CN103012513A - Preparation method and application of methyl-hydroxyl stilbene glycoside crystal - Google Patents
Preparation method and application of methyl-hydroxyl stilbene glycoside crystal Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a methyl-hydroxyl stilbene glycoside crystal. The methyl-hydroxyl stilbene glycoside is a compound (E)-1-(3-O-beta-D-glucopyranose-5-hydroxyl phenyl)-2-(4-methoxyphenyl) ethylene. The pharmaceutical composition has an extensive use in preparation of medicines for preventing and curing cardiovascular and cerebrovascular diseases. The preparation method of the crystal is simple and easy to apply and low in production cost, can be continuously operated and has strong practicability.
Description
Technical field
The invention belongs to medical technical field, relate to a kind of hydroxypropyl Zhigan crystal its preparation method and application thereof.
Background technology
Hydroxypropyl Zhigan is (E)-1-(3-O-β-D-Glucopyranose-5-hydroxy phenyl)-2-4-p-methoxy-phenyl) ethene, or be called 4 '-methoxyl group-5-hydroxyl-Stilbene-3 '-O-beta-glucoside, Rhapontin, deoxy-.
Hydroxypropyl Zhigan and extract thereof, drug regimen aspect the control ischemic cardio cerebrovascular diseases pharmacology and the patented mandate of drug efficacy study, its using value and preparation method (fat-soluble stilbene glycoside kind compound is at purposes and preparation thereof as the treatment ischemic cardio cerebrovascular diseases, 200510010757.X), the drug regimen patent of hydroxypropyl Zhigan injection formulations is open (a kind of pharmaceutical composition that contains hydroxypropyl Zhigan and preparation method thereof, 200710065812.4).
The present invention finds by research that the hydroxypropyl Zhigan crystal is oral can obviously reduce MCAO rat cerebral infarction scope, make its behavior disorder degree be improved significantly, to the significant provide protection of having of ischemic cardio cerebrovascular diseases.Preliminary safety research shows that the security of hydroxypropyl Zhigan crystal is fine: in the Acute oral tox-hty test in 24 hours single gavage mouse maximum dosage-feeding be 13.33 g/kg.bw, have no animal and toxic side effects and death occur.Therefore, this hydroxypropyl Zhigan crystal of almost non-toxic side effect has potential clinical value in disease aspect prevention and the treatment cardiac-cerebral ischemia.
In addition, stability study shows hydroxypropyl Zhigan crystal stability of the present invention and obviously is better than the crystal born in the unformed powder of hydroxypropyl Zhigan and the alcoholic solution with the mixed stability of conventional auxiliary material.Therefore, it is better than other hydroxypropyl Zhigan raw material in the preparation oral preparations.
There is no at present research or the report of hydroxypropyl Zhigan crystal habit.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of hydroxypropyl Zhigan crystal its preparation method and application thereof, of the present invention have the preparation method of crystal simple, and production cost is low, but operate continuously has extremely strong practicality.
Its technical scheme is as follows:
The invention provides a kind of feature of hydroxypropyl Zhigan crystal.
X-ray powder diffraction feature: use Cu-Ka irradiation, its typical powder x-ray diffraction figure sees Fig. 2,4.7,9.4,14.2,15.6,17.3,17.9,18.7,19.5,20.2,22.1,23.8,26.1,42.6,48.9 absorption peak is arranged to spend the X-ray powder diffraction spectrum that 2 θ represent.
The typical differential scanning calorimetry of hydroxypropyl Zhigan crystal of the present invention (Differential Scanning Calorimeter, DSC) collection of illustrative plates is seen accompanying drawing (accompanying drawing 3-8), and its DSC melting transition is at 229 ~ 232 ℃.
Typical infrared spectrogram (the Infrared Spectroscopy of hydroxypropyl Zhigan crystal of the present invention, IR) see Fig. 9, infrared absorption pattern is about 3420,2905,1596,1511,1455,1355,1303,1252,1177,1075,1024,963,836 cm
-1There is absorption peak at the place.Described hydroxypropyl Zhigan is (E)-1-(3-O-β-D-Glucopyranose-5-hydroxy phenyl)-2-(4-p-methoxy-phenyl) ethene.
A kind of preparation method of hydroxypropyl Zhigan crystal in the hydroxypropyl Zhigan material adding recrystallisation solvent with 98% above purity, adds activated carbon and boils decolouring in 3 minutes, and 4 ℃ leave standstill 4～24 hours crystallizatioies, suction filtration, P
2O
5Be dry 12 hours of siccative vacuum decompression, make the hydroxypropyl Zhigan crystal.
Described recrystallisation solvent contains the ketone of 3～4 carbon atoms and the mixture of ethyl acetate.
Described recrystallisation solvent is 20～45% acetone, 55%～80% ethyl acetate mixture,
The amount of described recrystallisation solvent is 4～24 times of raw material input amount.
The input amount of described activated carbon is raw material 0.5～2.5% of the weight that feeds intake.
The ketone of 3 ~ 4 carbon atoms and ethyl acetate are with the ketone of certain proportion 20～80%, 80%～20% ethyl acetate is recrystallisation solvent after mixing, adopt the crystal that refrigeration is left standstill or the method such as solvent evaporates can obtain to have the described feature of claim 1, therefore all can be used as the recrystallisation solvent of this crystal formation of hydroxypropyl Zhigan.Safe, the cheap and extraction rate of transform of considering production acetone is higher, and the present invention is 40% acetone most preferably, and 60% ethyl acetate mixture is as recrystallisation solvent.
