Summary of the invention
One object of the present invention is to disclose a kind of Chinese medicine composition of new treatment apoplexy; The object of the invention also is to disclose a kind of method of quality control of new Chinese medicine composition.
The bulk drug of pharmaceutical composition of the present invention is formed and proportioning following (by weight):
The A scheme:
Cape jasmine 150-220 weight portion pseudo-ginseng 70-110 weight portion.
Preferably:
Cape jasmine 170-200 weight portion pseudo-ginseng 80-100 weight portion.
The B scheme
Cape jasmine 150-220 weight portion arasaponin 3-9 weight portion
Preferably:
Cape jasmine 170-200 weight portion arasaponin 5-7 weight portion
The C scheme
Gardenoside 3.8-11.5 weight portion arasaponin 3-9 weight portion.
Preferably:
Gardenoside 6.3-8.9 weight portion arasaponin 5-7 weight portion.
Press practice of pharmacy, the invention described above preparation of pharmaceutical compositions can be become the various clinical pharmaceutical dosage form, comprise that oral formulations or non-enteron aisle are to formulation approximately.Said oral formulations is selected from a kind of in the middle of the tablet, capsule, pill, granule, supensoid agent, dripping pill, oral liquid; Said parenterai administration formulation is selected from a kind of in the middle of injection, aerosol, suppository or the subcutaneous administration formulation.
Medicine of the present invention also can add conventional drug excipient, as solvent, disintegrant, flavouring, antiseptic, colorant etc.
The preparation method that the B scheme is made parenteral solution is:
Get the cape jasmine of recipe quantity, be ground into meal, with the 65-80% ethanol percolation that 7-9 doubly measures, collect percolate, reclaim ethanol, be concentrated into the clear cream that 55-70 ℃ of relative density is 1.00-1.20, add 3-5 times of water gaging dilution, stir evenly, 0-4 ℃ refrigerates 45-50 hour, filter, 55-70 ℃ of relative density of filtrate decompression simmer down to is the clear cream of 1.00-1.20, and last activated-charcoal column is used earlier the distilled water wash-out, colourless to eluent, doubly measure the 65-80% ethanol elution with 9-11 again, collect eluent, decompression recycling ethanol also concentrates, drying gets the cape jasmine semi-manufacture; Get the cape jasmine semi-manufacture and dissolve, filter with an amount of water for injection; Other gets the arasaponin of recipe quantity, add an amount of water for injection dissolving, filter, pseudo-ginseng soup and cape jasmine soup mixing, regulate the pH value to 5.5-7.5,0-4 ℃ refrigerates 20-30 hour, and 0.45 μ m filter membrane filters, and filtrate adds to the full amount of water for injection, filter with 0.22 μ m filter membrane, the filtrate can becomes 10ml ampoule parenteral solution, 110-120 ℃ pressure sterilizing 20-40 minute, promptly.
The method of quality control that this composition is made injection comprises discriminating and/or assay and/or finger-print.
Discrimination method comprises a kind of in the following method and/or two kinds:
A. get this product 5ml, water bath method, residue add dissolve with ethanol and quantitative the commentaries on classics is dissolved in volumetric flask 5ml, and other gets the Gardenoside reference substance, adds ethanol and makes the solution that every 1ml contains 4mg, in contrast product solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version appendix in 2000 VI B); Draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with 8-11: 6-9: 1-3: 0.3-0.6 ethyl acetate-acetone-formic acid-water is developping agent, launches, and takes out, and dries, and spray is with 5% vanillic aldehyde sulfuric acid solution, heating; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color; B. get this product 10ml, in addition water saturated normal butyl alcohol jolting is extracted 2-4 time, and each 10ml merges n-butanol extracting liquid, with the saturated water liquid washing of normal butyl alcohol 2-4 time, 100ml at every turn; Get the n-butanol layer recovered under reduced pressure to doing, residue adds methyl alcohol 0.5ml makes dissolving, as need testing solution; Other gets ginsenoside Rb
1, the ginsenoside Rg
1And Panax Notoginseng saponin R
1Reference substance adds methyl alcohol and makes the mixed liquor that every ml contains 6mg, in contrast product solution; Test according to thin-layered chromatography (an appendix II of Chinese Pharmacopoeia version in 2000 B), draw above-mentioned two kinds of each 2ul of solution, put respectively on same silica gel g thin-layer plate, below environment temperature 15-25 ℃, with 11-15: 6-9: lower floor's solution that 1-3 chloroform-methanol-water is placed below 10 ℃ is developping agent, launches, take out, dry, spray is that the 1.0ml concentrated sulphuric acid adds absolute ethyl alcohol and makes into 10ml solution with 1 → 10 sulfuric acid ethanol liquid, and it is clear to be heated to the spot colour developing in 105-115 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
Content assaying method comprises a kind of in the following method and/or two kinds: a. Gardenoside, according to high performance liquid chromatography (" the about allusion quotation of a China " appendix VI of version D in 2000) mensuration; Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica; 13-17: the 80-90 acetonitrile-water is a moving phase; The detection wavelength is 238nm; Theoretical cam curve is calculated by the Gardenoside peak should be not less than 1500; It is an amount of that the preparation of reference substance solution, precision take by weighing the Gardenoside reference substance, adds methyl alcohol and make dissolving in right amount, makes the solution that every 1ml contains 0.14-0.15mg, promptly; The preparation of need testing solution, accurate this product 1ml that draws, water bath method, residue adds anhydrous alcohol solution, and ethanol liquid is transferred to another crucible, water bath method, residue is with dissolve with methanol and be transferred in the 100ml measuring bottle, adds methyl alcohol to scale, shakes up, promptly; Determination method, accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject high performance liquid chromatograph, measure, promptly; This product contains Gardenoside (C
17H
21O
10) every ml must not be less than 4.6-4.9mg; B. notoginsenoside is measured according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2000 D); Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica; With acetonitrile-water binary gradient is moving phase (seeing Table 1); The detection wavelength is 203nm; Theoretical cam curve is with the ginsenoside Rg
1The peak calculates should be not less than 2500; The reference substance solution preparation, precision takes by weighing notoginsenoside R, ginsenoside Rg
1, ginsenoside Rb
1Add dissolve with methanol in right amount, make every 1ml and contain Panax Notoginseng saponin R
11.5mg, the ginsenoside Rg
12.8mg, ginsenoside Rb
14.1mg the mixing reference substance solution; The need testing solution preparation, the accurate sample 5ml that draws extracts three times with the water-saturated n-butanol jolting, each 10ml; N-butanol layer merges, and with the washing of 10ml normal butyl alcohol saturation water once, abandons water layer, the n-butanol layer recovered under reduced pressure, and to doing, residue, shakes up to 5ml with dissolve with methanol, filters with miillpore filter 0.45 μ m, promptly; Determination method, the accurate reference substance solution 3 μ l that draw, 7 μ l, need testing solution 10 μ l inject high performance liquid chromatograph, calculate with the external standard two-point method, promptly; The every ml of this product contains Panax Notoginseng saponin R
1Must not be less than 0.18-1.25mg; The ginsenoside Rg
1Must not be less than 0.75-0.85mg; Ginsenoside Rb
1Must not be less than 1.8-2.5mg;
Table 1: acetonitrile-water binary gradient elution table
Time (min) | Flow velocity (ml/min) | A (water) | B (acetonitrile) |
0 20 | 1.0 1.0 | 80 60 | 20 40 |
Parenteral solution method of quality control of the present invention also can adopt the method for finger-print, this method comprise a kind of in the following method and or two kinds:
