CN1989985B - Chuanxiong rhizome effective ingredient, preparing method, preparation and use thereof - Google Patents
Chuanxiong rhizome effective ingredient, preparing method, preparation and use thereof Download PDFInfo
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- CN1989985B CN1989985B CN2005101353552A CN200510135355A CN1989985B CN 1989985 B CN1989985 B CN 1989985B CN 2005101353552 A CN2005101353552 A CN 2005101353552A CN 200510135355 A CN200510135355 A CN 200510135355A CN 1989985 B CN1989985 B CN 1989985B
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Abstract
The invention involves a Ligusticum wallichii effective composition, preparation method and its preparation and use. Among the effective components, in the weight percentage, the amount of neocnidilide is 45-55%, the amount of 3-(3,4,5-Trihydroxyphenyl) -2-propenoic acid is 25-35%. The preparation method of Ligusticum wallichii effective composition includes the following steps: disintegrating Ligusticum wallichii crude drugs, adding acetic ester and ethyl alcohol, heating and extracting, thickening the extract into concrete and integrating it with silica gel, separating them with positive phase silica gel column, first using petroleum ether and acetic ester as the mobile phase and getting eluate I, abandoning it, then replacing the chloroform and methanol as the mobile phase and getting eluate II, concentrating and drying the eluate II and getting sample; using preparative HPLC to separating again and getting s, then the sample is product. The chemical component of Ligusticum wallichii effective composition provided in the invention is simple and clear; it is easier to explaining its producing mechanism in pharmacological research and controlling the quality of drugs in production.
Description
Technical field
The present invention relates to a kind of Chinese medicine extract for the treatment of cardiovascular disease, relate in particular to the active component that from Rhizoma Chuanxiong, extracts, preparation method and preparation thereof and purposes, this active component mainly contains neocindilide (neocnidilide), (3-(3 for 3-(3,4,5-Trihydroxyphenyl)-2-propenoicacid, 4,5-Trihydroxyphenyl)-2-propenoic acid) two kinds of chemical compounds.
Background technology
Coronary heart disease and angina pectoris are harm humans health " first killers ", in recent years, along with the variation of China's population senescence and people's work, life, dietary structure and environment etc., the incidence rate of cardiovascular and cerebrovascular diseases such as coronary heart disease also increases year by year, and the people's physical and mental health in serious threat.In the natural product many active substances have resist myocardial ischemia, anoxia functions, some of them have been developed to treatment coronary heart disease and anginal new drug, as treat the medicine DIAOXINXUE KANG of coronary heart disease, it is by 8 kinds of pure Chinese medicinal preparations that steroid saponin is made that extract from Chinese medicine; Flavonihippophae is to be that raw material extracts the pure natural medical that processes with the Fructus Hippophae, and its effective ingredient is a Fructus Hippophae total flavones.Thereby from natural product, seek have resist myocardial ischemia, the active substance of anoxia physiologically active, be find, one of effective way of developing new drug.China's medicinal organism resource is very abundant, and its biological active substances is research and finds new drug guide chemicals, the natural treasure-house of developing new drug.At present, China extracts active substance from natural product, be used to be developed to treatment coronary heart disease, safety is good, toxicity is low new drug also seldom, from natural product, extract active substance, be developed to have and resist myocardial ischemia, be used for the treatment of coronary heart disease and anginal new drug, have significant application value and wide development prospect.