The very few crystallization excessive velocities that causes of the consumption of recrystallisation solvent obtains mixed crystal easily; The recrystallisation solvent consumption too much causes the crystal yield low.In crystallization trial, during with 4 ~ 24 times of solvent crystallizations, the hydroxypropyl Zhigan rate of transform is between 55 % ~ 86 %, and the crystal formation feature of bearing is identical, and as seen they all can be used as effective recrystallisation solvent of this crystal formation of hydroxypropyl Zhigan.Wherein, with 4 ~ 6 times of solvent crystallization speed.The rate of transform obviously reduces after V/W=12 times.Consider operability and the high rate of transform of manufacture, the solvent that preferably adds 8 ~ 12 times of volumes carries out crystalline.
Adopt decolorizing with activated carbon can further improve the purity of product before the crystallization, improve product colour.Because this product is phenolic constituent, the too much productive rate of activated carbon obviously reduces, and crossing at least, color and luster improves not obvious.This test is determined by experiment, activated carbon be raw material feed intake weight 0.5 ~ 2.5% the time better, can take into account yield and the decolorizing effect of product.
A kind of hydroxypropyl Zhigan crystalline pharmaceutical composition that contains contains hydroxypropyl Zhigan crystal of the present invention and one or more pharmaceutical excipients.
Composition of the present invention is tablet or the capsule preparations that contains 5-500 mg hydroxypropyl Zhigan crystal.
The application of hydroxypropyl Zhigan crystal of the present invention in preparation prevention and treatment cardiac-cerebral ischemia diseases medicine.
Beneficial effect of the present invention: the preparation method of hydroxypropyl Zhigan crystal of the present invention is simple, and production cost is low, but operate continuously has extremely strong practicality.
Description of drawings
Fig. 1 is that hydroxypropyl Zhigan of the present invention is (E)-1-(3-O-β-D-Glucopyranose-5-hydroxy phenyl)-2-(4-p-methoxy-phenyl) chemical structure of ethene;
Fig. 2 is that hydroxypropyl Zhigan crystal of the present invention typical case powder x-ray diffraction figure sees, 4.7,9.4,14.2,15.6,17.3,17.9,18.7,19.5,20.2,22.1,23.8,26.1,42.6,48.9 absorption peak is arranged to spend the X-ray powder diffraction spectrum that 2 θ represent.
Fig. 3 is the typical differential scanning calorimetry of hydroxypropyl Zhigan crystal of the present invention (DSC) collection of illustrative plates, and its DSC melting transition is at 229.0 ℃.
Fig. 4 is the typical differential scanning calorimetry of hydroxypropyl Zhigan crystal of the present invention (DSC) collection of illustrative plates, and its DSC melting transition is at 229.8 ℃.
Fig. 5 is the typical differential scanning calorimetry of hydroxypropyl Zhigan crystal of the present invention (DSC) collection of illustrative plates, and its DSC melting transition is at 230.0 ℃.
Fig. 6 is the typical differential scanning calorimetry of hydroxypropyl Zhigan crystal of the present invention (DSC) collection of illustrative plates, and its DSC melting transition is at 230.8 ℃.
Fig. 7 is the typical differential scanning calorimetry of hydroxypropyl Zhigan crystal of the present invention (DSC) collection of illustrative plates, and its DSC melting transition is at 231.4 ℃.
Fig. 8 is the typical differential scanning calorimetry of hydroxypropyl Zhigan crystal of the present invention (DSC) collection of illustrative plates, and its DSC melting transition is at 230.4 ℃.
Fig. 9 is typical infrared spectrogram (the Infrared Spectroscopy of the hydroxypropyl Zhigan crystal of the present invention of hydroxypropyl Zhigan crystal of the present invention, IR), infrared absorption pattern is about 3420,2905,1596,1511,1455,1355,1303,1252,1177,1075,1024,963,836 cm
-1There is absorption peak at the place.
To be hydroxypropyl Zhigan of the present invention obtain typical differential scanning calorimetry (DSC) collection of illustrative plates of crystal with 15%, 45% ethanol as recrystallisation solvent to Figure 10, do not have crystal characteristic of the present invention.
To be hydroxypropyl Zhigan of the present invention obtain typical differential scanning calorimetry (DSC) collection of illustrative plates of crystal with 75% ethanol as recrystallisation solvent to Figure 11, do not have crystal characteristic of the present invention.
To be hydroxypropyl Zhigan of the present invention obtain typical differential scanning calorimetry (DSC) collection of illustrative plates of crystal with 15% acetone as recrystallisation solvent to Figure 12, do not have crystal characteristic of the present invention.
To be hydroxypropyl Zhigan of the present invention obtain typical differential scanning calorimetry (DSC) collection of illustrative plates of crystal with 45% acetone as recrystallisation solvent to Figure 13, do not have crystal characteristic of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments technical scheme of the present invention is described in more detail.
Hydroxypropyl Zhigan of the present invention is (E)-1-(3-O-β-D-Glucopyranose-5-hydroxy phenyl)-2-(4-p-methoxy-phenyl) ethene, have chemical structure as shown in Figure 1.
Embodiment 1:X-ray diffraction, DSC curve and IR analyze
1, X-ray diffraction analysis
(1) detecting instrument: D/max-3AX-x ray diffractometer x
(2) testing conditions: Cu target K α 1 ray, voltage 35kV, electric current 25mA, 1 ° of divergent slit, 1 ° of anti-scatter slit receives slit 0.3mm, 0.3mm, 2 θ scopes: 3 °-60 °
(3) detect foundation: turn target polycrystal X-ray diffraction method general rule JY/T009-1996
(4) detected result: typical spectrogram such as Fig. 2.
