CN103980240A - New hydroxysafflor yellow pharmaceutical salt - Google Patents
New hydroxysafflor yellow pharmaceutical salt Download PDFInfo
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- CN103980240A CN103980240A CN201310048480.4A CN201310048480A CN103980240A CN 103980240 A CN103980240 A CN 103980240A CN 201310048480 A CN201310048480 A CN 201310048480A CN 103980240 A CN103980240 A CN 103980240A
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
- C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
- C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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Abstract
The invention provides a new hydroxysafflor yellow A pharmaceutical salt compound shown as formula I, particularly provides a new monomeric compound of potassium hydroxysafflor yellow A and ammonium hydroxysafflor yellow A, and provides a production method and medicinal use of the compound. The hydroxysafflor yellow A pharmaceutical salt monomeric compound is prepared from a medicinal material safflower as a raw material, by use of a strongly acidic H cation exchange resin, hydroxysafflor yellow A is transformed into an acid form, and then by pH adjustment, the hydroxysafflor yellow A pharmaceutical salt monomeric compound is generated from the hydroxysafflor yellow A. The purity higher than 98% can be guaranteed, and compared with the hydroxysafflor yellow A, the new hydroxysafflor yellow A pharmaceutical salt compound is a controllable monomeric compound which is safer, more effective and more stable in application in resisting platelet aggregation, coronary heart disease, angor pectoris, acute cerebral ischemia and other various blood circulation disorders. R is as defined by the specification.
Description
Technical field
The invention provides a kind of new Hydroxy Carthamus yellow pharmaceutical salts, specifically, provide a kind of hydroxyl radical carthamin yellow carthamus A pharmaceutical salts and preparation method thereof, lyophilized injectable powder and medicinal use.Belong to pharmaceutical chemistry field.
Background technology
Chinese medicine safflower is the dried floral of feverfew Carthamus tinctouiusL., is a kind of common activating blood herbs, can be used for the treatment of many disturbance of blood circulation diseases such as coronary heart diseases and angina pectoris.Hydroxyl radical carthamin yellow carthamus A (hydroxysafflor yellow A) is the compound with single cinnamophenone glycoside structure, it is the most effective water soluble part of safflower pharmacological effect, can suppress platelet aggregation and release that platelet activating factor is brought out, contestable ground suppresses the combination of platelet activating factor and platelet receptor, is the effective constituent promoting blood circulation and removing blood stasis of carthamin yellow.Research shows, it has many-sided cardiovascular pharmacological effect, can anti-freezing, promote fibrinolytic, antithrombotic to form, improve microcirculation etc.
Hydroxyl radical carthamin yellow carthamus A is as the highest component of content in carthamin yellow, and its pharmaceutical use is proven in cardiovascular application, and this mechanism is also very clear and definite.In prior art, disclose a large amount of hydroxyl radical carthamin yellow carthamus A production technique, comprised and take safflower as raw material, through steps such as water extraction, macroporous adsorbent resin separation, sephadex chromatography and ultrafiltration, obtained the hydroxyl radical carthamin yellow carthamus A of injection.Yet, the prepared hydroxyl radical carthamin yellow carthamus A of existing production technique, product purity is not high, substantially all there is more than 10% impurity, and the textural property of this class impurity is all failed qualitative, there is certain quality uncontrollability, and affected product, particularly the stability of injecting drug use and security.A kind of method of extracting refining hydroxyl radical carthamin yellow carthamus A from safflower is disclosed in CN102675379A, and specifically disclose process extraction, weak-base ion-exchange resin purifying, intermediate-polarity macroporous adsorption resin purifying and nonpolar macroporous adsorption resin purifying, lyophilize five steps from Chinese medicine safflower, also only obtaining content is more than 80% hydroxyl radical carthamin yellow carthamus A.
Summary of the invention
For overcoming the defect of prior art, the invention provides a kind of hydroxyl radical carthamin yellow carthamus A pharmaceutical salts new compound, its purity can guarantee to reach more than 98%, impurity number is controlled at below 5, safer and more effective compared to hydroxyl radical carthamin yellow carthamus A in the treatment of many disturbance of blood circulation diseases such as being applied to coronary heart diseases and angina pectoris, cerebral apoplexy by becoming, more stablize controlled new monomeric compound.
Technical solution of the present invention is as follows:
One of the object of the invention is to provide a kind of suc as formula the hydroxyl radical carthamin yellow carthamus A pharmaceutical salts shown in (I):
R represents K, NH
4or
Wherein, R1, R2, R3, R4 distinguish identical or different, give from being selected from hydrogen, alkyl; G, G ' represent Position Number.
Wherein, described " alkyl ", carbon atom 1-6 alkyl preferably, such as methyl, ethyl, n-propyl, sec.-propyl etc., is preferably methyl or ethyl.
Preferably, hydroxyl radical carthamin yellow carthamus A pharmaceutical salts described above, R is potassium, it is sylvite, shown in (II):
Preferably, hydroxyl radical carthamin yellow carthamus A pharmaceutical salts described above, R is NH
4, it is ammonium salt, shown in (III):
As another goal of the invention of the present invention, the preparation method of hydroxyl radical carthamin yellow carthamus A pharmaceutical salts described above is also provided, it comprises the extraction of flos carthami, the conversion of strongly-acid H type Zeo-karb, macroporous adsorbent resin separated, sephadex chromatography is separated and the step of ultrafiltration, it is characterized in that:
(1) raw material extracts: flos carthami is raw material, and water extraction obtains the extracting solution that contains hydroxyl radical carthamin yellow carthamus A;
(2) strongly-acid H type Zeo-karb conversion: the extracting solution that step (1) is made is crossed strongly-acid H type cation exchange resin column, collect elutriant, through regulating pH, hydroxyl radical carthamin yellow carthamus A generates hydroxyl radical carthamin yellow carthamus A pharmaceutical salts, collects the elutriant of hydroxyl carthamin yellow A-containing pharmaceutical salts;
(3) macroporous adsorbent resin is separated: the elutriant of the hydroxyl carthamin yellow A-containing pharmaceutical salts that step (2) is made is separated with macroporous adsorptive resins, take water as eluent, collect elutriant, concentrating under reduced pressure, obtains the crude product of hydroxyl radical carthamin yellow carthamus A pharmaceutical salts;
(4) dextrane gel is separated: the hydroxyl radical carthamin yellow carthamus A pharmaceutical salts crude product that step (3) is made is separated with sephadex chromatography, take water as eluent, collects hydroxyl carthamin yellow A-containing pharmaceutical salts elutriant;
(5) ultrafiltration: step (4) gained hydroxyl carthamin yellow A-containing pharmaceutical salts elutriant is concentrated by filtering or the daltonian ultra-filtration membrane of centrifugal rear employing molecular weight cut-off 8000-10000 carries out ultrafiltration and obtains ultrafiltrated, obtain hydroxyl radical carthamin yellow carthamus A pharmaceutical salts after dry.
Wherein, preferably, preparation method described above, wherein said hydroxyl radical carthamin yellow carthamus A pharmaceutical salts is suc as formula the hydroxyl radical carthamin yellow carthamus A potassium shown in (II), Zeo-karb described in step (2) is strongly-acid H type Zeo-karb, it is selected from 001*7 ion exchange resin or macropore HB-8 exchange resin, collects elutriant and regulates pH with potassium hydroxide, and hydroxyl radical carthamin yellow carthamus A generates hydroxyl radical carthamin yellow carthamus A potassium.
Wherein, preferably, preparation method described above, wherein said hydroxyl radical carthamin yellow carthamus A pharmaceutical salts is suc as formula the hydroxyl radical carthamin yellow carthamus A ammonium shown in (III), Zeo-karb described in step (2) is strongly-acid H type Zeo-karb, it is selected from 001*7 ion exchange resin or macropore HB-8 exchange resin, collects elutriant and regulates pH with ammonium hydroxide, and hydroxyl radical carthamin yellow carthamus A generates hydroxyl radical carthamin yellow carthamus A ammonium.
The strongly-acid H type Zeo-karb that the present invention is used; can adopt commercially available strongly-acid H type Zeo-karb; for example 001*7 ion exchange resin or macropore HB-8 exchange resin all can be purchased from Shanghai Huazhen Science and Technology Co., Ltd., and can regenerate with HCl, reuse.