Rhizoma Chuanxiong is the dry rhizome of samphire Ligusticum chuangxiong Hort, and the beginning is stated from Shennong's Herbal, classifies as top grade." figure is through book on Chinese herbal medicine " pointed out: " is victory with Shu Chuan ", so the name Rhizoma Chuanxiong.The Rhizoma Chuanxiong main product is the best in Guanxian county, Sichuan, Chongqing and Dujiangyan City and other places with Guanxian county product, and in Jiangxi, also there is cultivation on ground such as Hubei and Yunnan, wherein Jiangxi is produced and also claimed Ligusticum slnense, and Yunnan is produced and also claimed the cloud rhizome of chuanxiong.The Rhizoma Chuanxiong blood circulation promoting and blood stasis dispelling, activating QI to alleviate the depression, wind-expelling pain-stopping.Be used for the menoxenia due to the stagnation of QI and blood, dysmenorrhea amenorrhea, stagnation of QI due to depression of the liver and cause the pain in chest and hypochondrium of hematogenous blockage, headache, anemofrigid-damp arthralgia, treating swelling and pain by traumatic injury.Modern efficacy study shows that Rhizoma Chuanxiong can not only " blood-activating and qi-promoting, wind-expelling pain-stopping ", but also has the effect of separating harmony in the exterior dampness.The chemical constituent of Rhizoma Chuanxiong mainly contains alkaloid, volatilization wet goods, also has, multiple compositions such as lactone, phenols.Contain ligustrazine (chuanxiongzine), be tetramethylpyazine (tet ramethylpyrazine), loline or perlolyrine (perlo lyrine) are 1-(5-methylol-2-furyl)-9H-pyrido (3,4-b) indole (1-(5-hydroxymethyl-2-furyl)-9H-pyrido (3,4-b) indo le) etc.; Contain this lactone of Roripa (ligust ilide) in the volatile oil, vanillic acid (vanillic acid), vanillin (vanillin), spathulenol (spathulenol) etc. (model Yongchun etc., the time precious traditional Chinese medical science traditional Chinese medicines, 2003,14 (9): 574-576).
The application of Rhizoma Chuanxiong is very extensive, is that the preparation of principal agent is also too numerous to enumerate with the Rhizoma Chuanxiong, as removing blood stasis detumescence mixture, mind-easing tonic bolus with arborvitate seed, YANG invigorating also five pellets, Rhizoma Chuanxiong injection etc.But ferulic acid anticoagulant in the Rhizoma Chuanxiong, promote hematoblastic depolymerization, antithrombotic formation, remove the vascular smooth muscle spasm, antioxidation and improve membrane stability, and antiinflammatory, analgesia, antifertility, adjusting immunologic function, sharp liver are protected pharmacological action (Yang Guangde etc., Chinese patent medicines such as gallbladder, 2002,24 (6): 418-420).Its sodium salt sodium ferulate has protective effect (Ke Qibin etc., Clinical Anaesthesiology magazine, 2002,18 (8): 425-428 to spinal cord of rabbit, Myocardial Ischemia Reperfusion Injury.Chen Lifen etc., Medical University Of Chongqing's journal, 2003,28 (1): 49-52.), reduce (Wu Dazheng etc., the West China pharmaceutical journal of improving that the brain capillary permeability promotes inflammation thereby also have, 2001,16 (2): 98-100) and delay and treat cataractous effect (Qi Mingxin etc., Chinese Journal of Pathophysiology, 2002,18 (11): 1390-1393) etc.
Summary of the invention
The object of the present invention is to provide a kind of chuanxiong rhizome effective ingredient.
Another object of the present invention is to provide the preparation method of this chuanxiong rhizome effective ingredient.
The present invention also provides the preparation that contains the effective Rhizoma Chuanxiong of this Radix Salviae Miltiorrhizae and the purposes of this component.
The present invention is implemented by the following technical programs:
Chuanxiong rhizome effective ingredient of the present invention, by weight percentage, wherein the content of neocindilide (neocnidilide) is 45～55%, (content of 3-(3,4,5-Trihydroxyphenyl)-2-propenoic acid) is 25～35% to 3-(3,4,5-Trihydroxyphenyl)-2-propenoicacid.
Preferably, the content of neocindilide (neocnidilide) is 47～53%, and (content of 3-(3,4,5-Trihydroxyphenyl)-2-propenoic acid) is 27～33% to 3-(3,4,5-Trihydroxyphenyl)-2-propenoicacid.
Best, the content of neocindilide (neocnidilide) is 49～51%, and (content of 3-(3,4,5-Trihydroxyphenyl)-2-propenoic acid) is 29～31% to 3-(3,4,5-Trihydroxyphenyl)-2-propenoicacid.
The preparation method of chuanxiong rhizome effective ingredient of the present invention, comprise the following steps: and will add ethyl acetate and ethanol after the Rhizoma Chuanxiong pulverizing medicinal materials, heating extraction gets extracting solution, and extracting solution is condensed into extractum, and itself and silica gel mixed sample, with normal phase silicagel column it is separated, at first use petroleum ether and ethyl acetate, get eluent I as mobile phase, abandon it, change chloroform and methanol then as mobile phase, get eluent II, will get sample behind the eluent II concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography; The separation condition of preparative hplc: chromatographic column is a preparative column, and mobile phase is water and acetonitrile, gradient elution.