2, DSC tracing analysis
(1) detecting instrument: the German NETZSCH DSC of company 204 differential scanning calorimeter
(2) testing conditions: atmosphere: N
2, 20mL/min; Scanning sequence: be warming up to 250 ℃ from room temperature with 10 ℃/min, the record heating curve; Use aluminium quality sample dish.
(3) detected result: typical spectrogram such as Fig. 3-8.
3, IR analyzes
(1) detecting instrument: Perkin Elmer FT-IR spectrophotometer
(2) testing conditions: KBr compressing tablet
(3) detected result: typical spectrogram such as Fig. 9.
Embodiment 2: the preparation of hydroxypropyl Zhigan crystal
1, adopt different solvents to prepare the hydroxypropyl Zhigan crystal
Get P
2O
5For siccative vacuum decompression every part of 20g of hydroxypropyl Zhigan raw material (purity 98%) after dry 12 hours, add respectively 8 times (V/W=8), i.e. 120mL each crystallization solution shown in table 2-1; Adding 1.5%(is 0.3g) pin boiled 3 minutes with activity, filtered while hot, 4 ℃ left standstill 10 hours, crystallization solution suction filtration, P
2O
5Be dry 12 hours of siccative vacuum decompression, make the hydroxypropyl Zhigan crystal, calculate hydroxypropyl Zhigan crystal yield: the dry brilliant amount/hydroxypropyl Zhigan charging capacity of hydroxypropyl Zhigan * 100%.Analyze the DSC curve that each solvent obtains crystal.
The crystallization trial result is shown in table 2-1～2-4.
Table 2-1: the solvent crystallization test-results that contains alcohol-water
A: hydroxypropyl Zhigan crystal weight; B: hydroxypropyl Zhigan crystal yield; C:DSC spectrogram melt temperature
Table 2-2: the solvent crystallization test-results that contains acetone-water
A: hydroxypropyl Zhigan crystal amount; B: hydroxypropyl Zhigan crystal yield; C:DSC spectrogram melt temperature
Table 2-3: the solvent crystallization test-results that contains acetone and ethyl acetate
A: hydroxypropyl Zhigan crystal amount; B: hydroxypropyl Zhigan crystal yield; C:DSC spectrogram melt temperature
* the content of acetone in the solvent
Table 2-4: the solvent crystallization test-results that contains butanone-ethyl acetate
A: hydroxypropyl Zhigan crystal amount; B: hydroxypropyl Zhigan crystal yield; C:DSC spectrogram melt temperature
* the content of butanone in the solvent
In the said extracted test, with 15%, 45%, 75% ethanol; 15%, 45% acetone; 20%, 40%, 60%, 80% acetone and ethyl acetate mixed solution; When 20%, 40% butanone-ethyl acetate mixed solution is as recrystallisation solvent, can obtain different hydroxypropyl Zhigan crystal, remaining solvent is because very high to the solvent borne of hydroxypropyl Zhigan, 4 ℃ leave standstill 42 hours still can not crystallize out, can not crystallize out with a large amount of volatilizations of solvent.Each crystal dsc analysis spectrogram shows, 15%, 45%, 75% ethanol; 15%, 45% acetone obtains melt temperature at 215-223 ℃ mixed crystal as recrystallisation solvent, can not obtain crystal of the present invention, and its typical DSC collection of illustrative plates is seen Figure 10-13.20%, 30%, 60%, 80% acetone and ethyl acetate mixed solution; When 20%, 40% butanone-ethyl acetate mixed solution is as recrystallisation solvent, can obtain the monocrystalline of feature of the present invention, its typical DSC spectrogram is seen Fig. 3-8.Therefore, a certain proportion of mixed solution of acetone, butanone and ethyl acetate can be used as the recrystallisation solvent of this crystal formation of hydroxypropyl Zhigan.Consider production cycle and factor easy to operate, the present invention preferred 20～60% acetone, 80%～20% ethyl acetate mixture, wherein, 40% acetone most preferably, 60% ethyl acetate mixture.
2, adopt different volumes solution to prepare the hydroxypropyl Zhigan crystal
Get P
2O
5For siccative vacuum decompression every part of 20g of hydroxypropyl Zhigan raw material (purity 98%) after dry 12 hours, add respectively 4,6,8,12,18,24 times (V/W), namely 80,120,160,240,360mL 40% acetone-60% ethyl acetate mixture; Adding 1.5%(is 0.3g) pin boiled 3 minutes with activity, filtered while hot, 4 ℃ leave standstill and observe crystallization situation, suction filtration after 48 hours, P
2O
5Be dry 12 hours of siccative vacuum decompression, make the hydroxypropyl Zhigan crystal, calculate hydroxypropyl Zhigan crystal yield: the dry brilliant amount/hydroxypropyl Zhigan charging capacity of hydroxypropyl Zhigan * 100%.
Test-results is shown in table 2-5.
Table 2-5: different volumes solution crystallization test-results
A: hydroxypropyl Zhigan crystal amount; B: hydroxypropyl Zhigan crystal yield; C: fusing point; E: beginning crystallization time
In the said extracted test, during with 4 ~ 24 times of solvent crystallizations, the hydroxypropyl Zhigan rate of transform is between 55 % ~ 86 %, and the crystal formation feature of bearing is identical, and as seen they all can be used as effective recrystallisation solvent of this crystal formation of hydroxypropyl Zhigan.The rate of transform obviously reduces after V/W=12 times.Wherein, with 4 ~ 6 times of solvent crystallization speed.Consider operability and the high rate of transform of manufacture, the solvent that preferably adds 8 ~ 12 times of volumes carries out crystalline.