A. the preparation of need testing solution: the accurate parenteral solution 5ml that draws, extract twice with water saturated normal butyl alcohol jolting, for the first time 10ml; 5ml merges n-butanol extracting liquid for the second time; With the saturated water 10ml backwash of normal butyl alcohol once, discard water layer, the n-butanol extracting liquid recovered under reduced pressure is to doing, and residue is with dissolve with methanol and be settled in the 5ml volumetric flask, shakes up, and the 0.45um filter membrane filters, promptly; The preparation of reference substance solution: get the Gardenoside reference substance, the solution of making 0.5mg/ml with methyl alcohol is product solution in contrast; Assay method: detect wavelength 238nm, the acetonitrile-water gradient elution sees Table 2; Theoretical cam curve is calculated, and should not hang down 30000; Setting the need testing solution sample size is 10 μ l, writes down 60 minutes chromatogram, promptly; With the area at the retention time of the chromatographic peak (S peak) of Gardenoside and peak is 1 to calculate relative retention time and peak area ratio; Finger-print and technical parameter; Be 60 minutes writing time; The demarcation (relative retention time) of total fingerprint peaks: it is as follows to demarcate total fingerprint peaks according to the testing result of 10 batches of test samples: 1 (0.404) 2 (0.462) 3 (0.510) 4 (0.589) 5 (0.618) 6 (0.666) 7 (0.722) 8 (0.798) S (1.000) 9 (1.085) 10 (1.110) 11 (1.197) 12 (1.238) 13 (1.277); The ratio of total fingerprint peaks area: 1: S=1: (0.060-0.113); The part peak shape is described: separate when bad, peak 4 is the acromion at peak 5; Non-total peak area: test sample collection of illustrative plates and reference fingerprint are relatively; The non-total peak total area must not be greater than 5% of peak area;
Instrument reagent high performance liquid chromatograph: Waters 2695 pumps; Waters 2996 UV-detector; The Millcnnium32 chromatographic work station; Diamonsil C18 post (5 μ m, 4.6mm * 150mm); Acetonitrile is chromatographically pure (U.S. J.T.Baker); The Wahaha pure water; Methyl alcohol is chromatographically pure (Beijing prosperousization Fine Chemical Works);
Table 2 eluent gradient wash-out table
Time (min) | Flow velocity (ml/min) | Water % | Acetonitrile % |
0 8 21 35 40 45 50 60 | 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 | 95 92 88 88 70 40 0 0 | 5 8 12 12 30 60 100 100 |
B. the preparation of need testing solution: the accurate parenteral solution 10ml that draws, divide the secondary jolting to extract with water saturated normal butyl alcohol 20ml, merge n-butanol extracting liquid; Saturated water 300ml divides three backwashes with normal butyl alcohol, discards water layer, and the n-butanol layer recovered under reduced pressure is to doing, and residue is with dissolve with methanol and be settled in the 2ml measuring bottle, shakes up, and the 0.45um filter membrane filters, promptly; The preparation of tester: get the ginsenoside Rg
1Reference substance (available from China's product biological products assay institute approximately), the solution of making 2mg/ml with methyl alcohol is product solution in contrast; Assay method: detect wavelength 210nm, sensitivity 0.1AUFS: acetonitrile-water gradient elution, (seeing Table 3); Theoretical cam curve is by the ginsenoside Rg
1Calculate, should not hang down 30000; The accurate need testing solution 25 μ l that draw inject high performance liquid chromatograph, write down 70 minutes chromatogram, promptly; With the ginsenoside Rg
1The retention time of chromatographic peak (S peak) and the area at peak be 1 to calculate relative retention time and peak area ratio; Finger-print and technical parameter; Be 70 minutes writing time; The demarcation of total fingerprint peaks, (relative retention time): it is as follows to demarcate total fingerprint peaks according to the testing result of 10 batches of test samples: 1, (0.877) S, (1.000) 2, (1.027) 3, (1.555) 4, (1.592) 5, (1.652) 6, (1.675) 7, (1.708) 8, (1.744) 9, (1.799) 10, (2.005) 11, (2.042) 12, (2.074) 13, (2.111) 14, (2.150) 15, (2.212) 16, (2.235) 17, (2.364) 18, (2.391) 19, (2.420) 20, (2.440) 21, (2.771); The ratio of total fingerprint peaks area: S: 3: 8: 21=1: (0.222-0.412): (0.405-0.735): (1.428-2.380); Non-total peak area: test sample collection of illustrative plates and reference fingerprint are relatively; The non-total peak total area must not be greater than 5% of the total area;
Instrument reagent: Waters 2695 Waters 2996 UV-detector Millcnnium32 chromatographic work station Symmetryshild
TMRP
18(5 μ m, 4.6mm * 250mm); Other is that analysis is pure for Fisher acetonitrile (U.S.'s chromatographically pure) Fisher methyl alcohol (U.S.'s chromatographically pure) Wahaha pure water
Table 3 eluent gradient wash-out table
Time (min) | Flow velocity (ml/min) | A-water | The B-acetonitrile |
0 20 40 50 55 60 70 | 1.0 1.0 1.0 1.0 1.0 1.0 1.0 | 83 76 60 50 0 0 0 | 17 24 40 50 100 100 100 |
The clinical injection that cures mainly the acute ischemic headstroke of present composition preparation (vein relaxing is rescued injection of brain liquid), effect with removing pattogenic heat from the blood and toxic material from the body, disperse blood stasis and dredge collateral, by preventing the ischemic cascade reaction of headstroke, improve brain slight irrigation stream, reach the therapeutic purposes of anti-neuron infringement;
Rescue the relevant pharmacodynamics test research of injection of brain lyolysis poison effect with vein relaxing, adopt ferric trichloride to cause the thrombotic cerebral ischemia model of intraluminal middle cerebral artery occlusion in rats, studied vein relaxing and rescued rat nervous symptoms, brain water content, infraction cerebral morphology, brain tissue SOD and MDA content, cerebral infarction kitchen range Glu and NMDA expression, the Delayed Rectifier Potassium Current (I of injection of brain liquid this model
N) the mechanism of action; Experimental result demonstration vein relaxing is rescued the content that injection of brain liquid can obviously reduce the MDA of MCAT rat cerebral tissue, and increased SOD content reduces the expression of infraction brain tissue Glu and NMDA, increases Delayed Rectifier Potassium Current (I
N), reduce rat operation side brain water content, improve nervous symptoms;
Rescue the relevant pharmacodynamics test research of injection of brain liquid vein relaxing effect with vein relaxing, adopt ferric trichloride to cause the thrombotic cerebral ischemia model of intraluminal middle cerebral artery occlusion in rats, studied vein relaxing and rescued of the effect of injection of brain liquid this rat model cerebral blood flow, anesthetized dog cerebral blood flow, blood stasis hemorheology of rat, chick chorioallantoic membrane angiogenesis; The result shows that vein relaxing rescues injection of brain liquid and can promote the generation of neovascularity, obviously reduce Blood stasis rat whole blood viscosity and plasma viscosity, and red cell deformability is strengthened, and aggregation descends, and improves anesthetized dog, MCAT rat brain blood flow, thereby realizes its vein relaxing function;
Drug action and XUESHUANTONG that vein relaxing is rescued injection of brain liquid compare, in the determination experiment to MCAT rat cerebral tissue water cut, vein relaxing is rescued injection of brain liquid small dose group (comparing P<0.01 with model group) obviously because XUESHUANTONG group (comparing not statistically significant with model group); Vein relaxing rescues that dosage group SOD obviously raises in the injection of brain liquid, with XUESHUANTONG group P<0.05 relatively); Vein relaxing is rescued injection of brain liquid small dose group erythrodegeneration significantly increases (comparing P<0.05, P<0.01 with model group), and XUESHUANTONG does not have obvious effect to red blood cell deformation; Vein relaxing is rescued the heavy dose of group of injection of brain liquid blood vessel hyperplasia and significantly (is compared P<0.05, P<0.01 with model group), and a little less than the XUESHUANTONG effect; XUESHUANTONG can increase Delayed Rectifier Potassium Current (I
N), and that vein relaxing is rescued the effect of injection of brain liquid is not obvious, studies confirm that Delayed Rectifier Potassium Current (I at present
N) enhancing can promote the nerve cell apoptosis that ischemic causes; The effect that vein relaxing is rescued injection of brain liquid in other effect experiment all is better than XUESHUANTONG; Vein relaxing is rescued injection of brain liquid and is obviously had the effect that promotes the anesthetized dog cerebral blood flow; In a word, vein relaxing rescues injection of brain liquid and XUESHUANTONG compares, and the reduction dosage is arranged, thereby reach the purpose of attenuation synergistic;