Preferred chuanxiong rhizome effective ingredient preparation method, comprise the following steps: and to add ethyl acetate and ethanol (1: 0.8～1.2) after the Rhizoma Chuanxiong pulverizing medicinal materials, reflux 0.8～1.2 hour, extract 1～3 time, merging filtrate gets extracting solution, extracting solution is condensed into extractum, and itself and silica gel mixed sample, with normal phase silicagel column it is separated, at first use petroleum ether and ethyl acetate (47～53:, get eluent I 1) as mobile phase, abandon it, change chloroform and methanol (8～13: 1) as mobile phase, get eluent II, will get sample behind the eluent II concentrate drying then; Continue to separate the sample that obtains with preparative liquid chromatography; The separation condition of preparative hplc: chromatographic column is a preparative column, and mobile phase is water and acetonitrile, and gradient elution, flow velocity are 9～11ml/min, and column temperature is a room temperature.
Best chuanxiong rhizome effective ingredient preparation method comprises the following steps: and will add ethyl acetate and ethanol (1: 1) after the Rhizoma Chuanxiong pulverizing medicinal materials that reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution; Extracting solution is condensed into extractum, and itself and silica gel mixed sample, with normal phase silicagel column it is separated, at first use petroleum ether and ethyl acetate (50: 1) as mobile phase, get eluent I, abandon it, change chloroform and methanol (10: 1) then as mobile phase, get eluent II, will get sample behind the eluent II concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography; The separation condition of preparative hplc: chromatographic column is preparative column (Zorbax SB-C18; 21.2mm * 250mm), mobile phase is water (A) and acetonitrile (B), the gradient elution program is as follows: in the time of 0 minute, mobile phase A is that 75% aqueous solution, Mobile phase B are 25% acetonitrile solution; In the time of 40 minutes, mobile phase A is that 5% aqueous solution, Mobile phase B are 95% acetonitrile solution; In the time of 50 minutes, mobile phase A is that 5% aqueous solution, Mobile phase B are 95% acetonitrile solution; Flow velocity is 10ml/min, and column temperature is a room temperature; Sample 100% dissolve with ethanol separates through preparative liquid chromatography, collects solution at time period 16.2～24.0min, and solution obtains active component behind concentrate drying.
Chuanxiong rhizome effective ingredient of the present invention can be used as active component, adds the adjuvant of accepting on the pharmaceutics, makes preparation according to the preparation method of the preparation of putting down in writing on the pharmaceutics.
Chuanxiong rhizome effective ingredient of the present invention can also add the adjuvant of accepting on the pharmaceutics as one of active component, makes preparation according to the preparation method of the preparation of putting down in writing on the pharmaceutics.
Described preparation comprises injection, drip liquid, injectable powder, granule, tablet, electuary, powder, oral liquid, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, sucks agent, granule, pill, unguentum, sublimed preparation, spray, drop pill, disintegrating agent, oral cavity disintegration tablet, micropill etc.
Chuanxiong rhizome effective ingredient of the present invention and preparation thereof can be used in preparation treatment, angiocardiopathy preventing medicine.
Beneficial effect of the present invention is:
1. use normal phase silicagel column in the extraction and separation process of the present invention, can remove impurity such as desaccharide, protein, aminoacid effectively, improved content of effective, adopted preparative hplc simultaneously, can obtain effective ingredient fast and accurately.
2. chuanxiong rhizome effective ingredient chemical constituent provided by the invention is simply clear and definite, is easier to illustrate its mechanism of action on pharmacological research, is easier to the quality control of medicine aborning.
Description of drawings
Fig. 1 is the HPLC analysis chart of chuanxiong rhizome effective ingredient of the present invention.
The specific embodiment
Further describe flesh and blood of the present invention below in conjunction with embodiment, this embodiment only is used to the present invention is described and the present invention is not limited.
The preparation of embodiment one chuanxiong rhizome effective ingredient
Ethyl acetate and ethanol will be added after the 200g Rhizoma Chuanxiong pulverizing medicinal materials, heating extraction gets extracting solution, extracting solution is condensed into extractum 18.6g, and itself and silica gel are mixed sample, it is separated with normal phase silicagel column, at first use petroleum ether and ethyl acetate as mobile phase, get eluent I, abandon it, change chloroform and methanol then as mobile phase, get eluent II, will get sample 2.5g behind the eluent II concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography; The separation condition of preparative hplc: chromatographic column is a preparative column, and mobile phase is water and acetonitrile, gradient elution.Sample 100% dissolve with ethanol separates through preparative liquid chromatography, collects solution at time period 16.2～24.0min, obtains active component 0.14g behind the concentrate drying.