3, the different ratios activated carbon is on the impact of hydroxypropyl Zhigan crystal product color and yield
Get P
2O
5For siccative vacuum decompression every part of 10g of light yellow hydroxypropyl Zhigan raw material (purity 98%) after dry 12 hours in 160ml 40% acetone-60% ethyl acetate solution, add respectively 0.05,0.15,0.25,0.50,1.0g injection stage activated carbon (0.5%, 1.5%, 2.5%, 5.0% and 10%), parallel 3 parts, investigate the activated carbon add-on to the impact (seeing Table 2-6) of hydroxypropyl Zhigan crystal product.Solution after activity charcoal powder adds boils 5min, and millipore filtration is suction filtration 2 times while hot, and filter residue is with hot solvent washing (5ml * 3), washing lotion and filtrate merging, and room temperature leaves standstill 1hr, crystallization 10 hr, suction filtration, P
2O
5Be the observation of weighing after dry 12 hours of siccative vacuum decompression.The result is shown in table 2-6.
Table 2-6 activated carbon powder add-on is investigated (n=3, x)
Activated carbon add-on (g/g)=activated carbon adds weight (g)/crystallization raw material and adds (g)
After processing, the crystallization rate of recovery (%)=activated carbon powder obtains the amount (g) * 100% of the amount (g) of crystallization/input crystallization raw material
Table 2-6 test-results shows the increase along with the activated carbon input amount, and the purity of Quzhazhigan crystallization increases, and the crystallization rate of recovery obviously reduces.When the activated carbon input amount greater than 5.0%, it is not obvious that crystallized product purity increases, and the crystallization rate of recovery reduces obviously.Consider the factors such as decolouring and the crystallization rate of recovery according to test-results, select 0.5%-2.5% as the suitable amounts of decolorizing with activated carbon.
Embodiment 3: contain the preparation of hydroxypropyl Zhigan crystal tablet
By following prescription hydroxypropyl Zhigan is configured to every tablet of tablet that contains 100mg
Method for making: take by weighing the formula ratio vehicle and the hydroxypropyl Zhigan crystal mixes, add 1% Walocel MT 20.000PV ethanolic soln (concentration 50%) and be tackiness agent softwood processed, cross 40 mesh sieves and granulate, 60 ℃ of dryings are crossed the whole grain of 30 mesh sieves, add Magnesium Stearate and talcum powder and mix, compressing tablet, and get final product.
Embodiment 4: the stability test of different hydroxypropyl Zhigan crystal
Carry out the stability study of hydroxypropyl Zhigan crystal accelerated test.
1, key instrument
1.1 balance model METTER, TOLEDO, AG135;
1.2 climatic chamber SPX-150C;
1.3 illumination box SPE-250B-G;
1.4 electric heating constant-temperature blowing drying box GZX-9030MBE
The research of 2 influence factors
2.1 sample source
Test sample derives from the hydroxypropyl Zhigan crystal product that different solvents is born in the embodiment of the invention 1.Sample is shown in table 4-1:
Each crystallized sample source of table 4-1
2.2 test conditions
2.2.1 high temperature test: with sample exposed being positioned in the plate respectively, investigate 10 days in 60 ± 2 ° of C, the results are shown in Table 4-2.
2.2.2 high wet test: be positioned in the plate sample is exposed, put into respectively under 25 ° of C constant humidity 75 ± 5% of constant temperature and the condition examination 10 days, the results are shown in Table 4-3.
2.2.3 exposure experiments to light: under the room temperature condition, be positioned in the plate sample is exposed, with 4500 ± 500L * strong illumination, investigate 10 days, the results are shown in Table 4-4.
2.4 investigation project: outward appearance, moisture absorption weightening finish.
2.5 result:
Influence factor test-results (table 4-2,4-3,4-4) shows hydroxypropyl Zhigan crystal (S6-S11 of the present invention
) stability aspect outward appearance, water absorbability is better than the hydroxypropyl Zhigan product that obtains in aqueous alcohol solutions (S1-S3), aqueous acetone solution (S4-S5) solution.
60 ± 2 ° of C of table 4-2 place and investigate 10 days
Table 4-3 RH 75 ± 5% (25 ± 2 ° of C) place and investigate 10 days
Table 4-4 high light 4500 ± 500L x (25 ± 2 ° of C, RH60 ± 5%) places and investigates 10 days
Embodiment 5: oral hydroxypropyl Zhigan crystal is to the provide protection of cerebral ischemia
1 experiment purpose
Adopt middle cerebral artery occlusion in rat to pour into again the model that causes focal cerebral ischemia injury, the validity of research hydroxypropyl Zhigan crystal treatment cerebral apoplexy, its possible mechanism of action of Primary Study provides foundation for further developing.
2 experiment materials
2.1 medicine and reagent
Be subjected to reagent: the hydroxypropyl Zhigan crystal.Off-white color crystalline powder (being insoluble in water) is mixed with the suspendible liquid of desired concn for the rat oral gavage administrable with 0.5%CMC before use.
TCC (TTC), Solution on Chemical Reagents in Shanghai company produces, lot number: 20060118.
Protein quantification test kit, Nanjing build up bio-engineering research institute, lot number: 20080503.
MDA test kit, Nanjing build up bio-engineering research institute, lot number: 20080503.
SOD test kit, Nanjing build up bio-engineering research institute, lot number: 20080503.