The experimental result summary is as follows:
1. vein relaxing is rescued the influence of injection of brain liquid to MCAT rat nervous symptoms
Experiment has adopted ferric trichloride to cause the thrombotic rat cerebral ischemia model of arteria cerebri media, MCAT rat nervous symptoms evaluation results shows after the modeling: the rat that vein relaxing is rescued injection of brain liquid 83.2,41.6,20.8mg/kg group (20,10,5 times of quite clinical people's consumption respectively) is 12h after surgery, 24h, its nervous symptoms of 48h all has improve (P<0.01, P<0.05) in various degree; 2. vein relaxing is rescued the influence of injection of brain liquid to MCAT rat cerebral tissue water cut
The rat operation side brain water content that 48h after the modeling, vein relaxing rescue injection of brain liquid 83.2,41.6,20.8mg/kg group is starkly lower than model group, and the difference of comparing with model group has conspicuousness (P<0.01, p<0.05);
3. vein relaxing is rescued injection of brain liquid to the morphologic influence of MCAT rat cerebral tissue
Brain tissue Pathomorphology HE, the Nissl demonstration of dyeing, model group rat brain cortex ischemic focus cell quantity obviously reduces, the cell space atrophy, sex change, painted shallow; Vein relaxing is rescued three dosage groups of injection of brain liquid rat cerebral ischemia kitchen range cell quantity than the model group showed increased, and the cellular atrophy sex change obviously alleviates than model group; Graphical analysis shows that vein relaxing is rescued injection of brain liquid group positive cell surface density and model group relatively has statistical significance (P<0.01), and the prompting vein relaxing is rescued injection of brain liquid and had and alleviate the effect that cerebral ischemia institute neurocyte extremely damages;
4. vein relaxing is rescued the influence of injection of brain liquid to SOD of MCAT rat cerebral tissue and MDA
Vein relaxing is rescued injection of brain liquid 83.2,41.6mg/kg organizes the content that can obviously reduce the MDA of MCAT rat cerebral tissue, increased SOD content (P<0.01); The prompting vein relaxing is rescued injection of brain liquid and is had the Green Tea Extract damage, the effect of protection brain tissue;
5. vein relaxing is rescued the influence of injection of brain liquid to MCAT rat infraction brain tissue Glu and NMDA expression
Adopt ferric trichloride to cause thrombotic rat cerebral ischemia model of arteria cerebri media and immunohistochemical method, the result shows that vein relaxing rescues injection of brain liquid and can obviously reduce Glu, the NMDA expression at focus of infarct;
6. vein relaxing is rescued the influence of injection of brain liquid to Delayed Rectifier Potassium Current
Use the patch-clamp electrophysiological technique, detected vein relaxing and rescued the influence of injection of brain liquid to cardiac muscle cell's Delayed Rectifier Potassium Current, the result shows that vein relaxing is rescued injection of brain liquid to Delayed Rectifier Potassium Current (I
N) influence not obvious;
7. vein relaxing is rescued the influence of injection of brain liquid to MCAT rat brain volume of blood flow
MCAT rat cerebral tissue volume of blood flow obviously reduces, and vein relaxing is rescued the cerebral tissue blood flow of injection of brain liquid 83.2,41.6mg/kg group modeling 48h all apparently higher than model group (P<0.01), illustrates that this medicine is to the effect of having clear improvement of ischemic hindbrain tissue blood flow; Vein relaxing is rescued injection of brain liquid the rat Blood stasis that acute stress causes is improved significantly, and low dose of effect is the most obvious;
8. vein relaxing is rescued the influence of injection of brain liquid to the anesthetized dog cerebral blood flow (CBF)
Experiment is carried out 33 healthy hybrid dogs, and test shows: vein relaxing is rescued injection of brain liquid can significantly improve anesthetized dog cerebral blood flow (CBF) (P<0.05 or P<0.01); It acts on beginning onset in 10 minutes behind the injectable drug, and drug effect was maintained to after the administration 50 minutes; Vein relaxing is rescued injection of brain liquid 83.2, the 41.6mg/kg group does not relatively have significant difference with the positive drug XUESHUANTONG; Vein relaxing is rescued injection of brain liquid low dose group and positive drug XUESHUANTONG relatively, and significant difference (P<0.05) was arranged behind injectable drug in 45 minutes and 50 minutes;
9. vein relaxing is rescued the influence of injection of brain liquid to the stasis syndrome hemorheology of rat
Vein relaxing is rescued dosage in the injection of brain liquid (41.6mg/kg) can obviously reduce Blood stasis rat whole blood viscosity (with model group comparison P<0.01 with low dose of (20.8mg/kg), P<0.001, P<0.0001), plasma viscosity (but not seeing significant difference) on a declining curve, small dose group obviously reduce packed cell volume (comparing P<0.0001 with model group); Small dose group can make red cell deformability strengthen, and aggregation is down with (P<0.01, P<0.001);
10. vein relaxing is rescued the influence of injection of brain liquid to the chick chorioallantoic membrane angiogenesis
Vein relaxing is rescued injection of brain liquid 83.2,41.6, the 20.8mg/kg group all has the effect that promotes the chick chorioallantoic membrane angiogenesis;
Below to above-mentioned part experiment describe in detail:
Experimental example one vein relaxing is rescued the influence of injection of brain liquid to MCAT rat nervous symptoms
Experiment material
1. medicine and reagent
Be subjected to reagent: vein relaxing is rescued injection of brain liquid and provided by Kangyuan Pharmaceutical Industrial Co Ltd, Inner Mongolia, color: outward appearance is faint yellow, content: 10.39mg/ml, have removing pattogenic heat from the blood and toxic material from the body, and the function of disperse blood stasis and dredge collateral cures mainly the acute ischemic headstroke; Lot number: 2001042322; Clinical plan consumption: 4.16mg/kg, intravenous injection;
Positive control drug: XUESHUANTONG ZHUSHEYE is provided by Kangyuan Pharmaceutical Industrial Co Ltd, Inner Mongolia, color: white, and content: 50mg/ml has the function of promoting blood circulation, cures mainly the acute ischemic headstroke; Lot number: in defend the accurate word (1996) of medicine No. 001945; Clinical plan consumption: 10mg/kg, intravenous injection 1~2 time/day, 1 5ml;
Reagent and medicine: FeCl
36H
2O (A.R.), Beijing Chemical Plant's product is with the preparation of 1mol/L hydrochloric acid;
2. animal
The Wistar rat, the male and female dual-purpose, body weight 180~200g is provided by Chinese Academy of Medical Sciences's animal center breeding field, conformity certification number: the moving word of doctor 01-3008 number;
3. instrument
The XTT dissecting microscope, Beijing electric light scientific instrument factory product; SHZ-22 type water bath with thermostatic control oscillator, granary, Jiangsu medical apparatus and instruments factory product; AEG-220 type electronic analytical balance, day island proper Tianjin instrument company product;
Method and result
1. grouping and administration
60 rats are divided into six groups at random, be that sham operated rats, MCAT model group, vein relaxing are rescued injection of brain liquid 83.2mg/kg group, vein relaxing and rescued injection of brain liquid 41.6mg/kg group, vein relaxing and rescue injection of brain liquid 20.8mg/kg group (be equivalent to respectively clinical people's consumption 20,10,5 times), XUESHUANTONG 50mg/kg group (quite 5 times of people's consumption), 10 every group; Tail intravenously administrable after the modeling, a daily dose gives at twice, is administered five times altogether;
2. modeling method
The anesthesia of rats by intraperitoneal injection 10% chloral hydrate solution (350mg/kg); Press Tamura
[4]Deng method, improve a little; The rat right arm reclining is fixed, make an arc incision at paropia and external auditory meatus line mid point, be about 1.5cm, pinch off temporalis and excision, expose temporal bone, make a diameter 2.5mm bone window at cheekbone and temporo squamosum joint near oral-lateral 1mm place with dental burr, the cleaning residue exposes arteria cerebri media (between tractus olfactorius and venae cerebri inferiores); Put small pieces hollow plastic film protection blood vessel surrounding tissue; There is the small pieces quantitative filter paper of 50% ferric chloride solution, 10 μ l to apply on this section arteria cerebri media suction
