaceutical composition of the present invention can prevent and treat cerebral arterial thrombosis, mitigate cerebral infarct size.Drug effect is significantly better than conventional clinical application Ligustrazine Phosphate pill（P<0.01).
The Pharmaceutical composition of the present invention of table 4 is to line brush cerebral ischemic model result of the test
Note：* the P ＜ 0. 05 compared with model control group, * * P compared with model control group<0. 01 embodiments 13
Influence of the Pharmaceutical composition of the present invention to tri-chlorination iron processes cerebral ischemic model
(one）, experiment carry out as follows：
(1) packet and administration：Healthy male SD rat by 110 body weight in 180 200g by body weight is randomly divided into 9 groups.Blank control group, model control group, ligustrazine phosphat 120mg/ml groups（Medicine source be the same as Example 12), each composition component be two dosage groups of 80mg/ml, 40mg/ml, every group 12.Once a day oral administration, continuous 30 days, blank group, model control group took isometric edible oil.
(2) experimental method：In after last dose 1 hour, by the chloraldurate solution of 350mg/ml intraperitoneal injections 12% by rat anesthesia, lateral position is fixed, and is operated under surgical operation microscope.The eye corner of the eyes makees a vertical incision for being about lcm with external auditory meatus line midpoint, and exposure is simultaneously shelled from Temporal fleshes, bores a diameter about 2mm aperture with dental drill at the ware of oval foramen side front upper place 1, and meninx, exposure arteria cerebri media are punctured with fine needle（MCA), the filter paper bar for being moistened with 50% liquor ferri trichloridi is spread on arteria cerebri media, taken out after 30min, now arteria cerebri media becomes black or white.Sew up the incision.Broken end takes brain after 24h.Relevant partial content in embodiment 11 is shown in operation and computational methods etc. below.
Neuroethology test is carried out after operation 6h and 24h respectively.Standards of grading and the observation index of method be the same as Example 11（1).
(two）, observation index：The observation index of be the same as Example 11（1 )、 （2)
As a result（It is shown in Table 5) each group Pharmaceutical composition oral agents and the continuous prevention administration of positive drug group can obviously improve intraluminal middle cerebral artery occlusion in rats focal cerebral ischemia infarct size in 30 days compared with model group.Wherein present composition A drug effects are significantly better than conventional clinical application Ligustrazine Phosphate pill（P<0. 05)., have to escalated dose because the positive drug group of Isodose can not embody drug effect.And the 1/3 2/3 of present composition group dosage can show similar drug effect.Illustrate that Pharmaceutical composition oral agents long term administration of the present invention can prevent and treat cerebral arterial thrombosis, mitigate cerebral infarct size.Drug effect is better than or is not less than conventional clinical application Ligustrazine Phosphate pill.
The of table 5 pharmaceutical composition prevention administrations of the present invention 30 days are to cerebral ischemic model result of the test The composition E 9 40 4.33 ± 1.61 3.83 ± 1.27 4.34 ± 0.85 of 10 1 5.00 ± 1.15 Δ of ± 0.65 0.45 ± 0.52 0 model comparison of blank control 11 1 0.73,3.80 ± 1.40 Δ Δ, 5.17 ± 2.36 3.83 ± 1.53 3.92 ± 1.14 10 80 3.70 ± 1.34* of composition E of Δ Δ ligustrazine positive drug group 10 120 3.58 ± 1.44*, 308 ± 1.56 4.41 ± 0.72 10 80 3.58 ± 1.44* of composition A, 3.67 ± 1.50 383 ± 091 composition A, 9 40 4.00 ± 1.58 3.67 11 80 3.36 ± 1.57* of ± 1.80 3.23 ± 0.85* composition D, 3.55 ± 1.92 4.98 ± 1.15 11 40 3.92 ± 1.24* of composition D 3.30 ± 1.16 3.99 ± 2.06
Note：The Δ Δ Ρ compared with blank control group<0.01
The * Ρ compared with model control group<0.05
Embodiment 14
The acute toxicity test in mice of pharmaceutical composition of the present invention
(one）Experiment and computational methods：
It is disposable to be administered orally using SPF grades of Kun Ming mices.Using trial test result as test dose, agent is away from for 1:0.90.