As another object of the present invention, a kind of pharmaceutical composition is also provided, its hydroxyl radical carthamin yellow carthamus A pharmaceutical salts that comprises described in the claim 1-3 that treats significant quantity is that activeconstituents and pharmaceutically acceptable carrier are auxiliary material.
Hydroxyl radical carthamin yellow carthamus A pharmaceutical salts, can be prepared into suitable preparation and use.For example, can make: (1) injection hydroxyl radical carthamin yellow carthamus A pharmaceutical salts lyophilized injectable powder, every bottle contains 50mg-200mg, does not add auxiliary material or add 1: 0.5～1.5 N.F,USP MANNITOL; (2) hydroxyl radical carthamin yellow carthamus A pharmaceutical salts sodium chloride injection, every 100ml sodium chloride injection hydroxyl carthamin yellow A-containing pharmaceutical salts 50mg-200mg; (3) hydroxyl radical carthamin yellow carthamus A pharmaceutical salts glucose injection, every 100ml glucose injection hydroxyl carthamin yellow A-containing pharmaceutical salts 50mg-200mg.
Wherein, preferably pharmaceutical composition described above is lyophilized injectable powder, and prepares by the method comprising the steps:
(1) take flos carthami as raw material, adding temperature is the water extracting of 50～100 ℃, and extracting is with water extracting 2～3 times, each 0.5～24 hour, extracting water consumption was 10～30 times of safflower crude drug weight, the filtering dregs of a decoction after extracting, extracting solution is cooled to 5～30 ℃, standing 2～24 hours;
(2) extracting solution step (1) being made is crossed strongly-acid H type Zeo-karb, flow velocity 1～30ml/min, collects elutriant, through regulating pH, hydroxyl radical carthamin yellow carthamus A generates hydroxyl radical carthamin yellow carthamus A pharmaceutical salts, collects the elutriant of hydroxyl carthamin yellow A-containing pharmaceutical salts;
(3) elutriant step (2) being made is separated with macroporous adsorptive resins, take purified water as eluent, and elution flow rate 10～30ml/min collects elutriant, and concentrating under reduced pressure, obtains hydroxyl radical carthamin yellow carthamus A pharmaceutical salts concentrated solution crude product;
(4) hydroxyl radical carthamin yellow carthamus A pharmaceutical salts concentrated solution crude product step (3) being obtained filters or is centrifugal rear separated with sephadex chromatography, take purified water as eluent, it is 1～10cm/h that elution flow rate is controlled linear rate of flow, collect hydroxyl carthamin yellow A-containing pharmaceutical salts elutriant, concentrating under reduced pressure obtains concentrated solution;
(5) by step (4) gained concentrated solution after filtration or the daltonian ultra-filtration membrane of centrifugal rear employing molecular weight cut-off 8000-10000 carry out ultrafiltration and obtain ultrafiltrated;
(6) by step (5) gained ultrafiltrated through lyophilize, obtain hydroxyl radical carthamin yellow carthamus A pharmaceutical salts fine work;
(7) hydroxyl radical carthamin yellow carthamus A pharmaceutical salts fine work step (6) being obtained is dissolved in water for injection, after adopting the millipore filtration of 0.22 μ m or the daltonian ultrafiltration membrance filter of molecular weight cut-off 8000-10000, be sub-packed in bottle, after lyophilize, obtain hydroxyl radical carthamin yellow carthamus A pharmaceutical salts freeze-dried powder;
Wherein, described Zeo-karb is with 001*7 ion exchange resin or macropore HB-8 exchange resin;
Described macroporous adsorbent resin is to use macroporous adsorbent resin HZ801; Described sephadex chromatography is to use sephadex lh-20.
As embodiment of the present invention, the described hydroxyl radical carthamin yellow carthamus A potassium of a kind of formula (II) and the preparation method of lyophilized injectable powder thereof are provided, it comprises the extraction of flos carthami, the conversion of strongly-acid H type Zeo-karb, macroporous resin separated, sephadex chromatography is separated and the step of ultrafiltration, it is characterized in that: (1) take flos carthami as raw material, adding appropriate temperature is the water extracting of 50～100 ℃, extracting is with water extracting 2～3 times, each 0.5～24 hour, extracting water consumption was 10～30 times of safflower crude drug weight.The filtering dregs of a decoction after extracting, are cooled to 5～30 ℃ by extracting solution:, standing 2～24 hours;
(2) extracting solution is crossed to strongly-acid H type Zeo-karb, flow velocity 1～30ml/min, collects elutriant, with KOH, regulates PH to 4-7, obtains hydroxyl carthamin yellow A-containing potassium elutriant;
(3) macroporous adsorbent resin is separated: the elutriant that step (2) obtains is separated with macroporous adsorbent resin HZ801 post, the ratio that the column internal diameter of macroporous adsorptive resins and post are high is 1: 8～15, take purified water as eluent, elution flow rate 10～30ml/min, collect elutriant, concentrating under reduced pressure, obtains hydroxyl radical carthamin yellow carthamus A potassium concentrated solution crude product;
(4) sephadex chromatography is separated: the hydroxyl radical carthamin yellow carthamus A potassium concentrated solution crude product that step (3) obtains filters or be centrifugal rear separated with Sephadex LH-20 sephadex chromatography, the blade diameter length ratio of chromatography column is 1: 5～20, take purified water as eluent, it is 1～10cm/h that elution flow rate is controlled linear rate of flow, collect hydroxyl carthamin yellow A-containing potassium elutriant, concentrating under reduced pressure obtains concentrated solution;
(5) ultrafiltration: step (4) gained concentrated solution after filtration or the daltonian ultra-filtration membrane of centrifugal rear employing molecular weight cut-off 8000-10000 carry out ultrafiltration and obtain ultrafiltrated;
(6) freeze-drying: step (5) gained ultrafiltrated, through lyophilize, obtains hydroxyl radical carthamin yellow carthamus A potassium.
(7) hydroxyl radical carthamin yellow carthamus A potassium fine work step (6) being obtained, be dissolved in water for injection, after adopting the millipore filtration of 0.22 μ m or the daltonian ultrafiltration membrance filter of molecular weight cut-off 8000-10000, be sub-packed in bottle, after lyophilize, obtain hydroxyl radical carthamin yellow carthamus A potassium freeze-dried powder.
Described exchange resin is with 001*7 ion exchange resin or macropore HB-8 exchange resin;
Described macroporous resin separation is to use macroporous adsorbent resin HZ801;
Described sephadex chromatography separation is with sephadex lh-20;
Described ultrafiltration is with the daltonian ultra-filtration membrane of molecular weight cut-off 8000-10000.
As another embodiment of the present invention, the described hydroxyl radical carthamin yellow carthamus A ammonium of a kind of formula (III) and the preparation method of lyophilized injectable powder thereof are provided, it comprises the extraction of flos carthami, the conversion of storng-acid cation exchange resin, macroporous resin separated, sephadex chromatography is separated and the step of ultrafiltration, it is characterized in that: (1) take flos carthami as raw material, adding appropriate temperature is the water extracting of 50～100 ℃, extracting is with water extracting 2～3 times, each 0.5～24 hour, extracting water consumption was 10～30 times of safflower crude drug weight.The filtering dregs of a decoction after extracting, are cooled to 5～30 ℃ by extracting solution:, standing 2～24 hours;
(2) extracting solution is crossed to storng-acid cation exchange resin, flow velocity 1～30ml/min, the ammonium hydroxide for extracting solution of collection (being ammoniacal liquor) regulates between PH to 4-7, obtains the elutriant of hydroxyl carthamin yellow A-containing ammonium; (3) macroporous adsorbent resin is separated: the elutriant that step (2) obtains is separated with macroporous adsorbent resin HZ801 post, the ratio that the column internal diameter of macroporous adsorptive resins and post are high is 1: 8～15, take purified water as eluent, elution flow rate 10～30ml/min, collect elutriant, concentrating under reduced pressure, obtains hydroxyl radical carthamin yellow carthamus A ammonium salt concentrated solution crude product;
(4) sephadex chromatography is separated: the hydroxyl radical carthamin yellow carthamus A ammonium salt concentrated solution crude product that step (3) obtains filters or be centrifugal rear separated with Sephadex LH-20 sephadex chromatography, the blade diameter length ratio of chromatography column is 1: 5～20, take purified water as eluent, it is 1～10cm/h that elution flow rate is controlled linear rate of flow, collect hydroxyl carthamin yellow A-containing ammonium salt elutriant, concentrating under reduced pressure obtains concentrated solution;
(5) ultrafiltration: step (4) gained concentrated solution after filtration or the daltonian ultra-filtration membrane of centrifugal rear employing molecular weight cut-off 8000-10000 carry out ultrafiltration and obtain ultrafiltrated;
(6) freeze-drying: step (5) gained ultrafiltrated, through lyophilize, obtains hydroxyl radical carthamin yellow carthamus A ammonium salt.