The preparation of embodiment two chuanxiong rhizome effective ingredients
Ethyl acetate and ethanol (1: 0.8) will be added after the 500g Rhizoma Chuanxiong pulverizing medicinal materials, reflux 0.8～1.2 hour, extract 1～3 time, merging filtrate gets extracting solution, extracting solution is condensed into extractum 36.8g, and itself and silica gel mixed sample, with normal phase silicagel column it is separated, at first use petroleum ether and ethyl acetate (47: 1) as mobile phase, get eluent I, abandon it, change chloroform and methanol (8: 1) then as mobile phase, get eluent II, will get sample 5.8g behind the eluent II concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography; The separation condition of preparative hplc: chromatographic column is a preparative column, and mobile phase is water and acetonitrile, and gradient elution, flow velocity are 9～11ml/min, and column temperature is a room temperature.Sample 100% dissolve with ethanol separates through preparative liquid chromatography, collects solution at time period 16.2～24.0min, obtains active component 0.33g behind the concentrate drying.
The preparation of embodiment three chuanxiong rhizome effective ingredients
Ethyl acetate and ethanol (1: 1.2) will be added after the 500g Rhizoma Chuanxiong pulverizing medicinal materials, reflux 0.8～1.2 hour, extract 1～3 time, merging filtrate gets extracting solution, extracting solution is condensed into extractum 36.8g, and itself and silica gel mixed sample, with normal phase silicagel column it is separated, at first use petroleum ether and ethyl acetate (53: 1) as mobile phase, get eluent I, abandon it, change chloroform and methanol (13: 1) then as mobile phase, get eluent II, will get sample 5.8g behind the eluent II concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography; The separation condition of preparative hplc: chromatographic column is a preparative column, and mobile phase is water and acetonitrile, and gradient elution, flow velocity are 9～11ml/min, and column temperature is a room temperature.Sample 100% dissolve with ethanol separates through preparative liquid chromatography, collects solution at time period 16.2～24.0min, obtains active component 0.35g behind the concentrate drying.
The preparation of embodiment Sichuan rhizome of chuanxiong active component
Get Rhizoma Chuanxiong medical material 250g, it is pulverized the back add ethyl acetate and ethanol (1: 1), reflux 1 hour is extracted 2 times, filtrate merge extracting solution.Extracting solution is condensed into extractum gets 21g, with extractum and silica gel mixed sample, separate with normal phase silicagel column, at first use petroleum ether and ethyl acetate (50: 1) as mobile phase, get eluent I, change chloroform and methanol (10: 1) then as mobile phase, get eluent II, get the 4.3g sample behind the concentrate drying.Continue to separate the sample that obtains with preparative liquid chromatography.The separation condition of preparative hplc: chromatographic column is Agient preparative column (Zorbax SB-C18; 21.2mm * 250mm), mobile phase is water (A) and acetonitrile (B), the gradient elution program is as follows: in the time of 0 minute, mobile phase A is that 75% aqueous solution, Mobile phase B are 25% acetonitrile solution; In the time of 40 minutes, mobile phase A is that 5% aqueous solution, Mobile phase B are 95% acetonitrile solution; In the time of 50 minutes, mobile phase A is that 5% aqueous solution, Mobile phase B are 95% acetonitrile solution; Flow velocity is 10ml/min, and column temperature is a room temperature.Sample 100% dissolve with ethanol separates through preparative liquid chromatography, collects solution at time period 16.2～24.0min, obtains active component 0.21g behind the concentrate drying.