(6), take off filter paper behind the 30min, use the normal saline flushing local organization, layer-by-layer suture steams again and raises; Sham operated rats is except that not dripping the ferric chloride solution the same model group of all the other operating procedures;
3.MCAT the evaluation of rat nervous symptoms
(12h, 24h 48h), press Bederson etc. to different time after surgery
(7)Method and improved, animal is carried out behavior scoring; 1. carry the about chi of mouse tail built on stilts, observe forelimb flexing situation; Stretch to ground as two forelimb symmetries, be designated as 0 fen; As the flexing that shoulder flexing, elbow flexing, shoulder inward turning or existing wrist elbow appear in the offside forelimb of performing the operation has inward turning person again, is designated as 1,2,3 and 4 fen; 2. animal is placed on the level and smooth ground, push away both shoulders respectively, check resistance to side shifting; Be designated as 0 fen as bilateral resistance equity and strong person; As resistance descender when the operation offside promotes, according to decline degree difference be divided into gently, in, weigh 3 and spend, be designated as 1,2,3 fen respectively; 3. animal two forelimbs are put on the wire netting, observed the muscular tension of two forelimbs; Bilateral muscular tension equity and strong person are 0 minute; Be designated as 1,2,3 fen according to operation offside muscle of anterior limb tension force decline degree difference equally; 4. carry the about chi of mouse tail built on stilts, animal has ceaselessly to operation offside revolver, is designated as 1 fen; According to above standard scoring, full marks are 11 minutes, and mark is high more, and the behavior disorder of animal is serious more; To behavior detect the marking value organize between relatively, the t check; The results are shown in Table 1;
Table 1 vein relaxing is rescued injection of brain liquid to the influence of MCAT rat nervous symptoms (X ± SD)
Group | Dosage mg/kg | N | The mental symptom scoring |
12h | 24h | 48h |
Sham operated rats MCAT model vein relaxing is rescued brain group XUESHUANTONG group | - - 83.2 41.6 20.8 50 | 10 10 10 10 10 10 | 0 5.5±0.55 4.33±0.82** 4.4±0.55** 4.67±0.55 3.67±0.52** | 0 4.67±0.52 3.83±0.41** 3.83±0.75** 4±0.63* 4±0.63* | 0 4±0.63 3.17±0.75* 3.33±0.82* 3.33±1.03 3.33±0.52* |
Annotate: each group is compared * P<0.05, * * P<0.01 with model group;
The result shows, removes sham operated rats and does not see that abnormal behavior changes, and MCAT model group rat is 12h after surgery, and hemiplegia sample symptom all appears in 24h, 48h, mainly show as in the operation offside forelimb to receive, and the shoulder inward turning, muscle of anterior limb tension force reduces, and the shoulder drag descends; Vein relaxing rescue injection of brain liquid 20.8mg/kg organize after surgery the 48h nervous symptoms improve not obvious outside, the rat that vein relaxing is rescued each dosage group of injection of brain liquid and XUESHUANTONG group is 12h after surgery, 24h, its nervous symptoms of 48h all have improve (P<0.01, P<0.05) in various degree;
Experimental example two vein relaxings are rescued the influence of injection of brain liquid to MCAT rat cerebral tissue water cut
Experiment material
1. medicine and reagent
Be subjected to reagent, positive control drug with test 20.1;
2. animal
The Wistar rat, the male and female dual-purpose, body weight 180~200g, is provided conformity certification number by 71 by Chinese Academy of Medical Sciences's animal center breeding field: the moving word of doctor 01-3008 number;
3. instrument
The XTT dissecting microscope, Beijing electric light scientific instrument factory product; AEG-220 type electronic analytical balance, day island proper Tianjin instrument company product; Df-206 type air dry oven, west city, Beijing medical apparatus and instruments factory product;
Method and result
Grouping, administration and operation are with test 20.1; 48 hours broken ends of postoperative are got brain, cut brain center section (front and back are respectively removed 3 millimeters), divide right and left, and blot surface moisture with filter paper, respectively weighing left and right sides brain sheet weight in wet base; Place baking oven again, 105 ℃ are toasted 48 hours to constant weight, and accurately the weighing dry weight is calculated water cut
[8], with control sides comparison on the same group, and compare between group, carry out the t check; The results are shown in Table 2;
Table 2 vein relaxing is rescued injection of brain liquid to the influence of MCAT rat cerebral tissue water cut (X ± SD)
Group | Dosage (mg/kg) | N | Water cut (%) |
Left side brain water content | Right brain water content |
Sham operated rats MCAT model group vein relaxing is rescued brain group XUESHUANTONG group | - - 83.2 41.6 20.8 50 | 10 10 10 10 10 10 | 77.30±0.79 77.04±0.48** 76.55±1.33** 76.77±0.78** 77.51±2.16** 77.04±0.48** | 77.09±0.57
△△ 78.90±0.96 78.09±1.03 77.96±0.91 75.11±2.03
△△ 78.90±0.96
|
Annotate: compare with model group
△ △P<0.01; Compare * * P<0.01 with offside brain water content on the same group;
The result shows, postoperative 48h, sham operated rats is not seen the big brain water content abnormal change in the left and right sides, model group rat operation side brain water content obviously increases, the rat operation side brain water content that vein relaxing is rescued each dosage group of injection of brain liquid, XUESHUANTONG group obviously is less than model group, compare with model group and to have significant difference (P<0.01), but each administration group operation side brain water content is compared with left brain and obviously increased (P<0.01);
Experimental example 3 vein relaxings are rescued the influence of injection of brain liquid to SOD of MCAT rat cerebral tissue and MDA
Experiment material
1, medicine and reagent
Be subjected to reagent: XUESHUANTONG ZHUSHEYE, vein relaxing are rescued injection of brain liquid with experiment 20.1;
MDA, SOD reagent close available from Nanjing and build up bio-engineering research institute; Lot number: 20011031;
2, animal
The Wistar rat, male, body weight 190~210g, 60, for Chinese Academy of Medical Sciences's animal center breeding field provides, conformity certification number: the moving word of doctor 01-3008 number;
3, instrument
721 type spectrophotometers, Shanghai the 3rd analytical instrument factory product;
Method and result
Grouping, administration, modeling method are with experiment 20.1; Modeling, 24 hours broken ends of administration are got brain, and decerebellation, olfactory bulb and brain stem add 9 times of physiological saline and be prepared into 10% brain homogenate, and be standby;
1, the mensuration of SOD
Close explanation by SOD reagent and measure the SOD vigor, the results are shown in Table 3;
2, the mensuration of MDA
Close explanation by MDA reagent and measure MDA content, the results are shown in Table 3;
Table 3 vein relaxing is rescued injection of brain liquid to the influence of SOD of MCAT rat cerebral tissue and MDA (X ± SD)
Group | Dosage mg/kg | N | The MDAmmol/g brain tissue | The SODnu/mg brain tissue |
Sham operated rats model group vein relaxing is rescued brain group XUESHUANTONG group | - - 83.2 41.6 20.8 50 | 10 10 10 10 10 10 | 0.54±0.11** 0.87±0.21 0.56±0.1** 0.59±0.12** 0.79±0.21 0.57±0.07** | 144±20.20** 92.17±21.54 127.67±20.55** 138.33±27.27** 98.5±29.04 114.5±21.86** |
Annotate: each group is compared * * P<0.01 with model group;
The result shows that rat is after closing arteria cerebri media with fixed attention, and its superoxide dismutase (SOD), MDA all have significant change, model group rat MDA content is apparently higher than sham operated rats, SOD is starkly lower than sham operated rats, and vein relaxing is rescued injection of brain liquid can obviously reduce MDA content, increased SOD vigor (P<0.01); Point out this medical instrument that the effect of Green Tea Extract injury protection brain tissue is arranged;
Experimental example four-way network is rescued the influence of injection of brain liquid to rats with cerebral ischemia brain cortex focus of infarct Glu and NMD expression
Experiment material
1, medicine and reagent