(7) hydroxyl radical carthamin yellow carthamus A ammonium salt fine work step (6) being obtained, be dissolved in water for injection, after adopting the millipore filtration of 0.22 μ m or the daltonian ultrafiltration membrance filter of molecular weight cut-off 8000-10000, be sub-packed in bottle, after lyophilize, obtain hydroxyl radical carthamin yellow carthamus A ammonium salt freeze-dried powder.
Described ion exchange resin is with 001*7 ion exchange resin or macropore HB-8 exchange resin.
As another goal of the invention of the present invention, hydroxyl radical carthamin yellow carthamus A pharmaceutical salts described above application in preparing medicine is also provided, wherein said medicine has the platelet aggregation of anti-PAF or ADP induction, is used for the treatment of or prevents to relate to damage disease due to myocardial ischemia, cerebral ischemia, thrombosis.The clinical application dosage of hydroxyl radical carthamin yellow carthamus A pharmaceutical salts of the present invention is 50-200mg/ every day.
The present invention be take flos carthami as raw material, prepared novel monomeric medicine hydroxyl radical carthamin yellow carthamus A pharmaceutical salts, purity can guarantee to reach more than 98%, impurity number is controlled at below 5, safer and more effective compared to hydroxyl radical carthamin yellow carthamus A in the treatment that is applied to many disturbance of blood circulation diseases such as coronary heart diseases and angina pectoris by becoming, more stablize controlled monomeric compound.
Repetition test research of the present invention is found, hydroxyl radical carthamin yellow carthamus A in flos carthami extracting solution does not exist with sour form, prior art regulates pH value by carthamin yellow extracting solution, hydroxyl radical carthamin yellow carthamus A can not transform and generate hydroxyl radical carthamin yellow carthamus A pharmaceutical salts, therefore can not get hydroxyl radical carthamin yellow carthamus A pharmaceutical salts monomeric compound.The unexpected discovery of the present invention, carthamin yellow extracting solution obtains hydroxyl radical carthamin yellow carthamus A truly after transforming with strongly-acid H type Zeo-karb, with sour form, then by different alkali, regulate pH, conversion obtains the corresponding salt of hydroxyl radical carthamin yellow carthamus A, obtains single hydroxyl radical carthamin yellow carthamus A pharmaceutical salts monomeric compound.
Pharmacodynamic study test
Pharmacodynamic study test one:
The provide protection of hydroxyl radical carthamin yellow carthamus A potassium to acute myocardial infarction of rat
Tested medicine
Safflower Yellow Injection (50mg/ bottle), source: Zhejiang Yongning Pharmaceutical Co., Ltd, content: 50mg/ bottle hydroxyl carthamin yellow A-containing 42.5mg.
Hydroxyl radical carthamin yellow carthamus A potassium (obtained according to embodiment 1)
Animal: 24 of male SD rats, body weight 250～350g.
Test grouping and dosage setting:
Test method
1. the preparation of acute myocardial infarction of rat model: with 3% vetanarcol 45mg/kg intraperitoneal anesthesia.Right side Femoral arterial and venous cannulation is used as respectively monitoring of blood pressure and intravenously administrable; The capable Artificial controlled mechanical ventilation of trachea cannula, 60 times/min of frequency, the about 10ml/kg of air flow.The subcutaneous pin electrode of inserting of four limbs is with recording ecg.About 0.5cm place longitudinal incision skin outside left border of sternum, blunt separation subcutis, separated, ligation cut off pectoralis major, musculus pectoralis minor, intercostal muscle successively, at the strongest external-open chest of left border of sternum the 4th intercostal apex beat, cut off pericardium, with mosquito forceps clamping pericardium, in wall of the chest both sides, form pericardium bed, expose left ventricle vascular surface.6-0 silk thread is through the superficial layer of myocardium (being anterior descending coronary) at about 2mm place under left auricle of heart.After threading, stablize 15min.So, occur that irregular pulse or systolic arterial pressure continue to surpass 5min lower than 70mmHg (9.31KPa) and abolish this animal, the administration of threading 15min posterior vein, ligation coronary artery after administration 10min, ligation time remaining 4 hours.
2. irregular pulse scoring: to occurring in 30min after coronary ligation that irregular pulse severity marks.
3. Myocardial Enzymologic inspection: the about 2ml of off-test extracting vein blood, the centrifugal 5min of 4000/min at 4 ℃, gets supernatant liquor and do Myocardial Enzymologic inspection.Mensuration project: LDH, AKP, CK.
4. myocardial infarction district area estimation: after coronary ligation after 4h in the puncture of left ventricle antetheca, after injection burnt black ink, from putting to death animal, take out heart, with physiological saline, clean, remove blood stains and reject blood vessel, fatty Deng Fei cardiac muscular tissue, with thieving paper, suck moisture, weigh.From the apex of the heart to the heart, bottom is parallel is cut into about 2mm left and right thin slice by ventricle, perfusion area (ink perfusion part) is separated with ischemic hazardous area (without ink perfusion part), weighed in ischemic hazardous area and insert in 0.05% NBT solution, 15min dyes in 37 ℃ of constant water bath box.NBT can make matrix, coenzyme and the deoxygenase etc. in living tissue dye hyacinthine, and the forfeiture such as these metabolism matrix, enzyme in necrotic tissue, therefore not painted.Can see non-infarcted region and be dyed mazarine by NBT, infarcted region is not colored.Cut off the non-infarcted myocardium being colored in each cardiac muscle, the stalk heart cardiac muscle that end is colored is weighed, and calculates the weight percent (weight/hazardous area, infarcted region weight * 100%) that infarcted myocardium accounts for hazardous area cardiac muscle.
Statistical procedures
Testing data represents with mean ± standard deviation, adopts variance analysis to carry out statistical test, and P < 0.05 represents significant difference.
One, on ARR impact after Acute Myocardial Ischemia in Rats
After rat coronary ligation, 5min starts to have irregular pulse to occur, continues to 30min, and 10min peaks left and right.This test-results shows that carthamin yellow and hydroxyl radical carthamin yellow carthamus A potassium intravenously administrable all can reduce ARR severity, but the effect of hydroxyl radical carthamin yellow carthamus A potassium is better.
Each experimental group irregular pulse scoring
| Group | n | Irregular pulse scoring |
| Physiological saline group | 8 | 3.25±0.7 |
| Safflower Yellow Injection group | 8 | 1.63±0.7* |
| Hydroxyl radical carthamin yellow carthamus A potassium | 8 | 1.38±0.5* |
Two, relatively carthamin yellow and hydroxyl radical carthamin yellow carthamus A potassium affect the impact of each experimental group on myocardial infarct size to rat myocardial infarction model scope
* P < 0.05vs group 1
Three, Safflower Yellow Injection on rat myocardial infarction model after the impact of Serum LDH, AKP, CK
This test-results shows that Safflower Yellow Injection and hydroxyl radical carthamin yellow carthamus A potassium intravenously administrable all can suppress the rising of rat blood serum LDH, CK, but hydroxyl radical carthamin yellow carthamus A potassium more remarkable effect.
Each experimental group Serum LDH, AKP, CK value
* p < 0.05vs group 1
Conclusion (of pressure testing)
1. Safflower Yellow Injection and hydroxyl radical carthamin yellow carthamus A potassium all can obviously reduce ARR severity after rat coronary ligation, but the better effects if of hydroxyl radical carthamin yellow carthamus A potassium.
2. Safflower Yellow Injection and the comparison of hydroxyl radical carthamin yellow carthamus A potassium, it is better that hydroxyl radical carthamin yellow carthamus A potassium reduces rat myocardial infarction model range effect.
3. with Safflower Yellow Injection comparison, the curative effect that hydroxyl radical carthamin yellow carthamus A potassium suppresses rat blood serum LDH, CK is more obvious.
Pharmacodynamic study test two:
Hydroxyl radical carthamin yellow carthamus A potassium intravenous administration has preventive and therapeutic effect to acute cerebral ischemia.