The analysis of embodiment five chuanxiong rhizome effective ingredients
(1) neocindilide (neocnidilide) in the Rhizoma Chuanxiong extract, the 3-(3,4,5-Trihydroxyphenyl)-2-propenoicacid (assay (high performance liquid chromatography) of 3-(3,4,5-Trihydroxyphenyl)-2-propenoic acid)
Chromatographic conditionChromatographic column Agilent Zorbax SB-C18 post (4.6mm * 150mm, 5 μ m); Adopt gradient elution, mobile phase A is 0.2% glacial acetic acid aqueous solution mutually, and Mobile phase B is mutually for containing the acetonitrile solution of 0.2% glacial acetic acid; The gradient elution program is as follows: in the time of 0 minute, mobile phase A is that 90% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 10% 0.2% glacial acetic acid; In the time of 10 minutes, mobile phase A is that 50% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 50% 0.2% glacial acetic acid; In the time of 30 minutes, mobile phase A is that 5% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 95% 0.2% glacial acetic acid; Flow velocity 0.5mLmin
-1It is long to detect the wavelength all-wave; 30 ℃ of column temperatures; ELSD condition: 100 ℃ of drift tube temperatures; Nitrogen flow rate 1.4L/min
The preparation of need testing solutionTake by weighing this product, in volumetric flask, be diluted to scale, shake up, promptly with dissolve with methanol solution.
Assay methodThe accurate need testing solution of drawing injects chromatograph of liquid, measures, promptly.
(2) above-mentioned Rhizoma Chuanxiong extract HPLC-ELSD finger printing
Assay methodReferring to the neocindilide (neocnidilide) in (1) above-mentioned Rhizoma Chuanxiong extract, 3-(3,4,5-Trihydroxyphenyl)-2-propenoicacid (3-(3,4,5-Trihydroxyphenyl)-2-propenoic acid) assay (high performance liquid chromatography).The record chromatograph time is 30 minutes.
Analysis resultThe HPLC-ELSD analysis of spectra of chuanxiong rhizome effective ingredient is seen Fig. 1, and 1 among Fig. 1,2, number peak are respectively neocindilide (neocnidilide), 3-(3,4,5-Trihydroxyphenyl)-2-propenoicacid (3-(3,4,5-Trihydroxyphenyl)-2-propenoic acid).The relative retention time at two peaks is followed successively by 15.4,16.4.
Among the present invention, the structural formula of neocindilide (neocnidilide) is:
Among the present invention, 3-(3,4,5-Trihydroxyphenyl)-2-propenoicacid (structural formula of 3-(3,4,5-Trihydroxyphenyl)-2-propenoic acid) is:
Embodiment six chuanxiong rhizome effective ingredient preparations
Get chuanxiong rhizome effective ingredient 0.5g and 10.5g Polyethylene Glycol-6000 mix homogeneously of embodiment four, heating and melting moves in the drop pill drip irrigation behind the change material, and in ℃ liquid paraffin of medicine liquid droplet to 6～8, oil removing makes 400 of drop pill.
Embodiment seven chuanxiong rhizome effective ingredient preparations
Get chuanxiong rhizome effective ingredient 0.5g, glucose 4.5g, sodium thiosulfate 0.9g and the distilled water 1ml of embodiment four, behind the said components mix homogeneously, lyophilization, 500 of packing, promptly.
Embodiment eight chuanxiong rhizome effective ingredient preparations
Get Lignum Dalbergiae Odoriferae oil 1.5g, join in the saturated hydroxypropyl of 13ml, stirring and dissolving filters, the filter leaf cold drying, Lignum Dalbergiae Odoriferae oil and the clathrate powder of hydroxypropyl.Except that above-mentioned Lignum Dalbergiae Odoriferae oil closes the clathrate powder of hydroxypropyl, get Rhizoma Chuanxiong extract 0.5g, mannitol 5.5g, calcium disodium edetate 0.9g and the distilled water 2ml of embodiment four again, behind the said components mixing, lyophilization, 300 of packing, promptly.
The pharmacological evaluation of embodiment nine chuanxiong rhizome effective ingredients
1 pharmacological model: neonatal rat myocardial cell hypoxic-ischemic model
Former generation myocardial cell cultivationAfter 1～3 day SD neonatal rat that is born was put to death, it was dirty to core, and is cut into 1mm after PBS liquid cleans
3About fragment.With 0.125% trypsin Sigma, USA)+0.05% (Gibco is USA) in 37 degree digestion 3～4 times for collagenase II.The differential adherent method was separated into fibrocyte after 1.5 hours, and cell suspension is transferred to (every hole about 4 * 10 in 48 orifice plates
5Cell).(Gibco, (Falcon, USA) cell of cultivating 3-6 days supplies medicine efficacy screening USA) to add 10% hyclone through DMEM.Screen and replaced with serum-free medium in preceding 1 day.Cultivate 3～6 days myocardial cell, PBS washing back adds the pastille culture fluid.Place 95%N
2And 5%CO
2Anoxia is 6 hours in the incubator.Then at CO
2Reoxygenation is 3 hours in the incubator.Get cell conditioned medium liquid and measure LDH content.