Be subjected to reagent: vein relaxing is rescued injection of brain liquid and is provided by Kangyuan Pharmaceutical Industrial Co Ltd, Inner Mongolia, cures mainly the acute ischemic headstroke;
Positive control drug: XUESHUANTONG ZHUSHEYE is provided by Kangyuan Pharmaceutical Industrial Co Ltd, Inner Mongolia, cures mainly the acute ischemic headstroke, intravenous injection, 2 times/day, 1 5ml; Lot number: 2000091112;
Reagent and medicine: FeCl
36H
2O (A.R.), Beijing Chemical Plant's product is with the preparation of 1mol/L hydrochloric acid;
Immunohistochemistry reagent: the anti-Glu of rabbit, NMDA (1: 200, Santa cruz company), biotinylation goat anti-rabbit igg and ABC compound (1: 200, Histostatn-sp kit, ZYMED company);
2, animal
The Wistar rat, the male and female dual-purpose, body weight 180~200g is provided by Chinese Academy of Medical Sciences's animal center breeding field, conformity certification number: the moving word of doctor 01-3008 number;
3, instrument
The POLYVAR universal microscope, U.S.'s product; C
8Very color pathological image analyser, BJ University of Aeronautics ﹠ Astronautics's product;
Test method
1. grouping and administration
60 rats are divided into six groups at random, be that sham operated rats, MCAT model group, vein relaxing are rescued injection of brain liquid 83.2mg/kg group, vein relaxing and rescued injection of brain liquid 41.6mg/kg group, vein relaxing and rescue injection of brain liquid 20.8mg/kg group (be equivalent to respectively clinical people's consumption 20,10,5 times), XUESHUANTONG 50mg/kg group (quite 5 times of people's consumption), 10 every group; Tail intravenously administrable after the modeling, a daily dose gives at twice, is administered five times altogether;
2. modeling method
The anesthesia of rats by intraperitoneal injection 10% chloral hydrate solution (350mg/kg); Press Tamura
[3]Deng method, improve a little; The rat right arm reclining is fixed, make an arc incision at paropia and external auditory meatus line mid point, be about 1.5cm, pinch off temporalis and excision, expose temporal bone, make a diameter 2.5mm bone window at cheekbone and temporo squamosum joint near oral-lateral 1mm place with dental burr, the cleaning residue exposes arteria cerebri media (between tractus olfactorius and venae cerebri inferiores); Put small pieces hollow plastic film protection blood vessel surrounding tissue; There is the small pieces quantitative filter paper of 50% ferric chloride solution, 10 μ l to apply on this section arteria cerebri media suction
(4), take off filter paper behind the 30min, use the normal saline flushing local organization, layer-by-layer suture steams again and raises; Sham operated rats is except that not dripping the ferric chloride solution the same model group of all the other operating procedures;
3. animal is handled
After the last administration one hour (being postoperative 48 hours), broken end is got brain, fix with 10% neutral formalin respectively, and paraffin embedding, immunohistochemical staining is made in section (getting optic chiasma front and back brain sheet), and the microscopic observation tissue morphology changes, and does graphical analysis;
4. the immunohistochemical staining method adopts the ABC method, and step is as follows: 1.. and 1% methyl alcohol H is gone in section
2O
2Tuck in, room temperature 30min is 2.. protease K digesting, 37 ℃ of 30min are 3.. normal sheep serum, room temperature 30min is 4.. the anti-Glu of rabbit, NMDA (1: 200, Santa cruz company), 5. 4 ℃ spent the night. biotinylation goat anti-rabbit igg and ABC compound (1: 200, Histostatn-sp kit, ZYMED company), 6. .0.05%DAB-0.1%H of 37 ℃ of 2h
2O
2The liquid colour developing; 1., 2., 3., 5., 6. all wash 3 times before the step, wash 5min with 0.01MPBS at every turn; Negative control saves one and resists or resist with normal sheep serum replacement one;
5. graphical analysis and statistical procedures
To Glu, NMDA SABC result, analyze with the very color pathological image analytic system of CMIAS8 (Chinese Aero-Space university graphical analysis center), add up the surface density of each; Statistics are checked with t between group;
Experimental result
1. vein relaxing is rescued the influence that injection of brain liquid is expressed rats with cerebral ischemia brain cortex focus of infarct Glu
Normal rat brain cortex Glu is expressed in the kytoplasm of cell, and the nuclear district is negative, and cellular morphology is cone-shaped more, and has projection, and cell is methodically arranged; The Glu cell quantity is than normal rat showed increased in the model group rat brain cortex focus of infarct, and Glu expresses also and obviously strengthens, and cellular morphology is the cone-shaped of atrophy sex change; Glu expression ratio model group obviously weakens in positive drug group, heavy dose of group, the middle dosage group rat brain, and wherein middle dosage group Glu expresses the most weak, and cell quantity is minimum; The same model group of the expression of small dose group Glu; The graphical analysis statistics sees Table 4;
Table 4 vein relaxing rescue the influence that injection of brain liquid expresses MCAT rat cerebral cortex focus of infarct Glu (
X± SD)
Group | Dosage (mg/kg) | N | The positive cell surface density |
Sham operated rats MCAT model group vein relaxing is rescued injection of brain liquid group XUESHUANTONG group | - - 83.2 41.6 20.8 50 | 9 9 9 9 9 9 | 0.09±0.02** 0.13±0.02 0.09±0.02** 0.07±0.02** 0.06±0.02** 0.10±0.10** |
Annotate: compare * * P<0.01 with model group
2. vein relaxing is rescued the influence that injection of brain liquid is expressed rats with cerebral ischemia brain cortex focus of infarct NMDA
Normal rat brain cortex NMDA is expressed in the kytoplasm of cell, and the nuclear district is negative, and cellular morphology is cone-shaped and circle more, and cell is methodically arranged; The expression of the interior NMDA of rat model brain cortex focus of infarct is obviously strong than normal rat, and cellular morphology is swelling and shrinkage deformation form; Positive drug group and heavy dose of group, middle dosage group, small dose group rat NMDA expression ratio model group weaken, and the expression of wherein heavy dose of group, middle dosage group NMDA is the most weak, and heavy dose of group NMDA cell is cone-shaped, and the atrophy sex change; Middle dosage group NMDA positive cell is small circular more; Statistics sees Table 5;
Table 5 vein relaxing is rescued the influence that injection of brain liquid expresses MCAT rat cerebral cortex NMDA (X ± SD)
Group | Dosage (mg/kg) | N | The positive cell surface density |
Sham operated rats MCAT model group vein relaxing is rescued injection of brain liquid group XUESHUANTONG group | - - 83.2 41.6 20.8 50 | 9 9 9 9 9 9 | 0.08±0.02* 0.11±0.02 0.09±0.02** 0.07±0.02** 0.05±0.01** 0.07±0.01* |
Annotate: compare * P<0.05 with model group, * * P<0.01
Conclusion: vein relaxing is rescued injection of brain liquid by reducing Glu, the NMDA expression at focus of infarct, reverses swelling, the sex change of cell, thereby reaches therapeutic purposes;
Experimental example five-way network is rescued the influence experiment material of injection of brain liquid to the anesthetized dog cerebral blood flow (CBF)
1. animal
33 of healthy hybrid dogs, the male and female dual-purpose, body weight 12~15kg, tonneau animal used as test plant provides by the Haidian District, Beijing City, animal conformity certification numbering: 024-069 number;
2. medicine
Vein relaxing is rescued injection of brain liquid: Kangyuan Pharmaceutical Industrial Co Ltd, Inner Mongolia (Gan Qi card pharmaceutical factory, the Inner Mongol) produces; Lot number: 2001042322; Specification: 20ml/ only; 263mg panaxoside/20ml;
Blank medicine: physiological saline;
XUESHUANTONG ZHUSHEYE (positive drug): Kangyuan Pharmaceutical Industrial Co Ltd, Inner Mongolia (Gan Qi card pharmaceutical factory, the Inner Mongol) produces; Lot number: 2000091112; Specification: 250mg/5ml
Medicine stoste places 4 ℃ of stored refrigerated;
3. medicine preparation
When each experiment begins, carry out the medicine preparation according to the weight of animals:
XUESHUANTONG ZHUSHEYE: 0.5ml stoste/kg; (25mg/kg)
Vein relaxing is rescued injection of brain liquid low dose group: and 0.394ml stoste/kg (the 5.18mg panaxoside/kg);
Vein relaxing is rescued dosage group in the injection of brain liquid: and 0.788ml stoste/kg (the 10.4mg panaxoside/kg);