Tested medicine
Safflower Yellow Injection (50mg/ bottle), source: Zhejiang Yongning Pharmaceutical Co., Ltd, content: 50mg/ bottle hydroxyl carthamin yellow A-containing 42.5mg.
Hydroxyl radical carthamin yellow carthamus A potassium (obtained according to embodiment 1)
Experiment content is as follows:
1. the in vitro heart of pair dog, cerebrovascular selectivity: this experiment is fixed on Beagle dog willis' arteries and coronary artery ring on myocardium vessel measuring apparatus, adjust tension pick-up and also to bathing in cup liquid, add 10
-6the phyenlephrinium of mol/L makes blood vessel maintain moderate tension, then at interval of 5min, to bathing in cup liquid, by the dosage of every milliliter of 10mg, add injection hydroxyl radical carthamin yellow carthamus A potassium, until vascular circle reaction is very weak or no longer occur reaction (general dosing number of times is 4-5 time).Calculate vasoconstriction or diastole changing value.Experimental result, injection hydroxyl radical carthamin yellow carthamus A potassium act as 31.6% to the diastole of cardiac blood pipe ring, and the diastole of cerebrovascular ring is act as to 73.1%, illustrates that injection hydroxyl radical carthamin yellow carthamus A potassium has extraordinary selectivity and diastole effect to the cerebrovascular.
2. the impact on acute cerebral ischemia: experiment use SD rat, after intravenous injection hydroxyl radical carthamin yellow carthamus A potassium by conventional arteria cerebri media embolism (MCAO) line bolt legal system for acute cerebral ischemia model.After raising 24h, first it is carried out to neuroethology scoring, then sacrificed by decapitation rat, takes out brain, put into mould and be cut into 7, give TTC dyeing, survival cerebral tissue is dyed to redness, necrotic brain tissues is not painted, by pattern analysis computed in software necrotic brain tissues, accounts for Interhemispheric ratio.Test-results, brain infarction area solvent control group is 38%, nimodipine positive controls is 15.7%, the low middle Senior Three of an injection hydroxyl radical carthamin yellow carthamus A potassium dosage group is respectively 38.2%, 27.6% and 21.9%, has and significantly alleviates the effect that acute cerebral ischemia causes cerebral tissue necrosis with the relatively middle high dosage of solvent control group.
3. the impact on rat brain vascular permeability: rat intravenous injection administration, once a day, continuous 7 days, after last administration by rat anesthesia, intravenous injection Evans Blue 50mg/kg, ligation bilateral common carotid arteries after 5 minutes, sacrificed by decapitation animal after 3 hours, take out brain, after weighing, be soaked in formamide soln, put 72h in 45 ℃ of thermostat containers, now the Evans Blue in the cerebrovascular can leach in formamide soln, with spectrophotometer detect the Evans Blue amount of separating out in formamide soln number represent the height of cerebrovascular permeability.Experimental result shows: the effect of overflowing of the minimizing Evans Blue that in injection hydroxyl radical carthamin yellow carthamus A potassium, high dose group has a highly significant from the cerebrovascular, illustrates that this medicine has better effect to reducing vascular permeability.
4. the impact on dog cerebral blood flow (CBF): laboratory animal is used Beagle dog, dog is isolated a side external jugular vein, internal jugular vein and vertebral artery with operation after vetanarcol anesthesia, ligation external jugular vein, place flow probe at internal jugular vein and vertebral artery, the volume of blood flow of two place's probes records is added the full cerebral blood supply amount of 2 representative of taking advantage of, and experiment end taking-up brain is weighed and calculated every 100g cerebral tissue volume of blood flow.Test-results, injection hydroxyl radical carthamin yellow carthamus A potassium after intravenously administrable in two groups of high dosage be significantly increased the effect of volume of blood flow, but volume of blood flow increase is held time shorter (about 15min), this experiment prompting is from now in the clinical method administration that should select intravenous drip while being used for the treatment of acute cerebral ischemia.
5. whether the impact on acute cerebral hypoxia: experiment is carried out with Kunming mouse and SD rat, and animal is put into anaerobic environment, records the survival time, can increase the tolerance to acute anoxia after understanding animal-use drug.Experiment is found: mouse is in airtight container, the solvent control group survival time is 32min, and the mouse survival time of the low middle Senior Three of an injection hydroxyl radical carthamin yellow carthamus A potassium dosage group is respectively 36,37,36min, learns by statistics and process that relatively there were significant differences (P < 0.05～0.01) with solvent control group; Rat experiment carries out in the environment of the nitrogen containing 97% and 3% oxygen, and animal arrives breath stopped after putting into container, and respectively survival time of group is, solvent control group average 3 minutes and 43 seconds; Positive controls (nimodipine) is 5 minutes and 38 seconds, compares difference highly significant between the two; The low middle Senior Three of an injection hydroxyl radical carthamin yellow carthamus A potassium dosage group is respectively 3 minutes 20 seconds, 4 minutes and 30 seconds and 4 minutes and 9 seconds, and the relatively middle high dose group survival time significant difference of solvent control group.
6. antiplatelet is built up: experiment is divided into instrument detection and experiment made on the living two portions, 1) instrument detection method: rabbit vein injection hydroxyl radical carthamin yellow carthamus A potassium, continuous 5 days once a day, last administration finishes in 2 hours from heart extracting blood 4ml, and blood obtains being rich in hematoblastic serum through low-speed centrifugal; Through high speed centrifugation, obtain anaemia platelet serum, thrombocyte is built up inductor and is selected adenosine diphosphate (ADP) (adenosine diphosphate; ADP) and two kinds of platelet aggregation activation factors (Platelet-Activating Factor PAF), with platelet aggregation instrument, test.In the platelet aggregation test of the anti-ADP of hydroxyl radical carthamin yellow carthamus A potassium injection liquid and PAF induction, all show extraordinary antiplatelet aggregative activity, between three dosage, demonstrate good amount-result relation and exist.2) intravital method: form short circuit with emulsion tube between rat arteriovenous, in emulsion tube, be fixed with operation silk thread, utilize the feature of platelet adhesion reaction, open short circuit makes blood flow 15 minutes across arteriovenous through emulsion tube, taking-up silk thread is weighed, deducting silk thread dry weight is the thrombocyte weight sticking on silk thread, experimental result, the thrombocyte weight solvent control group adhering on silk thread is 14.8 ± 1.57mg, positive controls is 8.62 ± 2.79mg, the adhesion thrombocyte weight of the low middle Senior Three of an injection hydroxyl radical carthamin yellow carthamus A potassium dosage group is respectively 13.6 ± 1.89mg, 9.90 ± 1.53mg and 8.91 ± 1.34mg, middle high dose group and solvent control group relatively differ very obvious, illustrate that injection hydroxyl radical carthamin yellow carthamus A potassium has the effect of extraordinary anti-rat platelet aggregation.
7. the impact on blood viscosity: rabbit vein administration, once a day, continuous 5 days, directly detects with blood rheological instrument after last administration heart extracting blood anti-freezing in 2 hours.Test-results, compare hydroxyl radical carthamin yellow carthamus A potassium treatment group along with the increase of dosage with solvent control group, blood viscosity is low to be cut, in cut and height is cut three indexs and also decreased, reduction amplitude increases with dosage, high dose group effect is better than positive control drug nimodipine injection liquid group, illustrates that injection hydroxyl radical carthamin yellow carthamus A potassium has remarkable effect to reducing blood viscosity.
Two, stability test
Test objective: observe hydroxyl radical carthamin yellow carthamus A potassium, hydroxyl radical carthamin yellow carthamus A ammonium and hydroxyl radical carthamin yellow carthamus A stability
Tested medicine
Hydroxyl radical carthamin yellow carthamus A is pressed CN102675379A method self-control purity 89.9%
Hydroxyl radical carthamin yellow carthamus A ammonium self-control (pressing embodiment 3 preparations) purity 99.4%
Hydroxyl radical carthamin yellow carthamus A potassium self-control (pressing embodiment 1 preparation) purity 99.4%
Test-results shows that the stability comparison test of hydroxyl radical carthamin yellow carthamus A pharmaceutical salts monomeric compound of the present invention and hydroxyl radical carthamin yellow carthamus A illustrates that the stability of hydroxyl radical carthamin yellow carthamus A pharmaceutical salts is better.