Drug solution preparation method is as follows：Accurately weigh sedanolide 2.501g, ligustilide 2.502g, and Gastrodin 0.7502g, appropriate 2% sodium carboxymethylcellulose hydrotropy is added after mixing, the solution that concentration is 0.0575g/g is made after fully grinding well；Each group dosage respectively as shown in table 7 2% sodium carboxymethylcellulose of dosage the aqueous solution）Dilution, per 20g bodyweight ps administration 0.8ml;Continuous Observation 48 hours.
Animal is randomly divided into 9 groups, every group 20, male and female half and half by the body weight after fasting 16 hours；Agent is away from 1 between group:0.9;The tested material of above-mentioned various concentrations is pressed into 0.8ml/20g gavages respectively, the toxic reaction that each group animal occurs after tested material, Continuous Observation 48 hours are given in observation at once.Record the toxic reaction and death condition of animal.Mouse oral LD is calculated with Bilss methods5.Dosage and 95% fiducial limit.
Calculation formula is as follows：Draw number:
Median lethal dose: LD5.=antl.G [X+Shang (5-） ] l.gLD5.Standard error： S .=
LD5.95% average fiducial limit： L95=LD5Q±4.5 LD50 - J
b ~∑M) LD5.99% average fiducial limit： L99=LD5。±5.9 LD50 · "
b^n∑w
As a result（It is shown in Table the Mouse oral LD for 6) showing pharmaceutical composition of the present invention5.It is worth for 0.7276g/kg; LD5.95% average credible be limited to 0,7276 ± 0.0470g/kg(scope: 0.6806-0.7746 g/kg); LD5.99% average credible be limited to 0.7276 ± 0.0616g/kg (scopes: 0.6660-0· 7892g/kg).Agent is away from 1 between group:0.9.
This experiment is the acute toxicity test of chemical grade sterling.As a result show that pharmaceutical composition security of the present invention is good, valuable reference data is provided for clinical medicine dose.
The Mouse oral LD of table 65.Dosage and 95% fiducial limit experimental result
i=log (1/0.9) =0.0458
b = 12.3675
LD50=0.6326g · kg"1
L95=0.6326±0.0274g · kg—1(scope: 0.6052-0.6600 g · kg—1)
L99=0.6326±0.0359g · kg— 1(scope: 0.5967-0.6685g - kg"1)
S¾0=0.0096

Claims (1)

  1. Claims
    1st, a kind of pharmaceutical composition for preventing and treating cerebral arterial thrombosis, its constituent and its percentage by weight are：Sedanolide 19 72%
    Ligustilide 17 67%
    Gastrodin 2 29%
    Balance of solvent
    2nd, according to the pharmaceutical composition described in claim 1, it is characterised in that described solvent refers to sodium carboxymethylcellulose, tween -80 or vegetable oil etc..
    3rd, according to the pharmaceutical composition described in claim 2, it is characterised in that described vegetable oil refer to olive oil, ' one or more in the vegetable oil such as peanut oil, corn oil, soybean oil, sunflower oil, sesame oil, castor oil, apricot kernel oil or persic oil.
    4th, according to the pharmaceutical composition described in claim 1 or 2, it is characterised in that its formulation can be injection, spray, capsule, dripping pill, tablet, granule, emulsion, dispersant, sustained release agent etc..
    5th, application of any described pharmaceutical composition of claim 14 in treatment cerebral arterial thrombosis medicine is prepared.
    6th, the preparation method of the pharmaceutical composition described in claim 1, proportionally takes sedanolide, ligustilide, Gastrodin and solvent, is then well mixed.