Vein relaxing is rescued injection of brain liquid high dose group: and 1.577ml stoste/kg (the 20.7mg panaxoside/kg);
Draw the stoste of corresponding milliliter number, be mixed into the drip-feed liquid that cumulative volume is 100ml with an amount of physiological saline, water-bath is heated to 37 ℃ of laggard row veins instillation;
4. experimental apparatus and apparatus
TA-4000 polygraph (U.S. GouldInc company product), MF-27 electromagnetic flowmeter (Japanese photoelectricity company product), animal surgery apparatus one cover;
5. experimental procedure animal used as test operation
The animal pneumoretroperitoneum of weighing is injected 5% yellow Jackets 30mg/kg, and anesthesia back position is fixed, and it is standby to separate the left side femoral vein; Cervical incision separates right common carotid artery, and intubate (anti-freezing in the heparin solution pipe) directly writes down Blood pressure of carotid artery; Separate left common carotid artery, separate the ligation external carotid artery, arteria carotis communis is embedded the electromagnetic flowmeter probe, and (ф=2mm), fix perpendicular to arteria carotis communis surveys the ICAF amount fully and uses; Dry for preventing probe, drip physiological saline and keep moistening; Lay ECG electrode, record standard limbs II lead electrocardiogram; Respiration probes is placed nostril place's recording respiration frequency and depth of respiration;
6. animal used as test administration
Stablized after the operation 30 minutes, and write down every index, as contrasting before the administration; During the experiment beginning, carry out the preparation of soup according to preceding method; Dog is at the uniform velocity instiled through the left side femoral vein and studies medicine accordingly, and record instils the start time, and the control medicine at the uniform velocity dripped off in 20 minutes;
7. observation index
Measure immediately when instiling beginning (0 minute), every 5 minutes records once, write down 15~30 seconds at every turn, chart drive speed is 10mm/s during record, and record interval chart drive speed is 50mm/h, and the polygraph monitor is monitored synchronously; Observed and recorded drug effect to the beginning of instiling finished in back 60 minutes; The following physical signs of synchronous recording:
1) ICAF amount
2) arterial pressure
Experimental result
The TA-4000 polygraph on the recording chart of special use, by artificial counting, is converted to numeral with the figure of record with Experiment Data Records, is attached to each experimental record back with the form of form;
Experimental result is used the SAS statistical software, carries out the t check to have determined whether significant difference with regard to the every index between each group;
Vein relaxing rescue injection of brain liquid to the influence of anesthetized dog cerebral blood flow (CBF) referring to table 6;
Table 6 vein relaxing is rescued the influence (ml/min) of injection of brain liquid to the anesthetized dog cerebral blood flow (CBF)
Time after the administration (branch) | XUESHUANTONG (n=6) | Vein relaxing is rescued brain high dose (n=7) | Vein relaxing is rescued dosage in the brain (n=7) | Vein relaxing is rescued brain low dosage (n=6) | Physiological saline (n=7) |
0 5 10 15 20 25 30 35 40 45 50 55 60 | 110.7±2.2 117.5±6.6 124.2±8.8
** 126.7±10.8
** 128.3±13.7
** 130.8±16.6
** 115.3±53.5 132.2±14.5
** 129.2±10.2
** 130.0±8.4
** 130.2±7.8
** 122.5±13.6 119.2±17.4
| 109.1±2.6 116.7±5.0 121.7±7.3
** 128.3±16.7
** 131.4±16.0
** 130.7±12.1
** 131.4±8.9
** 130.4±8.1
** 126.9±6.9
** 126.1±8.7
** 126.1±12.8
** 119.3±13.7 118.4±14.6
| 106.6±6.1 112.0±7.3 119.4±9.4
* 122.7±11.3
** 125.4±12.7
** 127.6±11.7
** 126.9±10.9
** 122.9±15.5 126.1±8.5
** 126.7±6.9
** 125.6±6.8
** 122.3±10.2 106.4±39.7
| 110.8±3.4 116.7±5.2 120.8±8.6
* 122.2±7.1
** 123.8±8.6
** 124.0±7.9
** 125.5±7.4
** 123.8±6.0
** 120.2±2.6 115.8±4.9
△ 115.3±11.9
△ 112.8±7.5
** 108.7±11.9
| 111.9±2.0 111.8±2.9 110.3±5.9 110.3±4.5 111.3±6.6 110.9±6.6 110.6±5.6 111.3±8.3 113.3±8.7 113.0±7.9 110.9±7.9 110.3±6.1 110.9±7.2 |
*Compare P<0.05 with control group;
*Compare P<0.01 with control group;
△Compare P<0.05 with XUESHUANTONG;
Experiment shows: vein relaxing is rescued injection of brain liquid can significantly improve anesthetized dog cerebral blood flow (CBF) (P<0.05 or P<0.01); It acts on beginning onset in 10 minutes behind the injectable drug, and drug effect was maintained to after the administration 50 minutes; Vein relaxing is rescued injection of brain liquid high dose group and middle dosage group and positive drug XUESHUANTONG does not relatively have significant difference; Vein relaxing is rescued injection of brain liquid low dose group and positive drug XUESHUANTONG relatively, and significant difference (P<0.05) was arranged behind injectable drug in 45 minutes and 50 minutes;
2. vein relaxing is rescued the influence of injection of brain liquid to anaesthetized dog blood pressure
Vein relaxing rescue injection of brain liquid to the influence of anesthetized dog systolic pressure referring to table 7;
Table 7 vein relaxing is rescued the influence (mmHg) of injection of brain liquid to the anesthetized dog systolic pressure
Time after the administration (branch) | XUESHUANTONG (n=6) | Vein relaxing is rescued brain high dose (n=7) | Vein relaxing is rescued dosage in the brain (n=7) | Vein relaxing is rescued brain low dosage (n=6) | Physiological saline (n=7) |
0 5 10 15 20 25 30 35 40 45 50 55 60 | 148.3±12.5 146.3±13.6 149.8±16.7 148.7±15.2 147.7±16.5 148.2±16.0 144.3±11.2 144.3±11.5 147.2±12.6 146.7±13.8 144.0±10.4 146.7±16.9 148.2±12.1 | 149.6±12.5 152.1±11.8 153.0±13.3 154.7±13.5 152.7±12.6 151.0±13.1 151.9±12.4 150.4±13.1 147.6±12.6 147.4±13.0 146.7±14.7 145.4±16.3 146.8±14.9 | 140.6±16.8 138.0±11.7 139.0±11.7 140.0±10.8 140.6±10.7 139.1±10.9 139.1±10.4 139.9±9.5 140.0±13.7 141.7±10.7 141.4±12.2 140.6±12.9 141.1±13.2 | 146.8±19.6 147.9±18.9 149.3±18.1 149.3±18.8 149.3±19.5 149.5±18.3 148.8±18.6 150.3±19.3 148.2±19.1 144.6±22.3 147.3±19.9 143.5±17.2 145.4±18.6 | 137.2±17.0 138.0±18.4 137.4±17.3 136.9±16.6 137.1±15.9 136.7±18.3 137.9±16.6 138.1±17.6 138.4±16.6 138.7±16.3 138.8±17.0 137.8±17.0 138.2±17.1 |
Experiment shows: vein relaxing is rescued injection of brain liquid does not have significant difference (P>0.05) to the influence of anesthetized dog systolic pressure
Vein relaxing rescue injection of brain liquid to the influence of anesthetized dog diastolic pressure referring to table 8;
Table 8 vein relaxing is rescued the influence (mmHg) of injection of brain liquid to the anesthetized dog diastolic pressure
Time after the administration (branch) | XUESHUANTONG (n=6) | Vein relaxing is rescued brain high dose (n=7) | Vein relaxing is rescued dosage in the brain (n=7) | Vein relaxing is rescued brain low dosage (n=6) | Physiological saline (n=7) |
0 5 10 15 20 25 30 35 40 45 50 55 60 | 116.3±10.2 115.0±8.6 119.5±14.4 116.0±7.9 116.8±10.6 117.7±9.8 116.0±8.0 115.7±8.0 116.7±8.4 117.2±9.2 114.8±8.5 118.7±13.3 117.3±10.1 | 121.3±10.5 119.7±11.6 120.0±10.9 119.4±13.9 118.1±12.3 117.0±12.8 117.6±13.0 118.4±12.6 115.6±12.8 117.0±12.2 116.0±14.9 115.5±16.0 117.0±13.9 | 112.9±14.2 111.2±10.4 112.1±10.6 112.4±10.0 112.8±10.6 112.0±10.7 112.1±10.6 113.0±9.4 112.1±12.1 113.7±10.5 113.0±12.1 112.6±13.5 112.9±13.4 | 119.7±13.2 120.0±12.9 121.3±13.5 120.5±12.7 119.7±13.1 120.8±12.4 119.7±13.0 122.8±11.9 121.8±12.8 117.8±16.7 118.7±17.0 117.7±15.5 116.3±15.8 | 109.6±13.1 110.8±14.7 110.3±13.5 110.3±13.5 109.9±13.1 110.7±14.1 110.3±14.0 110.4±14.7 110.9±13.2 111.1±12.2 111.1±13.5 110.7±13.7 110.7±13.7 |
Experiment shows: vein relaxing is rescued the high, medium and low dosage group of injection of brain liquid does not have significant difference (P>0.05) to the influence of anesthetized dog diastolic pressure
Experimental example six vein relaxings are rescued the influence of injection of brain liquid to the chick chorioallantoic membrane angiogenesis
Experiment material and method
1. medicine:
Injection Chinese medicine XUESHUANTONG, vein relaxing are rescued brain and are provided by professor Niu Xin, and concentration is 50mg notoginseng total saponin/ml solution; This soup is divided into greatly (50mg notoginseng total saponin/ml), in (25mg notoginseng total saponin/ml), little (three concentration of 12.5mg notoginseng total saponin/ml);
2. hatch:
Fresh white skin kind egg is available from Changping livestock corporation, select no damaged crackle, the approximate kind egg that air chamber is arranged of size, after the ethanol with 75% is sterilized its surperficial wiping, greatly head-up, tilt to put into 37.8 ℃ of incubators of having sterilized, place water pond in the incubator to keep certain humidity in the case; For preventing embryo's adhesion, promote the amnion motion, every day, turning egg(s) was 4-5 time
[3]
3. window:
With reference to paying method such as think of a way
[4]And improved; Plant when egg is hatched to the 5th day and take out, on candler, check the vascular development situation; Select clear, well-developed kind of the egg of blood vessel, between two trunks in the lower right of embryo head, draw the window's position of fixed one 1cm * 1cm, and also draw a mark at the air chamber place; In the immigration super-clean bench, with finish position kind egg shell with 75% ethanol wiping sterilization after, bore an aperture at kind of an egg air chamber place to be used for decompression with dental burr earlier, cut the shell that picture is decided the window's position with the little reduction of dental burr again, remove the place's chorion of windowing gently with the ophthalmology tweezer, expose white shell membrane, carefully remove membrana putaminis behind a little physiological saline again and expose chorioallantoic membrane;
4. dosing:
According to results of study such as Zhang Shucheng
[5], we adopt gelfoam as adding drug carrier; Sterile gelatin sponge (available from China-Japan Friendship Hospital) is cut into the fritter of 5mm * 2mm * 2mm, after adding the soup of 35 μ l with the filtration of 0.22 μ m sterile filters, place on the chorioallantoic membrane of window middle position, seal the aperture at window and air chamber place with aseptic adhesive tape after, put back to and continued in the incubator to hatch 48 hours; And control group (stroke-physiological saline solution, chicken embryo of 35 μ l/), the positive group of XUESHUANTONG are set, vein relaxing is rescued injection of brain liquid 83.2mg/kg, 41.6mg/kg, 20.8mg/kg group; All do 20 chicken embryos for every group;
5. get film:
Open the adhesive tape on the chicken embryo fenestella, after kind of an egg bottom is bored an aperture, is waited to flow out the egg white about 1ml, in window, drip immobile liquid (methyl alcohol: acetone=1: 1), fixing 15-20min at room temperature; Afterwards, eggshell is broken into two with one's hands to both sides, content is all poured in the double dish, carefully chorioallantoic membrane is taken off with eye scissors and ophthalmology tweezer, puts into the plate that fills clear water and launches, and apply ointment or plaster on filter paper the preservation of drying in the shade;
6. count:
Under anatomical lens, observe vessel growth situation and counting on the chorioallantoic membrane; Counting region: with the administration be the center in the scope of diameter 1.5cm, be divided into large, medium and small blood vessel and count according to the diameter of blood vessel;
7. meter is learned and is handled:
We carry out statistical procedures with the physiological saline group with the t check with count results;
Experimental result
The data statistics result is as shown in table 9:
Table 9 vein relaxing is rescued injection of brain liquid to the angiopoietic influence of chick chorioallantoic membrane
Group | Dosage mg/kg | Chorioallantoic membrane blood vessel number/embryo (x ± s) |
Trunk | Medium vessels | Little blood vessel |
The control group vein relaxing is rescued brain group XUESHUANTONG group | 83.2 41.6 20.8 50 | 2.33±1.53 2.00±0.82 1.71±0.76 2.29±1.11 2.01±0.55 | 11.00±1.00 8.86±2.27 7.86±2.12* 9.43±2.30 6.80±3.11* | 43.67±6.35 35.43±6.60 36.14±2.34** 36.71±3.86* 30.60±6.77** |
Annotate: n=20, compare with control group: * P<0.05 * * P<0.01
The result shows that vein relaxing rescues each dosage group of injection of brain liquid, and particularly heavy dose of group and XUESHUANTONG relatively have the effect that promotes angiogenesis;
Following embodiment all can realize the effect of above-mentioned experimental example.
Embodiment
Embodiment 1 injection
Cape jasmine 175g arasaponin 5.5g
Get the cape jasmine of recipe quantity, be ground into meal, the percolation under the photograph liquid extract item (" appendix IO of Chinese pharmacopoeia version in 2000), with 70% ethanol percolation of 8 times of amounts, collect percolate, reclaim ethanol, being concentrated into relative density is the clear cream of 1.10 (60 ℃ of mensuration), adds 4 times of water gaging dilutions, stir evenly, 0-4 ℃ refrigerates 48 hours, filters, and filtrate decompression simmer down to relative density is the clear cream of 1.10 (60 ℃ of mensuration), last activated-charcoal column, earlier use the distilled water wash-out, colourless to eluent, again with 10 times of amount 70% ethanol elutions, collect eluent, decompression recycling ethanol also concentrates, and drying gets the cape jasmine semi-manufacture; Get the cape jasmine semi-manufacture and dissolve, filter with an amount of water for injection; Other gets the arasaponin of recipe quantity, add an amount of water for injection dissolving, filter, pseudo-ginseng soup and cape jasmine soup mixing, regulate the pH value to 6.0-7.0,0-4 ℃ refrigerates 24 hours, and 0.45 μ m filter membrane filters, and filtrate adds to the full amount of water for injection, filter with 0.22 μ m filter membrane, the filtrate can becomes 10ml ampoule parenteral solution, 110 ℃ of pressure sterilizings 30 minutes, promptly.Function with cure mainly: removing pattogenic heat from the blood and toxic material from the body, disperse blood stasis and dredge collateral.Cure mainly the ishemic stroke disease.Be used for the apoplexy hemiplegia, hemianesthesia, dizziness and headache, tongue say not smoothgoing by force or in silence, dispute is crooked, and mind dusk covers; Very the person is unexpected falls forward, and mind is muddle-headed, and a name gurgling weith sputum is talked in the nose snore, dimly red tongue, the card that disturb on the wind fire such as wiry and rolling pulse, the stasis of blood hinders train of thought.
Usage and consumption: once-a-day, a 154mg, two weeks of the course of treatment.
Specification: 10ml/ props up, and 154mg is (with Gardenoside, ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside R
1Content sum meter).
Embodiment 2 injections
Cape jasmine 190g arasaponin 7g
Get the cape jasmine of recipe quantity, be ground into meal,, collect percolate with 75% ethanol percolation of 7 times of amounts, reclaim ethanol, be concentrated into 65 ℃ of relative densities and be 1.10 clear cream, add 5 times of water gagings dilutions, stir evenly, 0-4 ℃ refrigerates 49 hours, filters, 60 ℃ of relative densities of filtrate decompression simmer down to are 1.10 clear cream, and last activated-charcoal column is used earlier the distilled water wash-out, colourless to eluent, with 10 times of amount 70% ethanol elutions, collect eluent again, decompression recycling ethanol also concentrates, and drying gets the cape jasmine semi-manufacture; Get the cape jasmine semi-manufacture and dissolve, filter with an amount of water for injection; Other gets the arasaponin of recipe quantity, add an amount of water for injection dissolving, filter, pseudo-ginseng soup and cape jasmine soup mixing, regulate the pH value to 6.0-7.0,0-4 ℃ refrigerates 24 hours, and 0.45 μ m filter membrane filters, and filtrate adds to the full amount of water for injection, filter with 0.22 μ m filter membrane, the filtrate can becomes 10ml ampoule parenteral solution, and 115 ℃ of pressure sterilizings 30 minutes promptly get injection.Usage and consumption: once-a-day, a 154mg, two weeks of the course of treatment.Specification: 10ml/ props up, and 154mg is (with Gardenoside, ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside R
1Content sum meter).
Embodiment 3 tablets
Cape jasmine 175g pseudo-ginseng 90g.
Get cape jasmine, the pseudo-ginseng of recipe quantity, be ground into meal, with 75% ethanol percolation of 7 times of amounts, collect percolate, reclaim ethanol, be concentrated into 65 ℃ of relative densities and be 1.10 clear cream, add 5 times of water gaging dilutions, stir evenly, 0-4 ℃ refrigerates 49 hours, filter, 60 ℃ of relative densities of filtrate decompression simmer down to are 1.10 clear cream, and often the regulation grain is made particle 100 grams, add excipient, compressing tablet (the pelletizing press sheet machine is once finished).Every heavy 0.5 gram.
Embodiment 4 capsules
Gardenoside 70g arasaponin 60g
Often regulation becomes capsule.