Embodiment
Embodiment 1: hydroxyl radical carthamin yellow carthamus A potassium (in general formula I, R is potassium)
Take safflower, add the deionized water of 12.5 times of medicinal material weight, in 100 ℃ of extraction 20-25 minute, filter, the deionized water that filter residue adds 10 times of medicinal material weight repeats to extract once by above-mentioned condition again, filters.Merge secondary raffinate, be cooled to room temperature, after using whizzer centrifugal, extracting centrifugal liquid is standby.Above-mentioned centrifugate is slowly added to the 001*7 strongly-acid H type Zeo-karb that processed balance is good, post blade diameter length ratio is 1: 10, column volume is 500ml, flow velocity is 3ml/min, collect effluent liquid, with KOH, regulate PH to 4-7, be then slowly added in macroporous adsorbent resin separator column, post blade diameter length ratio is 1: 12, and loading flow velocity is per minute 10ml.After end of the sample, use the deionized water of normal temperature with the flow velocity wash-out of per minute 20ml.Elutriant, in 60 ℃ of concentrating under reduced pressure, obtains hydroxyl radical carthamin yellow carthamus A potassium crude product concentrated solution.In safflower, per kilogram safflower obtains concentrated solution 100ml.Gel LH-20 post on hydroxyl radical carthamin yellow carthamus A potassium crude product concentrated solution, the blade diameter length ratio of post is 1: 5, and applied sample amount is column volume 10%, and elution flow rate is per minute 5ml, collects hydroxyl carthamin yellow A-containing potassium part.Collect liquid after 60 ℃ of concentrating under reduced pressure, obtain hydroxyl radical carthamin yellow carthamus A potassium fine work concentrated solution, in safflower, per kilogram safflower obtains concentrated solution 35～50ml, through lyophilize, obtains flaxen hydroxyl radical carthamin yellow carthamus A potassium fine work powder, purity is 98.5%, and yield is counted 0.55% left and right by safflower.
The infrared spectra of hydroxyl radical carthamin yellow carthamus A potassium (IR), mass spectrum MS, nucleus magnetic resonance
1h-NMR,
13c-NMR data are as follows:
1. infrared absorption spectrum
Instrument model: Bruker VECTOR-22 type infrared absorption spectrometer
IR (pressing potassium bromide troche)
2. mass spectrum
Instrument model: the multistage ion trap mass spectrometry system of U.S. FINNIGAN company LC-MS (LCQ-DECAXP)
Test condition: ESI
Mass spectrum MS
+c?ESI651.06(M)
+
-c?ESI611.24(M-K)
-
3. proton nmr spectra and carbon are composed
Instrument model: BRUCKERAVANCE III500 type NMR spectrometer with superconducting magnet
Test condition: solvent: DMSO, interior mark: TMS
Hydroxyl radical carthamin yellow carthamus A potassium
1h mono-NMR data
| Proton sequence number (H ownership) | Chemical shift δ (ppm) | Proton number |
| 8 | 7.27 | 1 |
| 9 | 7.39 | 1 |
| 11，15 | 7.41 | 2 |
| 12，14 | 6.75 | 2 |
| 3-OH | 18.65 | 1 |
| 4-OH | 4.71 | 1 |
| 5-OH | Disappear | Be substituted |
| 13-OH | 9.75 | 1 |
| Sugar moieties | ? | ? |
| G1 | 3.61 | 1 |
| G2 | 2.84 | 1 |
| G3 | 3.06 | 1 |
| G4 | 3.27 | 1 |
| G5 | 3.01 | 1 |
| G6 | 3.36～3.25 | 2 |
| G’1 | 4.14 | 1 |
| G’2 | 4.03 | 1 |
| G’3 | 3.08 | 1 |
| G’4 | 3.04 | 1 |
| G’5 | 2.92 | 1 |
| G’6 | 3.58 | 2 |
| Hydroxyl on sugar | 4.41～4.79 | 8 |
Hydroxyl radical carthamin yellow carthamus A sylvite
13c mono-NMR data
| Carbon sequence number | Chemical shift (ppm) |
| 1 | 189.6 |
| 2 | 106.7 |
| 3 | 195.9 |
| 4 | 86.1 |
| 5 | 183.2 |
| 6 | 99.7 |
| 7 | 179.3 |
| 8 | 123.9 |
| 9 | 135.9 |
| 10 | 127.9 |
| 11(15) | 129.7 |
| 12(14) | 116.0 |
| 13 | 158.8 |
| G1 | 85.9 |
| G2 | 70.4 |
| G3 | 78.8 |
| G4 | 70.3 |
| G5 | 81.2 |
| G6 | 61.7 |
| G’1 | 74.4 |
| G’2 | 69.2 |
| G’3 | 79.7 |
| G’4 | 71.6 |
| G’5 | 80.8 |
| G’6 | 62.2 |
By prepared hydroxyl radical carthamin yellow carthamus A potassium fine work, be dissolved in water for injection, after adopting the millipore filtration of 0.22 μ m or the daltonian ultrafiltration membrance filter of molecular weight cut-off 8000-10000, be sub-packed in bottle, after lyophilize, obtain hydroxyl radical carthamin yellow carthamus A potassium freeze-dried powder.
Embodiment 2: hydroxyl radical carthamin yellow carthamus A potassium (in formula I, R is potassium)
Take safflower, add the deionized water of 12.5 times of medicinal material weight, in 100 ℃ of extraction 20-25 minute, filter, the deionized water that filter residue adds 10 times of medicinal material weight repeats to extract once by above-mentioned condition again, filters.Merge secondary raffinate, be cooled to room temperature, after using whizzer centrifugal, extracting centrifugal liquid is standby.Above-mentioned centrifugate is slowly added to the HB-8 macropore strong acid H type Zeo-karb that processed balance is good, post blade diameter length ratio is 1: 10, column volume is 500ml, flow velocity is 3ml/min, collect effluent liquid, with KOH, regulate PH to 4-7, be then slowly added in macroporous adsorbent resin separator column, post blade diameter length ratio is 1: 12, and loading flow velocity is per minute 10ml.After end of the sample, use the deionized water of normal temperature with the flow velocity wash-out of per minute 20ml.Elutriant, in 60 ℃ of concentrating under reduced pressure, obtains hydroxyl radical carthamin yellow carthamus A potassium crude product concentrated solution.In safflower, per kilogram safflower obtains concentrated solution 100ml.Gel LH-20 post on hydroxyl radical carthamin yellow carthamus A potassium crude product concentrated solution, the blade diameter length ratio of post is 1: 5, and applied sample amount is column volume 10%, and elution flow rate is per minute 5ml, collects hydroxyl carthamin yellow A-containing potassium part.Collect liquid after 60 ℃ of concentrating under reduced pressure, obtain hydroxyl radical carthamin yellow carthamus A potassium fine work concentrated solution, in safflower, per kilogram safflower obtains concentrated solution 35～50ml, through lyophilize, obtains flaxen hydroxyl radical carthamin yellow carthamus A potassium fine work powder, purity is 98.6%, and yield is counted 0.50% left and right by safflower.
Embodiment 3: (in formula I, R is NH to the hydroxyl radical carthamin yellow carthamus A ammonium of formula III
4)
Take safflower, add the deionized water of 12.5 times of medicinal material weight, in 100 ℃ of extraction 20-25 minute, filter, the deionized water that filter residue adds 10 times of medicinal material weight repeats to extract once by above-mentioned condition again, filters.Merge secondary raffinate, be cooled to room temperature, after using whizzer centrifugal, extracting centrifugal liquid is standby.Above-mentioned centrifugate is slowly added to the 001*7 strongly-acid H type Zeo-karb that processed balance is good, post blade diameter length ratio is 1: 10, column volume is 500ml, flow velocity is 3ml/min, collect effluent liquid, with ammoniacal liquor, regulate PH to 4-7, be then slowly added in macroporous adsorbent resin separator column, post blade diameter length ratio is 1: 12, and loading flow velocity is per minute 10ml.After end of the sample, use the deionized water of normal temperature with the flow velocity wash-out of per minute 20ml.Elutriant, in 60 ℃ of concentrating under reduced pressure, obtains hydroxyl radical carthamin yellow carthamus A ammonium crude product concentrated solution.In safflower, per kilogram safflower obtains concentrated solution 100ml.Gel LH-20 post on hydroxyl radical carthamin yellow carthamus A ammonium crude product concentrated solution, the blade diameter length ratio of post is 1: 5, and applied sample amount is column volume 10%, and elution flow rate is per minute 5ml, collects hydroxyl carthamin yellow A-containing potassium part.Collect liquid after 60 ℃ of concentrating under reduced pressure, obtain hydroxyl radical carthamin yellow carthamus A ammonium fine work concentrated solution, in safflower, per kilogram safflower obtains concentrated solution 35～50ml, through lyophilize, obtains flaxen hydroxyl radical carthamin yellow carthamus A ammonium fine work powder, purity is 99.3%, and yield is counted 0.45% left and right by safflower.