    7th, according to the preparation method described in claim 6, it is characterised in that the preparation method of described sedanolide, comprise the following steps：
    (1) dry Ligusticum wallichii root block is crushed to diameter：！3 particles drawn, are subsequently placed in extraction kettle；
    (2) temperature adjustment knob of extraction kettle is set to 42 58QC, heating；When extraction kettle temperature is 42 58flDuring C, C0 is opened2Air intake pump；
    (3) extract and separate, C02Air-flow enters separation reactor I after extraction kettle, enters back into separation reactor I I；It is 18 32Mpa to reach extraction kettle pressure;Wherein separation reactor I pressure is 8 14Mpa, and temperature is 45 55DC, extraction time is 60 120 min, C02Flow is 220 350kg/h;Separating still Π pressure is 4 8Mpa, and temperature is 35 45 °C, and extraction time is 60： 120 min;
    (4) first time extracts and fed intake again into extraction kettle after the completion of separating the Ligusticum wallichii root block particle of same particle diameter, repeat step（ 2 ) 〜 （3) second, is carried out to extract and separate；
    (5) after extracting and separate for the second time, the discharging opening from separation reactor I and separation reactor I I disposably releases entire contents respectively；Correct page (then the 91st article of ＞ (6) above two content is respectively placed in separatory funnel until layering, supernatant liquid is collected after layering, the I levels for respectively obtaining ligustilide separate product（Come from separation reactor I) and II grades of separation products（Come from separation reactor I I);
    (7) aspiration step（6) 7ml of mixture 3 of the I levels separation product of ligustilide or II levels separation product either two kinds of products of ligustilide adds petroleum ether：Ethyl acetate（95：5) mixed solution shakes up to 8 12ml；Sample introduction is to Biotage 40+M positive liquid chromatogram silicagel columns；Its mobile phase is A petroleum ethers, B ethyl acetate；Flow velocity 50ml/min;Detection wavelength 280nm;Gradient elution, 0.01 12minA： B= 95：5,15 30minA： B =85： 15;Eluent is collected between 18 25min；
    (8) repeat step（7), I grades and/or II grades separation product sample introductions are finished successively, must be eluted
    、―、―
    Y instrument；
    (9) by step（8) eluent rotary evaporation in 30 40 °C of water-baths removes solvent, produces sedanolide Primary purification product；
    (10) aspiration step（9) 7ml of Primary purification product 3 in adds methanol and is dissolved to the anti-phase preparation liquid phase systems sample introductions of Shimadzu LC-8A；Wherein chromatographic column is Phenomenex Luna Ι Ο μ η ι, 250mmx50mm I.D.;Mobile phase is water（) and methanol A（B), flow velocity is 80ml/min, isocratic elution, A： B =40： 60；Detection wavelength is 280nm;Eluent is collected between 18 26min；
    (11) by step（10) gained eluent rotary evaporation in 30 40 °C of water-baths removes methanol；Residue is extracted with ethyl acetate 35 times, extract is obtained；
    (12) by above-mentioned extract, rotary evaporation removes solvent in 30 40 °C of water-baths, obtains sedanolide.8th, according to the preparation method described in claim 6, it is characterised in that the preparation method of described ligustilide, comprise the following steps：
    (1) dry Ligusticum wallichii root block is crushed to diameter l 3mm particle, is subsequently placed in extraction kettle；
    (2) temperature adjustment knob of extraction kettle is set to 42 58 °C, heated；When extraction kettle temperature is 42 58 °C, open（02Air intake pump；
    (3) extract and separate, C02Air-flow enters separation reactor I after extraction kettle, enters back into separation reactor I I；It is 18 32Mpa to reach extraction kettle pressure;Wherein separation reactor I pressure is 8 14MPA, temperature is 45 55 °C, and extraction time is 60 120 min, C02Flow is 220 350kg/h;Separation reactor I I pressure is 4 8Mpa, and temperature is 35 45 °C, and extraction time is 60 120 min;
    (4) first time extracts and fed intake again into extraction kettle after the completion of separating the Ligusticum wallichii root block particle of same particle diameter, repeat step（2 ) 〜 （3) second, is carried out to extract and separate；
    18
    Correcting page, (detailed rules and regulations are raised91) (5) after extracting and separate for the second time, the discharging opening from separation reactor I and separating still Π disposably releases entire contents respectively；
    (6) above two content is respectively placed in separatory funnel until layering, supernatant liquid is collected after layering, the I levels for respectively obtaining ligustilide separate product（Come from separation reactor I) and II grades of separation products（Come from separation reactor I I);
    (7) aspiration step（6) 7ml of mixture 3 of the I levels separation product of ligustilide or II levels separation product either both products of ligustilide adds petroleum ether：Ethyl acetate (95：5) mixed solution shakes up to 8 12ml；Sample introduction is to BiotaGe 40+M positive liquid chromatogram silicagel columns；Its mobile phase is A petroleum ethers, B ethyl acetate；Flow velocity 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) rotary evaporation removes solvent in 30 40 °C of water-baths, and products obtained therefrom is ligustilide.
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CN1569848A (en) * 2003-07-14 2005-01-26 中国中医研究院中药研究所 Ligustilide for treating ischemic heart disease and its preparing method
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