2.2 animal
Male SD rat, about body weight 300g, Beijing Vital River Experimental Animals Technology Co., Ltd. provides.
2.3 instrument
The COOLPIX955 digital camera, Japanese Nikon company product.
Pathological image gathers and analytical system, BJ University of Aeronautics ﹠ Astronautics's image center product.
722 grating spectrophotometers, Shanghai the 3rd analytical instrument factory produces.
3 experimental techniques
3.1 model preparation and dosage regimen
The SD rat at one night of fasting, freely drinks water.Test abdominal injection 35% Chloral Hydrate on the same day (350 mg/kg) anesthesia, dorsal position is fixed, cut skin along the neck median line, expose right carotid, careful removal arteria carotis communis branches to circumvascular nerve and the manadesma of basis cranii section, separate successively External Carotid Artery Branch occipital artery, superior thyroid artery, lingual artery and maxilla arteria maxillaris, ligation is cut off; Separate internal carotid artery, and carefully separate with vagus nerve, from root tie wings jaw artery.Insert 3 from the external carotid artery free end
#Nylon wire (diameter 0.285 mm) imports to internal carotid artery from the external carotid artery distal end with nylon wire, is inserted to Willis ring arteria cerebri media place, and with effective blocking-up arteria cerebri media, the nylon wire length of insertion is apart from arteria carotis communis crotch 18～20 mm.Then with the external carotid artery free end together with nylon wire in the chamber in the lump ligation, in case hemorrhage.Layer-by-layer suture subcutaneous fascia and skin, the local penicillin that drips is protected from infection.Be more effective blocking-up arteria cerebri media, nylon yarn before use, circle is burnt at its tip on the flame limit blunt, and with 0.1% poly-l-lysine parcel, at 60 ℃ of oven for baking 1 h, because poly-l-lysine can impel cell and albumen to adhere to frosting, and can make electronegative nylon wire surface become positively charged, thereby attract the anionic site of endotheliocyte, make nylon wire and inner skin surface tight adhesion, in case blood flow is from silk thread leakage current on every side.The sham operated rats animal is only isolated internal carotid artery.
Behind MCAO 2 h, the right side upper limbs has no the rat of obvious hemiplegia and subsequently all rejectings from test of died.MCAO begins rear 2 h, carefully extracts the nylon wire of internal carotid artery Endovascular out, and internal carotid artery is poured into again.Animal is steam again and raises.
Test is established 9 groups, 10 every group.Sham operated rats and model control group gavage give CMC, and 5 are subjected to reagent group gavage to give hydroxypropyl Zhigan, and dosage is respectively 7.5,15,30,60,120 mg/kg.Above-mentionedly respectively organize equal administration 4 times, be respectively 1 h, 12 h after the again perfusion, 24 h, 36 h, each gavage volume is 5ml/kg, vein constant speed gasing injection administration volume be 2 ml/ only, infusion 1 h.
3. 2 nerve function lesion degree evaluations
Respectively at pouring into again 1 h (before the administration), 12 h, 24 h, 36 h, 48 h, according to table 5-1 standards of grading, adopt blind method that animal is carried out the behavior disorder scoring, be used for estimating the nerve function lesion degree, total points is 16 minutes, and mark is higher, shows that animal nerve functional lesion degree is more serious.
Table 5-1 MCAO rat nerve function lesion degree standards of grading
3. 3 infarction sizes are measured
After pouring into again 48 h, broken end is got brain, place physiological saline flush away bloodstain, blot with filter paper, freezing rapidly, be cut into 2 mm slabs along coronal-plane, the interval is got above-mentioned half brain sheet and is put into 2% TTC dye liquor, 37 ℃ of temperature of lucifuge are incubated 30 min and are dyeed, then after 10% formaldehyde solution is fixed a week, adopt COOLPIX955 digital camera imaging system that digital image is stored in the computer, and with ias v 4.0 software measurement infarcted region and the full brain area of BJ University of Aeronautics ﹠ Astronautics's image center, calculating cerebral infarction scope (Infarct area accounts for full brain area percentage).
3.2 statistical procedures
Data all be expressed as mean ± standard deviation (± s).Non-paired t test carries out statistical procedures between the employing group, relatively the significance of mean difference.
4 experimental results
4.1 the impact on neural function
After rat oral gavage gave hydroxypropyl Zhigan, the neurological dysfunction scoring was improved in varying degrees, and certain dose-dependently is arranged.With model control group relatively, the again perfusion 48 h behavior disorders of the hydroxypropyl Zhigan group of 5 dosage scoring 9.4%(P that descended respectively〉0.05), 15.6%(P 0.05), 19.8%(P＜0.05), 38.5%(P＜0.01), 38.5%(P＜0.001).Effective dosage ranges is 30～120 mg/kg.
Table 5-2 hydroxypropyl Zhigan is on the impact (± s, n=10) of MCAO ischemia-reperfusion rat neural function
Annotate: 1. compare with sham operated rats:
+++P＜0.001; 2. compare with model group:
*P＜0.05,
*P＜0.01,
* *P＜0.001.
4. 2 impacts on the cerebral infarction scope
Gavage gives hydroxypropyl Zhigan all can obviously dwindle rat cerebral infarction scope due to cerebral ischemia re-pouring injured, and effect is increased with dosage and strengthened.Compare with model control group, the cerebral infarction scope of the hydroxypropyl Zhigan group of 5 dosage has been dwindled respectively 13.3%(P〉0.05), 28.3%(P＜0.05), 28.9%(P＜0.05), 31.7%(P＜0.05), 31.4%(P＜0.05).Effective dosage ranges is 15～120 mg/kg.