The infrared spectra of hydroxyl radical carthamin yellow carthamus A ammonium (IR), mass spectrum MS, nucleus magnetic resonance
1h-NMR,
13c-NMR data are as follows:
1. infrared absorption spectrum
Instrument model: Bruker VECTOR-22 type infrared absorption spectrometer
IR (pressing potassium bromide troche)
| Absorption peak cm -1 | Oscillatory type | Group | Intensity |
| 3361 | υ -OH | -OH | br?s |
| 1650 | υ C＝O | -C＝O | s |
| 1624 | υ C＝C | -C＝C- | s |
| 16041515 | υ C＝C | C 6H 6 | s |
| 1440 | δ -CH2 | -CH 2 | m |
| 11711077 | υ C-O | -C-OH | s |
| 1005 | ? | ? | ? |
2. mass spectrum
Instrument model: the multistage ion trap mass spectrometry system of U.S. FINNIGAN company LC-MS (LCQ-DECAXP)
Test condition: ESI
Mass spectrum MS
+c?ESI613.17(M)
+
-c?ESI611.22(M-H)
-
3. proton nmr spectra and carbon are composed
Instrument model: BRUCKERAVANCE III500 type NMR spectrometer with superconducting magnet
Test condition: solvent: DMSO, interior mark: TMS
Nucleus magnetic resonance
Hydroxyl radical carthamin yellow carthamus A ammonium
1h mono-NMR data
| Proton sequence number (H ownership) | Chemical shift δ (ppm) | Proton number |
| 8 | 7.26 | 1 |
| 9 | 7.39 | 1 |
| 11，15 | 7.40 | 2 |
| 12，14 | 6.76 | 2 |
| 3-OH | 18.65 | 1 |
| 4-OH | 4.76 | 1 |
| 5-ONH4 | 6.99-7.23 | 4 |
| 13-OH | 9.74 | 1 |
| Sugar moieties | ? | ? |
| G1 | 3.61 | 1 |
| G2 | 2.81 | 1 |
| G3 | 3.06 | 1 |
| G4 | 3.27 | 1 |
| G5 | 3.00 | 1 |
| G6 | 3.35-3.24 | 2 |
| G’1 | 4.15 | 1 |
| G’2 | 4.04 | 1 |
| G’3 | 3.06 | 1 |
| G’4 | 3.04 | 1 |
| G’5 | 2.91 | 1 |
| G’6 | 3.60 | 2 |
| Hydroxyl on sugar | 4.41～4.77 | 8 |
Hydroxyl radical carthamin yellow carthamus A ammonium
13c mono-NMR data
| Carbon sequence number | Chemical shift (ppm) |
| 1 | 189.3 |
| 2 | 107.0 |
| 3 | 196.0 |
| 4 | 86.1 |
| 5 | 183.7 |
| 6 | 189.2 |
| 7 | 179.0 |
| 8 | 123.8 |
| 9 | 135.9 |
| 10 | 127.9 |
| 11(15) | 129.7 |
| 12(14) | 116.0 |
| 13 | 158.8 |
| G1 | 85.8 |
| G2 | 70.5 |
| G3 | 78.9 |
| G4 | 70.4 |
| G5 | 81.2 |
| G6 | 61.9 |
| G’1 | 74.4 |
| G’2 | 69.2 |
| G’3 | 79.7 |
| G’4 | 71.7 |
| G’5 | 80.9 |
| G’6 | 62.3 |
Embodiment 4: hydroxyl radical carthamin yellow carthamus A ammonium (is that in formula I, R is NH
4)
Take safflower, add the deionized water of 12.5 times of medicinal material weight, in 100 ℃ of extraction 20-25 minute, filter, the deionized water that filter residue adds 10 times of medicinal material weight repeats to extract once by above-mentioned condition again, filters.Merge secondary raffinate, be cooled to room temperature, after using whizzer centrifugal, extracting centrifugal liquid is standby.Above-mentioned centrifugate is slowly added to the HB-8 macropore strong acid H type Zeo-karb that processed balance is good, post blade diameter length ratio is 1: 10, column volume is 500ml, flow velocity is 3ml/min, collect effluent liquid, with ammoniacal liquor, regulate PH to 4-7, be then slowly added in macroporous adsorbent resin separator column, post blade diameter length ratio is 1: 12, and loading flow velocity is per minute 10ml.After end of the sample, use the deionized water of normal temperature with the flow velocity wash-out of per minute 20ml.Elutriant, in 60 ℃ of concentrating under reduced pressure, obtains hydroxyl radical carthamin yellow carthamus A ammonium crude product concentrated solution.In safflower, per kilogram safflower obtains concentrated solution 100ml.Gel LH-20 post on hydroxyl radical carthamin yellow carthamus A ammonium crude product concentrated solution, the blade diameter length ratio of post is 1: 5, and applied sample amount is column volume 10%, and elution flow rate is per minute 5ml, collects hydroxyl carthamin yellow A-containing ammonium part.Collect liquid after 60 ℃ of concentrating under reduced pressure, obtain hydroxyl radical carthamin yellow carthamus A ammonium fine work concentrated solution, in safflower, per kilogram safflower obtains concentrated solution 35～50ml, through lyophilize, obtains flaxen hydroxyl radical carthamin yellow carthamus A ammonium fine work powder, purity is 99.4%, and yield is counted 0.50% left and right by safflower.
Embodiment 5: hydroxyl radical carthamin yellow carthamus A organic amine salt (hydroxyl radical carthamin yellow carthamus A triethylamine) (be in formula I, R1 is hydrogen, and R2, R3, R4 are ethyl)
Take safflower, add the deionized water of 12.5 times of medicinal material weight, in 100 ℃ of extraction 20-25 minute, filter, the deionized water that filter residue adds 10 times of medicinal material weight repeats to extract once by above-mentioned condition again, filters.Merge secondary raffinate, be cooled to room temperature, after using whizzer centrifugal, extracting centrifugal liquid is standby.Above-mentioned centrifugate is slowly added to the 001*7 strongly-acid H type Zeo-karb that processed balance is good, post blade diameter length ratio is 1: 10, column volume is 500ml, flow velocity is 3ml/min, collect effluent liquid, with triethylamine, regulate PH to 4-7, be then slowly added in macroporous adsorbent resin separator column, post blade diameter length ratio is 1: 12, and loading flow velocity is per minute 10ml.After end of the sample, use the deionized water of normal temperature with the flow velocity wash-out of per minute 20ml.Elutriant, in 60 ℃ of concentrating under reduced pressure, obtains hydroxyl radical carthamin yellow carthamus A organic ammonium crude product concentrated solution.In safflower, per kilogram safflower obtains concentrated solution 100ml.Gel LH-20 post on hydroxyl radical carthamin yellow carthamus A organic ammonium crude product concentrated solution, the blade diameter length ratio of post is 1: 5, and applied sample amount is column volume 10%, and elution flow rate is per minute 5ml, collects hydroxyl carthamin yellow A-containing organic ammonium part.Collect liquid after 60 ℃ of concentrating under reduced pressure, obtain hydroxyl radical carthamin yellow carthamus A organic ammonium fine work concentrated solution, in safflower, per kilogram safflower obtains concentrated solution 35～50ml, through lyophilize, obtain flaxen hydroxyl radical carthamin yellow carthamus A organic ammonium fine work powder, purity is 99.0%, and yield is counted 0.45% left and right by safflower.