Dosage regimenExtract the chuanxiong rhizome effective ingredient that obtains according to embodiment four and be made into 10 with sugar-free Hanks liquid
-4G/mL test liquid, liposoluble constituent can add a small amount of DMSO hydrotropy (the DMSO final concentration is controlled at below 0.2%).The final concentration of each component in culture fluid is controlled at 10
-5G/mL.
The lactic acid dehydrogenase assayLactic acid dehydrogenase content can be represented the cell injury degree in the cell culture fluid.The lactic acid dehydrogenase content that bleeds in the supernatant is high more, shows that cell injury is also remarkable.The determination of lactate dehydrogenase test kit builds up bio-engineering research institute available from Nanjing.Assay method is: get 30 microlitre cell conditioned medium liquid, add 50 microlitre substrate buffer and 10 microlitre lactic dehydrogenase enzyme cofactors, 37 degree vibrations 15 minutes add 50 microlitre 2,4 dinitrophenyl hydrazines again, 37 degree vibrations 15 minutes.Parallel blank.Extract reaction solution 50 microlitres and add 200 microlitre 0.4mol/L sodium hydroxide solutions, leave standstill 3～4 minutes after, measure shading value with microplate reader in 450nm.
All data are represented with the mean value variance.Adopt monolateral variance analysis and T check carrying out statistical analysis.Compare with model group, P＜0.05 is thought significantly effectively.Drug effect the results are shown in Table 1.According to myocardial cell lactic acid dehydrogenase (LDH) testing result, chuanxiong rhizome effective ingredient has the highly significant effect to reducing LDH release.
Table 1
2 pharmacological models: huve cell anoxia reoxygenation model
Former generation huve cell cultivationGet the healthy newborn umbilical cord, clean the interior residual bloodstain of vein blood vessel with 30～50mlHanks liquid.Look the 0.1% collagenase I that umbilical cord length adds respective amount, change incubator digestion 15～20 minutes over to.Subsequently Digestive system in the umbilical cord is carefully collected in the beaker, and with Hanks liquid with beaker rinse twice, collect in the lump and be sub-packed in centrifuge tube in the beaker, centrifugal 10 minutes of 900rpm.Supernatant is removed in suction, with the abundant dissolution precipitation of M199 culture fluid (contain 10% hyclone, 100U/ml is two anti-, 0.15mg/ml Heparin, 10uM VC).Gained cell suspension is sub-packed in 5cm
2Culture bottle places in the incubator and cultivates.Changed in second day and adopt the M199 culture fluid that contains 30ug/ml ECGS, changed a culture fluid and be paved with the bottom surface in per afterwards three days until cell.Used cell is former generation endotheliocyte.The culture bottle inner cell is seeded to 96 orifice plates before the modeling and cultivated one day, used cell concentration is 3.5～40,000/hole.During modeling, inhale and abandon old M199 culture fluid, adding pastille does not have phenol red M199 culture fluid, and every hole total amount is 100ul.Place 95%N
2And 5%CO
2Anoxia is 12 hours in the incubator, then at CO
2Reoxygenation is 2 hours in the incubator.Get cell conditioned medium liquid and measure LDH content and NO content.
Dosage regimenExtract the chuanxiong rhizome effective ingredient that obtains according to embodiment four and be made into 10 with sugar-free Hanks liquid
-4G/mL test liquid, liposoluble constituent can add a small amount of DMSO hydrotropy (the DMSO final concentration is controlled at below 0.2%).The final concentration of each component in no phenol red M199 culture fluid is controlled at 10
-5G/mL.
The lactic acid dehydrogenase assayLactic acid dehydrogenase content can be represented the cell injury degree in the cell culture fluid.The lactic acid dehydrogenase content that bleeds in the supernatant is high more, shows that cell injury is also remarkable.The determination of lactate dehydrogenase test kit builds up bio-engineering research institute available from Nanjing.Assay method is: get 30 microlitre cell conditioned medium liquid, add 50 microlitre substrate buffer and 10 microlitre lactic dehydrogenase enzyme cofactors, 37 degree vibrations 15 minutes add 50 microlitre 2,4 dinitrophenyl hydrazines again, 37 degree vibrations 15 minutes.Parallel blank.Extract reaction solution 50 microlitres and add 200 microlitre 0.4mol/L sodium hydroxide solutions, leave standstill 3～4 minutes after, measure shading value with microplate reader in 450nm.