Table 5-3 hydroxypropyl Zhigan is on the impact (± s, n=10) of MCAO ischemia-reperfusion rat cerebral infarction scope
Annotate: 1. compare with sham operated rats:
+++P＜0.001; 2. compare with model group:
*P＜0.05,
*P＜0.01.
5. conclusion: the ischemia-reperfusion rat oral gavage gives hydroxypropyl Zhigan (7.5,15,30,60,120 mg/kg) but dose-dependently improves impaired neural function, dwindles infarction size.Its effective dose is about 30mg/kg.
Embodiment 6: oral hydroxypropyl Zhigan is to the provide protection of core ischemia
1 test objective: select the index oral disposition hydroxypropyl Zhigan crystal such as anti-hypoxia, anti-arrhythmia, antithrombotic formation to carry out the many-sided research of core ischemia, for further Application and Development provides experimental basis.
2 experiment materials
2.1 sample
The hydroxypropyl Zhigan crystal prototype, off-white color crystalline powder (being insoluble in water) is mixed with the suspendible liquid of desired concn for the rat oral gavage administrable with 0.5%CMC before use.The Asprin enteric coated tablet, the 40mg/ sheet, Beijing dawn medicine company limited liability company,
The Racemic isoproterenol injection liquid, 1mg/2ml, Shanghai Hefeng Pharmaceutical Co., Ltd.; The vitamin-E capsule and pill, the 100mg/ grain, the summer door Oils,glyceridic,cod-liver factory; The adrenalin hydrochloride injection liquid, 1mg/ml, Foochow, Fujian pharmaceutical factory.
2 reagent
Collagen protein, Sigma company; Bromobenzene, analytical pure, Chinese Qingpu synthetic agent factory; Olive is pulled oil, chemical pure, Solution on Chemical Reagents in Shanghai company of Chinese Medicine group; Tetraoxypyrimidine, Sigma company; MDA measures test kit, Nanjing Jian Cheng bio-engineering research institute; GLU, TC, TG, HDL-C and LDL-C measure test kit, Sichuan Mai Ke Science and Technology Ltd..
1.3 instrument
The BS110S electronic analytical balance, company limited of German Sai Duoli department; Centrifuge 5810 R refrigerated centrifuges, eppendorf company; 722 spectrophotometers, Shanghai the 3rd analytical instrument factory; The TM1024 automatic clinical chemistry analyzer, Tokyo strain city commercial firm.
1.4 animal
SPF level ICR mouse is available from unming Medical College's Experimental Animal Center.
2 methods
2.1 on thrombotic impact in the Mice Body
Get 20～22g mouse, male and female half and half by sex and body weight random packet, see Table 6-1, table 6-2, table 6-3.Each treated animal every day according to dosage gastric infusion once, continuous 5 days.30min after the last administration, the inductor that every mouse is made by collagen protein-suprarenin by the dosage intravenous injection of 0.1ml/10g body weight is observed and interior each the treated animal hemiplegia incidence of record 15min, mortality ratio and recovery rate.The significance of X2 check group difference the results are shown in Table 6-1, table 6-2 and table 6-3.
Table 6-1 QZQ is on thrombotic impact in the Mice Body
Compare with the blank group: * P＜0.05; * P＜0.01
Experimental result shows, positive control ASP and hydroxypropyl Zhigan 200mg/kg group can obviously suppress thrombosis in the Mice Body due to the collagen and adrenalin; All the other each groups only have effect trend and no difference of science of statistics.
Table 6-2 QZQ is on thrombotic impact in the Mice Body
Compare with the blank group: * P＜0.05; * P＜0.01
Experimental result shows, positive control ASP, hydroxypropyl Zhigan 100mg/kg abdominal injection group can obviously suppress thrombosis in the Mice Body due to the collagen and adrenalin; All the other each groups only have effect trend and no difference of science of statistics.
Table 6-3 QZQ is on thrombotic impact (three) in the Mice Body
Compare with the blank group: * P＜0.05; * P＜0.01
Experimental result shows, positive control ASP, Herba Erigerontis injection (newly), network Thailand and oral group of QZQ-01-001Q 100mg/kg and abdominal injection three dosage groups all can obviously suppress thrombosis in the Mice Body due to the collagen and adrenalin; All the other each groups only have effect trend and no difference of science of statistics.
2.2 to the anti-scarce O of Racemic isoproterenol inducing mouse cardiac muscle
2Impact
Get 18～20g mouse, male and female half and half are divided into 10 groups at random by table-4.Each treated animal respectively according to dosage gastric infusion once, behind the 30min, abdominal injection Racemic isoproterenol 1mg/kg again.Behind the 15min, mouse is inserted in the airtight wide-necked bottle of 125ml, observe and record the animal breath stand-by time as myocardium hypoxia endurance time, the t check is the significance of group difference relatively.For reducing as far as possible systematic error, parallel running is adopted in this experiment.The results are shown in Table 6-4.
Table 6-4 QZQ is on the impact of the anti-scarce O2 of mouse cardiac muscle
Compare with the blank group: * P＜0.05; * P＜0.01
Experimental result shows that hydroxypropyl Zhigan oral administration gavage three dosage groups all can obviously prolong the mouse cardiac muscle hypoxia endurance time, and dose-dependently is arranged.