The method of quality control of embodiment 5 present composition injections
Discrimination method: a. gets this product 5ml, and water bath method, residue add dissolve with ethanol and quantitative the commentaries on classics is dissolved in volumetric flask 5ml, and other gets the Gardenoside reference substance, adds ethanol and makes the solution that every 1ml contains 4mg, in contrast product solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version appendix in 2000 VI B); Draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with 10: 7: 2: 0.5 ethyl acetate-acetone-formic acid-water is developping agent, launches, and takes out, and dries, and spray is heated with 5% vanillic aldehyde sulfuric acid solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color; B. get this product 10ml, in addition water saturated normal butyl alcohol jolting is extracted 3 times, and each 10ml merges n-butanol extracting liquid, with the saturated water liquid washing of normal butyl alcohol 3 times, each 100ml; Get the n-butanol layer recovered under reduced pressure to doing, residue adds methyl alcohol 0.5ml makes dissolving, as need testing solution; Other gets ginsenoside Rb
1, the ginsenoside Rg
1And Panax Notoginseng saponin R
1Reference substance adds methyl alcohol and makes the mixed liquor that every ml contains 6mg, in contrast product solution; Test according to thin-layered chromatography (appendix IIB of Chinese Pharmacopoeia version in 2000), draw above-mentioned two kinds of each 2ul of solution, put respectively on same silica gel g thin-layer plate, in environment temperature below 20 ℃, lower floor's solution of placing below 10 ℃ with 13: 7: 2 chloroform-methanol-water is developping agent, launches, take out, dry, spray is that the 1.0ml concentrated sulphuric acid adds absolute ethyl alcohol and makes into 10ml solution with 1 → 10 sulfuric acid ethanol liquid, and it is clear to be heated to the spot colour developing in 110 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
Content assaying method: a. Gardenoside, the photograph high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2000 D) measure; Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica; 15: 85 acetonitrile-waters are moving phase; The detection wavelength is 238nm; Theoretical cam curve is calculated by the Gardenoside peak should be not less than 1500; It is an amount of that the preparation precision of reference substance solution takes by weighing the Gardenoside reference substance, adds methyl alcohol and make dissolving in right amount, makes the solution that every 1ml contains 0.144mg, promptly; The preparation of need testing solution, accurate this product 1ml that draws, water bath method, residue adds anhydrous alcohol solution, and ethanol liquid is transferred to another crucible, water bath method, residue is with dissolve with methanol and be transferred in the 100ml measuring bottle, adds methyl alcohol to scale, shakes up, promptly; Determination method, accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject high performance liquid chromatograph, measure, promptly; This product contains Gardenoside (C
17H
24O
10) every ml must not be less than 4.7mg;
B. notoginsenoside is measured according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2000 D); Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica; With acetonitrile-water binary gradient is moving phase (seeing Table 1); The detection wavelength is 203nm; Theoretical cam curve is with the ginsenoside Rg
1The peak calculates should be not less than 2500; The reference substance solution preparation, precision takes by weighing notoginsenoside R, ginsenoside Rg
1, ginsenoside Rb
1Add dissolve with methanol in right amount, make every 1ml and contain Panax Notoginseng saponin R
11.5mg, the ginsenoside Rg
12.8mg, ginsenoside Rb
14.1mg the mixing reference substance solution; The need testing solution preparation, the accurate sample 5ml that draws extracts three times with the water-saturated n-butanol jolting, each 10ml; N-butanol layer merges, and with the washing of 10ml normal butyl alcohol saturation water once, abandons water layer, the n-butanol layer recovered under reduced pressure, and to doing, residue, shakes up to 5ml with dissolve with methanol, filters with miillpore filter 0.45 μ m, promptly; Determination method, the accurate reference substance solution 3 μ l that draw, 7 μ l, need testing solution 10 μ l inject high performance liquid chromatograph, calculate with the external standard two-point method, promptly; The every ml of this product contains Panax Notoginseng saponin R
1Must not be less than 0.2mg; The ginsenoside Rg
1Must not be less than 0.8mg; Ginsenoside Rb
1Must not be less than 2.0mg;
Table 1: acetonitrile-water binary gradient elution table
Time (min) | Flow velocity (ml/min) | A (water) | B (acetonitrile) |
0 20 | 1.0 1.0 | 80 60 | 20 40 |
Parenteral solution method of quality control of the present invention also can adopt the method for finger-print:
A. the preparation of need testing solution: the accurate parenteral solution 5ml that draws, extract twice with water saturated normal butyl alcohol jolting, for the first time 10ml; 5ml merges n-butanol extracting liquid for the second time; With the saturated water 10ml backwash of normal butyl alcohol once, discard water layer, the n-butanol extracting liquid recovered under reduced pressure is to doing, and residue is with dissolve with methanol and be settled in the 5ml volumetric flask, shakes up, and the 0.45um filter membrane filters, promptly; The preparation of reference substance solution: get the Gardenoside reference substance, the solution of making 0.5mg/ml with methyl alcohol is product solution in contrast; Assay method: detect wavelength 238nm, the acetonitrile-water gradient elution sees Table 2; Theoretical cam curve is calculated, and should not hang down 30000; Setting the need testing solution sample size is 10 μ l, writes down 60 minutes chromatogram, promptly; With the area at the retention time of the chromatographic peak (S peak) of Gardenoside and peak is 1 to calculate relative retention time and peak area ratio; Finger-print and technical parameter; Be 60 minutes writing time; The demarcation (relative retention time) of total fingerprint peaks: it is as follows to demarcate total fingerprint peaks according to the testing result of 10 batches of test samples: 1 (0.404) 2 (0.462) 3 (0.510) 4 (0.589) 5 (0.618) 6 (0.666) 7 (0.722) 8 (0.798) S (1.000) 9 (1.085) 10 (1.110) 11 (1.197) 12 (1.238) 13 (1.277); The ratio of total fingerprint peaks area: 1: S=1: (0.060-0.113); The part peak shape is described: separate when bad, peak 4 is the acromion at peak 5; Non-total peak area: test sample collection of illustrative plates and reference fingerprint are relatively; The non-total peak total area must not be greater than 5% of peak area;
Instrument reagent high performance liquid chromatograph: Waters 2695 pumps; Waters 2996 UV-detector; The Millcnnium32 chromatographic work station; Diamonsil C18 post (5 μ m, 4.6mm * 150mm); Acetonitrile is chromatographically pure (U.S. J.T.Baker); The Wahaha pure water; Methyl alcohol is chromatographically pure (Beijing prosperousization Fine Chemical Works);
Table 2 eluent gradient wash-out table
Time (min) | Flow velocity (ml/min) | Water % | Acetonitrile % |
0 8 21 35 40 45 50 60 | 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 | 95 92 88 88 70 40 0 0 | 5 8 12 12 30 60 100 100 |
B. the preparation of need testing solution: the accurate parenteral solution 10ml that draws, divide the secondary jolting to extract with water saturated normal butyl alcohol 20ml, merge n-butanol extracting liquid; Saturated water 300ml divides three backwashes with normal butyl alcohol, discards water layer, and the n-butanol layer recovered under reduced pressure is to doing, and residue is with dissolve with methanol and be settled in the 2ml measuring bottle, shakes up, and the 0.45um filter membrane filters, promptly; The preparation of tester: get the ginsenoside Rg
1Reference substance (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute), the solution of making 2mg/ml with methyl alcohol is product solution in contrast; Assay method: detect wavelength 210nm, sensitivity 0.1AUFS: the acetonitrile-water gradient elution sees Table 3; Theoretical cam curve is by the ginsenoside Rg
1Calculate, should not hang down 30000; The accurate need testing solution 25 μ l that draw inject high performance liquid chromatograph, write down 70 minutes chromatogram, promptly; With the ginsenoside Rg
1The retention time of chromatographic peak (S peak) and the area at peak be 1 to calculate relative retention time and peak area ratio; Finger-print and technical parameter; Be 70 minutes writing time; The demarcation of total fingerprint peaks, (relative retention time): it is as follows to demarcate total fingerprint peaks according to the testing result of 10 batches of test samples: 1, (0.877) S, (1.000) 2, (1.027) 3, (1.555) 4, (1.592) 5, (1.652) 6, (1.675) 7, (1.708) 8, (1.744) 9, (1.799) 10, (2.005) 11, (2.042) 12, (2.074) 13, (2.111) 14, (2.150) 15, (2.212) 16, (2.235) 17, (2.364) 18, (2.391) 19, (2.420) 20, (2.440) 21, (2.771); The ratio of total fingerprint peaks area: S: 3: 8: 21=1: (0.222-0.412): (0.405-0.735): (1.428-2.380); Non-total peak area: test sample collection of illustrative plates and reference fingerprint are relatively; The non-total peak total area must not be greater than 5% of the total area;
Instrument reagent: Waters 2695 Waters 2996 UV-detector Millcnnium32 chromatographic work station Symmetryshild
TMRP
18(5 μ m, 4.6mm * 250mm); It is that analysis is pure for the pure glassware for drinking water of Fisher acetonitrile (U.S.'s chromatographically pure) Fisher methyl alcohol (U.S.'s chromatographically pure) Wahaha
Table 3 eluent gradient wash-out table
Time (min) | Flow velocity (ml/min) | A-water | The B-acetonitrile |
0 20 40 50 55 60 70 | 1.0 1.0 1.0 1.0 1.0 1.0 1.0 | 83 76 60 50 0 0 0 | 17 24 40 50 100 100 100 |