Embodiment 6: hydroxyl radical carthamin yellow carthamus A organic ammonium (hydroxyl radical carthamin yellow carthamus A triethylamine) (be in formula I, R1 is hydrogen, and R2, R3, R4 are ethyl)
Take safflower, add the deionized water of 12.5 times of medicinal material weight, in 100 ℃ of extraction 20-25 minute, filter, the deionized water that filter residue adds 10 times of medicinal material weight repeats to extract once by above-mentioned condition again, filters.Merge secondary raffinate, be cooled to room temperature, after using whizzer centrifugal, extracting centrifugal liquid is standby.Above-mentioned centrifugate is slowly added to the HB-8 macropore strong acid H type Zeo-karb that processed balance is good, post blade diameter length ratio is 1: 10, column volume is 500ml, flow velocity is 3ml/min, collect effluent liquid, with triethylamine, regulate PH to 4-7, be then slowly added in macroporous adsorbent resin separator column, post blade diameter length ratio is 1: 12, and loading flow velocity is per minute 10ml.After end of the sample, use the deionized water of normal temperature with the flow velocity wash-out of per minute 20ml.Elutriant, in 60 ℃ of concentrating under reduced pressure, obtains hydroxyl radical carthamin yellow carthamus A organic ammonium crude product concentrated solution.In safflower, per kilogram safflower obtains concentrated solution 100ml.Gel LH-20 post on hydroxyl radical carthamin yellow carthamus A organic ammonium crude product concentrated solution, the blade diameter length ratio of post is 1: 5, and applied sample amount is column volume 10%, and elution flow rate is per minute 5ml, collects hydroxyl carthamin yellow A-containing organic ammonium part.Collect liquid after 60 ℃ of concentrating under reduced pressure, obtain hydroxyl radical carthamin yellow carthamus A organic ammonium fine work concentrated solution, in safflower, per kilogram safflower obtains concentrated solution 35～50ml, through lyophilize, obtain flaxen hydroxyl radical carthamin yellow carthamus A organic ammonium fine work powder, purity is 99.0%, and yield is counted 0.47% left and right by safflower.
Embodiment 7: hydroxyl radical carthamin yellow carthamus A organic ammonium (hydroxyl radical carthamin yellow carthamus A tetramethyl-ammonium) (be in formula I, R1, R2, R3, R4 are methyl)
Take safflower, add the deionized water of 12.5 times of medicinal material weight, in 100 ℃ of extraction 20-25 minute, filter, the deionized water that filter residue adds 10 times of medicinal material weight repeats to extract once by above-mentioned condition again, filters.Merge secondary raffinate, be cooled to room temperature, after using whizzer centrifugal, extracting centrifugal liquid is standby.Above-mentioned centrifugate is slowly added to the 001*7 strongly-acid H type Zeo-karb that processed balance is good, post blade diameter length ratio is 1: 10, column volume is 500ml, flow velocity is 3ml/min, collect effluent liquid, by Tetramethylammonium hydroxide, regulate PH to 4-7, be then slowly added in macroporous adsorbent resin separator column, post blade diameter length ratio is 1: 12, and loading flow velocity is per minute 10ml.After end of the sample, use the deionized water of normal temperature with the flow velocity wash-out of per minute 20ml.Elutriant, in 60 ℃ of concentrating under reduced pressure, obtains hydroxyl radical carthamin yellow carthamus A organic ammonium crude product concentrated solution.In safflower, per kilogram safflower obtains concentrated solution 100ml.Gel LH-20 post on hydroxyl radical carthamin yellow carthamus A organic ammonium crude product concentrated solution, the blade diameter length ratio of post is 1: 5, and applied sample amount is column volume 10%, and elution flow rate is per minute 5ml, collects hydroxyl carthamin yellow A-containing organic ammonium part.Collect liquid after 60 ℃ of concentrating under reduced pressure, obtain hydroxyl radical carthamin yellow carthamus A organic ammonium fine work concentrated solution, in safflower, per kilogram safflower obtains concentrated solution 35～50ml, through lyophilize, obtain flaxen hydroxyl radical carthamin yellow carthamus A organic ammonium fine work powder, purity is 99.0%, and yield is counted 0.45% left and right by safflower.
The mass spectrum MS of hydroxyl radical carthamin yellow carthamus A Tetramethylammonium hydroxide, nucleus magnetic resonance
1h-NMR, data are as follows:
1. mass spectrum
Instrument model: the multistage ion trap mass spectrometry system of U.S. FINNIGAN company LC-MS (LCQ-DECAXP)
Test condition: ESI
Mass spectrum MS
-c?ESI611.22(M-H)
2. proton nmr spectra
Instrument model: BRUCKER AVANCE III500 type NMR spectrometer with superconducting magnet
Test condition: solvent: DMSO, interior mark: TMS
Nucleus magnetic resonance
Hydroxyl radical carthamin yellow carthamus A Tetramethylammonium hydroxide
1h mono-NMR data
| Proton sequence number (H ownership) | Chemical shift δ (ppm) | Proton number |
| 8 | 7.25 | 1 |
| 9 | 7.39 | 1 |
| 11，15 | 7.41 | 2 |
| 12，14 | 6.76 | 2 |
| 3-OH | 18.66 | 1 |
| 4-OH | 4.73 | 1 |
| 5-ON(CH 3) 4 | 6.90-7.33 | 12 |
| 13-OH | 9.74 | 1 |
| Sugar moieties | ? | ? |
| G1 | 3.60 | 1 |
| G2 | 2.82 | 1 |
| G3 | 3.06 | 1 |
| G4 | 3.27 | 1 |
| G5 | 3.01 | 1 |
| G6 | 3.36-3.22 | 2 |
| G’1 | 4.15 | 1 |
| G’2 | 4.03 | 1 |
| G’3 | 3.07 | 1 |
| G’4 | 3.04 | 1 |
| G’5 | 2.91 | 1 |
| G’6 | 3.59 | 2 |
| Hydroxyl on sugar | 4.40～4.78 | 8 |
Embodiment 8: hydroxyl radical carthamin yellow carthamus A organic ammonium (hydroxyl radical carthamin yellow carthamus A tetramethyl-ammonium) (be in formula I, R1, R2, R3, R4 are methyl)
Take safflower, add the deionized water of 12.5 times of medicinal material weight, in 100 ℃ of extraction 20-25 minute, filter, the deionized water that filter residue adds 10 times of medicinal material weight repeats to extract once by above-mentioned condition again, filters.Merge secondary raffinate, be cooled to room temperature, after using whizzer centrifugal, extracting centrifugal liquid is standby.Above-mentioned centrifugate is slowly added to the HB-8 macropore strong acid H type Zeo-karb that processed balance is good, post blade diameter length ratio is 1: 10, column volume is 500ml, flow velocity is 3ml/min, collect effluent liquid, by Tetramethylammonium hydroxide, regulate PH to 4-7, be then slowly added in macroporous adsorbent resin separator column, post blade diameter length ratio is 1: 12, and loading flow velocity is per minute 10ml.After end of the sample, use the deionized water of normal temperature with the flow velocity wash-out of per minute 20ml.Elutriant, in 60 ℃ of concentrating under reduced pressure, obtains hydroxyl radical carthamin yellow carthamus A organic ammonium crude product concentrated solution.In safflower, per kilogram safflower obtains concentrated solution 100ml.Gel LH-20 post on hydroxyl radical carthamin yellow carthamus A organic ammonium crude product concentrated solution, the blade diameter length ratio of post is 1: 5, and applied sample amount is column volume 10%, and elution flow rate is per minute 5ml, collects hydroxyl carthamin yellow A-containing organic ammonium part.Collect liquid after 60 ℃ of concentrating under reduced pressure, obtain hydroxyl radical carthamin yellow carthamus A organic ammonium fine work concentrated solution, in safflower, per kilogram safflower obtains concentrated solution 35～50ml, through lyophilize, obtain flaxen hydroxyl radical carthamin yellow carthamus A organic ammonium fine work powder, purity is 99.0%, and yield is counted 0.45% left and right by safflower.
Claims (10)
1. one kind suc as formula the hydroxyl radical carthamin yellow carthamus A pharmaceutical salts shown in (I):
R represents K, NH
4or
Wherein, R1, R2, R3, R4 distinguish identical or different, are independently selected from separately hydrogen, alkyl; G, G ' represent Position Number.