2.4 chloroform is brought out the ARR impact of mouse
Get 25～28g male mice, be divided at random 10 groups, see Table-6.The respectively according to dosage oral administration gavage administration of each treated animal, behind the 30min, mouse is put into one by one the glass beaker of 500ml back-off, the built-in cotton balls (later every animal is injected chloroform 0.5ml) that contains the 2ml chloroform, after breath stopped, cut open immediately chest, the chamber of observing and recording each treated animal incidence of quivering, the X2 check is the significance of group difference relatively.The results are shown in Table 6-5.
Table 6-5 hydroxypropyl Zhigan brings out the ARR impact of mouse to chloroform
Compare with the blank group: * P＜0.05; * P＜0.01
Experimental result shows that hydroxypropyl Zhigan two dosage groups all can obviously be resisted the ventricular fibrillation that chloroform brings out mouse, show that antiarrhythmic effect is obvious.
3 results
Preliminary Results shows that the hydroxypropyl Zhigan crystal can obviously suppress thrombosis, reduces myocardial consumption of oxygen, anti-arrhythmia, may have therapeutic action to the acute ischemic cardiovascular disorder.
Embodiment 7: the studies on acute toxicity of hydroxypropyl Zhigan
1. research purpose
Adopt single gastric infusion maximum dosage-feeding test in the mouse 24 hours, observe the acute toxic reaction of hydroxypropyl Zhigan, for the security of tested medicine provides experimental basis.
2. experiment material
2.1 tested medicine: hydroxypropyl Zhigan crystal raw material
2.2 solvent contrast: sterilization pure water
2.3 laboratory animal: SPF level ICR mouse, available from unming Medical College's Experimental Animal Center.
3. test method
3.1 animal fasting: the animal fasting be can't help water 10 hours before the administration.
3.2 animal grouping and identification: after adaptability is raised, selective body weight average one (body weight difference be no more than mean body weight 20%), fur gloss, behavioral activity freely, 40 of the healthy animal that general status is good are used for test.After weighing, animal by sex and body weight segmentation, is divided at random again two groups, that is: solvent control group, hydroxypropyl Zhigan group, 20 every group, each 10 of male and female.Raise in SPF level experimentation on animals two Room, male and female are divided cage, 5 in every cage.Carry out mark with picric acid fur staining.Hang cage card sign on the cage box, indicate: thematic code name, thematic responsible official's name, cage number, group, animal kind system, number of animals, sex, observation beginning and ending time.With blank group, hydroxypropyl Zhigan group cage box from top to bottom, from left to right put on cage in order.
All the other 10 animals in time shift out use for laboratory and test in other.
3.3 dosage setting:
3.3.1 clinical plan situation: intend uncertainly temporarily with dosage, it is oral intending with route of administration.
3.3.2 trial test: take by weighing hydroxypropyl Zhigan crystal raw material 1.0g, be mixed with the milky white color contamination magma that concentration is 33.33mg/ml with the sterilization pure water, give mouse with 40ml/kg.bw maximum volume gavage, obvious abnormal response and death do not appear in animal after the administration.
3.4.3 official test: hydroxypropyl Zhigan is with peak concentration 33.33mg/ml, and maximum volume 40ml/kg.bw gavage gives mouse, and dosage is 13.33 g/kg.bw.
3.5 tested method for preparation of drug: take by weighing hydroxypropyl Zhigan crystal raw material 16g, place mortar, add a small amount of pure water, fully grind rear the immigration in the graduated cylinder with the mortar rod, in mortar, add again a small amount of pure water, move in the graduated cylinder behind the ground and cleaned mortar, so repeatedly clean 2～3 times, final graduated cylinder herb liquid total amount adds to 48ml, fully mixing.This liquid is milky white color contamination thick liquid, and concentration is 33.33mg/ml.
3.6 medication: first to the blank group, again to the hydroxypropyl Zhigan group, each administration is front with the abundant mixing of liquid.
3.7 route of administration and dosage: the clinical plan approach of hydroxypropyl Zhigan is oral, and the reagent route of administration is identical with being subjected to, this test and Selection gastric infusion.
3.8 administration frequency and time limit: administration is 1 time in 24 hours.
3.9 the detection method of various indexs and frequency:
3.9.1 generalized case: mouse stomach gave behind the hydroxypropyl Zhigan Continuous Observation 4 hours, and observe at least once later every day, totally 14 days.The main observation animal diet followed, outward appearance, behavior, secretory product, movement, death condition and toxic reaction symptom and time of origin thereof, severity, time length, reversible and time of recovery etc. whether.
3.9.2 body weight: weighed in the 3rd, 7,14 day before the administration and after the administration.
3.9.3 anatomic observation: to the animal of observation period death with observe and take off the animal that cervical vertebra puts to death after finishing and dissect, the volume, color, quality etc. of observing each internal organs, organ have or not unusual.
3.9.4 histopathological examination: occur in time drawing materials and carrying out histopathological examination when unusual to internal organs, organ such as anatomic observation.
4. statistical method: the weight of animals is carried out statistical procedures with the many groups mean analysis in the DAS ver1.0 statistical software.
5. test-results
5.1 toxic reaction: after mouse stomach gave hydroxypropyl Zhigan, animal behavior, diet, outward appearance, secretory product, movement etc. are Non Apparent Abnormality all.
5.2 body weight: blank group Mouse Weight sustainable growth, the 3rd day body weight increases slower after the administration of hydroxypropyl Zhigan group male mice, with the blank group significant difference (p＜0.05) is arranged relatively, the 7th, 14 day two treated animal weight ratios are than there was no significant difference after the administration.(seeing Table 7-1).