Drug effect resultAll data are represented with the mean value variance.Adopt monolateral variance analysis and T check carrying out statistical analysis.Compare with model group, P＜0.05 is thought significantly effectively.Drug effect the results are shown in Table 2.According to endotheliocyte lactic acid dehydrogenase testing result, chuanxiong rhizome effective ingredient has the highly significant effect to reducing LDH release.
Table 2
Claims (4)
1. a chuanxiong rhizome effective ingredient is characterized in that, adopts following method preparation:
To add ethyl acetate and ethanol after the Rhizoma Chuanxiong pulverizing medicinal materials, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution; Extracting solution is condensed into extractum, and itself and silica gel mixed sample, with normal phase silicagel column it is separated, at first use petroleum ether and ethyl acetate as mobile phase, get eluent I, abandon it, change chloroform and methanol then as mobile phase, get eluent II, will get sample behind the eluent II concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography; The separation condition of preparative hplc: chromatographic column is a preparative column, and mobile phase is water and acetonitrile, and the gradient elution program is as follows: in the time of 0 minute, mobile phase A is that 75% aqueous solution, Mobile phase B are 25% acetonitrile solution; In the time of 40 minutes, mobile phase A is that 5% aqueous solution, Mobile phase B are 95% acetonitrile solution; In the time of 50 minutes, mobile phase A is that 5% aqueous solution, Mobile phase B are 95% acetonitrile solution; Flow velocity is 10ml/min, and column temperature is a room temperature; Sample 100% dissolve with ethanol separates through preparative liquid chromatography, collects solution at time period 16.2～24.0min, and solution obtains active component behind concentrate drying;
The content of neocindilide is 45～55% in the described active component, and the content of 3-(3,4,5-Trihydroxyphenyl)-2-propenoicacid is 25～35%.
2. the method for claim 1, wherein ethyl acetate and alcoholic acid volume ratio are 1: 1, and the volume ratio of petroleum ether and ethyl acetate is 50: 1, and the volume ratio of chloroform and methanol is 10: 1.
3. be the pharmaceutical formulation that active component is made with the described chuanxiong rhizome effective ingredient of claim 1, it is characterized in that the Rhizoma Chuanxiong component adds the adjuvant of pharmaceutics acceptance and makes pharmaceutical formulation according to the preparation method that pharmaceutics allows.
4. the application of the described chuanxiong rhizome effective ingredient of claim 1 in preparation treatment and angiocardiopathy preventing medicine.
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Effective date of registration: 20070511 Address after: Hedong District of Beichen Puji road in Tianjin No. 2 city of the modern Chinese Medicine Applicant after: Tianjin Tianshili Pharmaceutical Co., Ltd. Address before: White road, Beichen science and Technology Park in Tianjin City Xinyi Liaohe Road No. 1 Applicant before: Tianshili Modern Traditional Chinese Medicine Research & Devleopment Co., Ltd., |
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Owner name: TASLY PHARMACEUTICAL GROUP CO., LTD. Free format text: FORMER NAME: TIANJIN TASLY PHARMACEUTICAL CO., LTD. |
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Address after: 300402 Tianjin City Hedong District of Beichen Puji Road No. 2 city of the modern Chinese Medicine Patentee after: Tasly Pharmaceutical Group Co., Ltd. Address before: 300402 Tianjin City Hedong District of Beichen Puji Road No. 2 city of the modern Chinese Medicine Patentee before: Tianjin Tianshili Pharmaceutical Co., Ltd. |
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Address after: 300402 Tianjin City Hedong District of Beichen Puji Road No. 2 city of the modern Chinese Medicine Patentee after: Tasly Pharmaceutical Group Limited by Share Ltd Address before: 300402 Tianjin City Hedong District of Beichen Puji Road No. 2 city of the modern Chinese Medicine Patentee before: Tasly Pharmaceutical Group Co., Ltd. |
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| CF01 | Termination of patent right due to non-payment of annual fee | ||
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Granted publication date: 20101215 Termination date: 20191231 |