2. hydroxyl radical carthamin yellow carthamus A pharmaceutical salts according to claim 1, it is sylvite, shown in (II):
3. hydroxyl radical carthamin yellow carthamus A pharmaceutical salts according to claim 1, it is ammonium salt, shown in (III):
4. a method of preparing the hydroxyl radical carthamin yellow carthamus A pharmaceutical salts as described in claim 1-3, it comprises the extraction of flos carthami, the conversion of strongly-acid H type Zeo-karb, macroporous adsorbent resin separated, sephadex chromatography is separated and the step of ultrafiltration, it is characterized in that:
(1) raw material extracts: flos carthami is raw material, and water extraction obtains the extracting solution that contains hydroxyl radical carthamin yellow carthamus A;
(2) strongly-acid H type Zeo-karb conversion: the extracting solution that step (1) is made is crossed strongly-acid H type cation exchange resin column, collect elutriant, adjust after pH, hydroxyl radical carthamin yellow carthamus A generates hydroxyl radical carthamin yellow carthamus A pharmaceutical salts, collects the elutriant of hydroxyl carthamin yellow A-containing pharmaceutical salts;
(3) macroporous adsorbent resin is separated: the elutriant of the hydroxyl carthamin yellow A-containing pharmaceutical salts that step (2) is made is separated with macroporous adsorptive resins, take water as eluent, collect elutriant, concentrating under reduced pressure, obtains the crude product of hydroxyl radical carthamin yellow carthamus A pharmaceutical salts;
(4) dextrane gel is separated: the hydroxyl radical carthamin yellow carthamus A pharmaceutical salts crude product that step (3) is made is separated with sephadex chromatography, take water as eluent, collects hydroxyl carthamin yellow A-containing pharmaceutical salts elutriant;
(5) ultrafiltration: step (4) gained hydroxyl carthamin yellow A-containing pharmaceutical salts elutriant is concentrated by filtering or the daltonian ultra-filtration membrane of centrifugal rear employing molecular weight cut-off 8000-10000 carries out ultrafiltration and obtains ultrafiltrated, obtain hydroxyl radical carthamin yellow carthamus A pharmaceutical salts after dry.
5. preparation method according to claim 4, wherein said hydroxyl radical carthamin yellow carthamus A pharmaceutical salts is suc as formula the hydroxyl radical carthamin yellow carthamus A potassium shown in (II), Zeo-karb described in step (2) is strongly-acid H type Zeo-karb, it is selected from 001*7 ion exchange resin or macropore HB-8 exchange resin, collect elutriant and regulate pH with potassium hydroxide, hydroxyl radical carthamin yellow carthamus A generates hydroxyl radical carthamin yellow carthamus A potassium.
6. preparation method according to claim 4, wherein said hydroxyl radical carthamin yellow carthamus A pharmaceutical salts is suc as formula the hydroxyl radical carthamin yellow carthamus A ammonium shown in (III), Zeo-karb described in step (2) is strongly-acid H type Zeo-karb, it is selected from 001*7 ion exchange resin or macropore HB-8 exchange resin, collect elutriant and regulate pH with ammonium hydroxide, hydroxyl radical carthamin yellow carthamus A generates hydroxyl radical carthamin yellow carthamus A ammonium.
7. a pharmaceutical composition, its hydroxyl radical carthamin yellow carthamus A pharmaceutical salts that comprises described in the claim 1-3 that treats significant quantity is that activeconstituents and pharmaceutically acceptable carrier are auxiliary material.
8. pharmaceutical composition according to claim 7, it is the preparations such as lyophilized injectable powder, infusion solutions.
9. pharmaceutical composition according to claim 8, it is lyophilized injectable powder, and prepares by the method comprising the steps:
(1) take flos carthami as raw material, add temperature to be
water extracting, extracting is with water extracting
inferior, each
hour, extracting water consumption is safflower crude drug weight
doubly, after extracting, the filtering dregs of a decoction, are cooled to extracting solution
standing
hour;
(2) extracting solution step (1) being made is crossed strongly-acid H type Zeo-karb, flow velocity 1～30ml/min, collect elutriant, adjust after pH, hydroxyl radical carthamin yellow carthamus A generates hydroxyl radical carthamin yellow carthamus A pharmaceutical salts, the elutriant that collection comprises the hydroxyl radical carthamin yellow carthamus A pharmaceutical salts described in claim 1-3 any one;
(3) elutriant step (2) being made is separated with macroporous adsorptive resins, take purified water as eluent, and elution flow rate 10～30ml/min collects elutriant, and concentrating under reduced pressure, obtains hydroxyl radical carthamin yellow carthamus A pharmaceutical salts concentrated solution crude product;
(4) hydroxyl radical carthamin yellow carthamus A pharmaceutical salts concentrated solution crude product step (3) being obtained filters or is centrifugal rear separated with sephadex chromatography, take purified water as eluent, it is 1～10cm/h that elution flow rate is controlled linear rate of flow, collect hydroxyl carthamin yellow A-containing pharmaceutical salts elutriant, concentrating under reduced pressure obtains concentrated solution;
(5) by step (4) gained concentrated solution after filtration or the daltonian ultra-filtration membrane of centrifugal rear employing molecular weight cut-off 8000-10000 carry out ultrafiltration and obtain ultrafiltrated;
(6) by step (5) gained ultrafiltrated through lyophilize, obtain hydroxyl radical carthamin yellow carthamus A pharmaceutical salts fine work;
(7) hydroxyl radical carthamin yellow carthamus A pharmaceutical salts fine work step (6) being obtained is dissolved in water for injection, after adopting the millipore filtration of 0.22 μ m or the daltonian ultrafiltration membrance filter of molecular weight cut-off 8000-10000, be sub-packed in bottle, after lyophilize, obtain hydroxyl radical carthamin yellow carthamus A pharmaceutical salts freeze-dried powder;
Wherein, described strongly-acid H type Zeo-karb is with 001*7 ion exchange resin or macropore HB-8 exchange resin;
Described macroporous adsorbent resin is to use macroporous adsorbent resin HZ801; Described sephadex chromatography is to use sephadex lh-20.
10. the application of the hydroxyl radical carthamin yellow carthamus A pharmaceutical salts described in claim 1-3 any one in preparing medicine, wherein said medicine has the platelet aggregation of anti-PAF or ADP induction, is used for the treatment of or prevents to relate to damage disease due to myocardial ischemia, cerebral ischemia, thrombosis.
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| CN201310048480.4A CN103980240B (en) | 2013-02-07 | 2013-02-07 | New Hydroxy Carthamus yellow pharmaceutical salts |
| IN10592DEN2014 IN2014DN10592A (en) | 2013-02-07 | 2014-02-26 | |
| US14/417,811 US9624254B2 (en) | 2013-02-07 | 2014-02-26 | Hydroxysafflor yellow pharmaceutical salts |
| PCT/CN2014/000180 WO2014121666A1 (en) | 2013-02-07 | 2014-02-26 | New hydroxysafflor yellow pharmaceutical salts |
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Cited By (3)
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| CN104513216A (en) * | 2014-12-27 | 2015-04-15 | 杭州奥默医药股份有限公司 | Analogue of hydroxysafflor yellow A as well as preparation and application of analogue of hydroxysafflor yellow A |
| CN104540811A (en) * | 2013-06-08 | 2015-04-22 | 浙江永宁药业股份有限公司 | New hydroxysafflor yellow pharmaceutical salts |
| WO2022247860A1 (en) * | 2021-05-26 | 2022-12-01 | 台州永健医药科技有限公司 | Novel crystal form of hydroxysafflor yellow a and preparation method therefor |
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| CN102702150A (en) * | 2012-06-19 | 2012-10-03 | 浙江永宁药业股份有限公司 | Preparation method and application of hydroxysafflor yellow A |
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| CN102702150A (en) * | 2012-06-19 | 2012-10-03 | 浙江永宁药业股份有限公司 | Preparation method and application of hydroxysafflor yellow A |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104540811A (en) * | 2013-06-08 | 2015-04-22 | 浙江永宁药业股份有限公司 | New hydroxysafflor yellow pharmaceutical salts |
| CN104513216A (en) * | 2014-12-27 | 2015-04-15 | 杭州奥默医药股份有限公司 | Analogue of hydroxysafflor yellow A as well as preparation and application of analogue of hydroxysafflor yellow A |
| WO2022247860A1 (en) * | 2021-05-26 | 2022-12-01 | 台州永健医药科技有限公司 | Novel crystal form of hydroxysafflor yellow a and preparation method therefor |
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Denomination of invention: New hydroxysafflower yellow medicinal salt Effective date of registration: 20210914 Granted publication date: 20160803 Pledgee: Industrial Commercial Bank of China Ltd. Taizhou Huangyan branch Pledgor: ZHEJIANG YONGNING PHARMACEUTICAL Co.,Ltd. Registration number: Y2021330001585 |