Table 7-1 hydroxypropyl Zhigan mouse stomach acute toxicity test body weight table (
, g)
Statistical analysis: * and blank relatively have significant difference P＜0.05
5.3 anatomic observation: the experimental observation phase finishes, and takes off cervical vertebra execution animal and carries out the gross anatomy observation, and the volume of each internal organs, organ, color, quality etc. are showed no obvious abnormalities.
6. results and analysis
To give the maximum dosage-feeding of mouse hydroxypropyl Zhigan be 13.33 g/kg.bw to the single gavage in 24 hours, and the 3rd day body weight increases slowly than the blank group after rarely seen male mice administration, has no animal and toxic side effects and death occur.
The present invention is 40% acetone most preferably, and 60% ethyl acetate mixture is as recrystallisation solvent.Embodiment 2.1 has illustrated; The amount of preferred described recrystallisation solvent is 8 ~ 12 times of raw material input amount.Illustrate among the embodiment 2.2.
The above only is best mode for carrying out the invention, according to the preparation process of routine, crystal of the present invention can be prepared into any drug oral preparation that is applicable to clinical application.Such as tablet, capsule, pill, powder, dispersion agent, granule etc.Anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.
Claims (10)
1. the crystal of a hydroxypropyl Zhigan, it is characterized in that, described crystal uses Cu-Ka irradiation, 4.7,9.4,14.2,15.6,17.3,17.9,18.7,19.5,20.2,22.1,23.8,26.1,42.6,48.9 the peak is arranged to spend the X-ray powder diffraction spectrum that 2 θ represent; Its DSC endothermic transition is at 229 ~ 232 ℃, and infrared absorption pattern is at 3420,2905,1596,1511,1455,1355,1303,1252,1177,1075,1024,963,836 cm
-1There is an absorption peak at the place, described hydroxypropyl Zhigan crystal according to claim 1, described hydroxypropyl Zhigan is (E)-1-(3-O-β-D-Glucopyranose-5-hydroxy phenyl)-2-(4-p-methoxy-phenyl) ethene.
2. the preparation method of hydroxypropyl Zhigan crystal claimed in claim 1 is characterized in that, the hydroxypropyl Zhigan raw material is added in the recrystallisation solvent, adds activated carbon and boils decolouring in 3 minutes, and 4 ℃ leave standstill 4 ~ 24 hours crystallizatioies, suction filtration, P
2O
5Be dry 12 hours of siccative vacuum decompression, make the hydroxypropyl Zhigan crystal.
3. the preparation method of hydroxypropyl Zhigan crystal according to claim 2 is characterized in that, described recrystallisation solvent is organic solvent, is mixed solvent, contains ketone and the ethyl acetate of 3 ~ 4 carbon atoms.
4. the preparation method of hydroxypropyl Zhigan crystal according to claim 3 is characterized in that, described organic solvent is 20～80% acetone, the mixing solutions of 80%～20% ethyl acetate.
5. the preparation method of hydroxypropyl Zhigan crystal according to claim 4 is characterized in that, described organic solvent is 40% acetone and 60% ethyl acetate mixture.
6. the preparation method of hydroxypropyl Zhigan crystal according to claim 2 is characterized in that, the amount of described recrystallisation solvent is 4 ~ 24 times of raw material input amount.
7. method according to claim 2 is characterized in that, the input amount of described activated carbon is raw material 0.5 ~ 2.5% of the weight that feeds intake.
8. one kind contains hydroxypropyl Zhigan crystalline pharmaceutical composition, it is characterized in that, contains hydroxypropyl Zhigan crystal claimed in claim 1 and one or more pharmaceutical excipients.
9. composition according to claim 7 is characterized in that, described composition is tablet or the capsule preparations that contains 5-500 mg hydroxypropyl Zhigan crystal.
10. the application of the described hydroxypropyl Zhigan crystal of claim 1 in preparation prevention and treatment cardiac-cerebral ischemia diseases medicine.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1398838A (en) * | 2001-07-26 | 2003-02-26 | 中国人民解放军军事医学科学院放射医学研究所 | Diphenylethylene compound and its prepn and application in preventing and treating diabetes |
| CN1686148A (en) * | 2005-04-20 | 2005-10-26 | 昆明翔昊科技有限公司 | Use of liposoluble stilbene glycoside kind compound in treating ischemic heart brain blood vessel disease and its preparation |
| CN101036663A (en) * | 2007-04-18 | 2007-09-19 | 昆明翔昊科技有限公司 | Medicinal composition containing hydroxypropyl Zhigan and its preparing process |
-
2013
- 2013-01-09 CN CN2013100079038A patent/CN103012513A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1398838A (en) * | 2001-07-26 | 2003-02-26 | 中国人民解放军军事医学科学院放射医学研究所 | Diphenylethylene compound and its prepn and application in preventing and treating diabetes |
| CN1686148A (en) * | 2005-04-20 | 2005-10-26 | 昆明翔昊科技有限公司 | Use of liposoluble stilbene glycoside kind compound in treating ischemic heart brain blood vessel disease and its preparation |
| CN101036663A (en) * | 2007-04-18 | 2007-09-19 | 昆明翔昊科技有限公司 | Medicinal composition containing hydroxypropyl Zhigan and its preparing process |
Non-Patent Citations (1)
| Title |
|---|
| BANKS H J ET AL: "New natural stilbene glucoside from Rheum rhaponticum (Polygonaceae)", 《AUSTRALIAN JOURNAL OF CHEMISTRY》, vol. 24, no. 11, 31 December 1971 (1971-12-31), pages 2427 - 2430 * |
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