[summary of the invention]
The purpose of this invention is to provide a kind of compositions that contains Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and ligustrazine or its pharmaceutically acceptable salt, its weight ratio is: 10~400 parts of 30~400 parts of Radix Notoginseng extracts, 10~400 parts of Radix Salviae Miltiorrhizae extracts, ligustrazine or its pharmaceutically acceptable salts.Be preferably: 30~200 parts of 100~200 parts of Radix Notoginseng extracts, 30~200 parts of Radix Salviae Miltiorrhizae extracts, ligustrazine or its pharmaceutically acceptable salts.Best preferred: 50 parts of 150 parts of Radix Notoginseng extracts, 50 parts of Radix Salviae Miltiorrhizae extracts, ligustrazine or its pharmaceutically acceptable salts.
More than form,, can make the preparation of 100~1000 consumptions,, can be made into 1000,1~10 of each consumption as injection as if being unit with the gram.As tablet, can be made into 1000, take 1~10 at every turn.
More than form to be by weight as proportioning, when producing, can or reduce according to the corresponding proportion increase, as large-scale production can be raw material with the kilogram, or be unit with the ton, small-scale production can be unit with the gram also, weight can increase or reduce, but the constant rate of weight proportion between each composition.
The ratio of above weight proportion obtains through science screening, and for especial patient, the ratio of can corresponding adjustment forming increases or reduce being no more than 100%.
Ligustrazine pharmaceutically acceptable salt mentioned above can be phosphate, hydrochlorate, lactate, sulfate, mesylate, benzoate, aspartate, tartrate, preferably phosphate and hydrochlorate.
The Panax Notoginseng saponin R that Radix Notoginseng extract mentioned above is contained
1Be not less than 2.0%, the ginsenoside Rg
1Be not less than 28.0%, ginsenoside Rb
1Be not less than 25.0%; Panax Notoginseng saponin R
1, the ginsenoside Rg
1And ginsenoside Rb
1Total content be not less than 55%.
Radix Notoginseng extract can obtain by the raw material of buying listing or the mode of groping preparation technology voluntarily, and the present invention has carried out preferably the preparation technology of this extract, and is as follows:
Get pseudo-ginseng, be ground into coarse powder, add alcohol reflux 3 times, each 3 hours, filter, filtrate recycling ethanol is to there not being the alcohol flavor, add water to an amount of (every 1ml is equivalent to the 2g crude drug), cold preservation 24 hours filters, filtrate is added on the low pole macroporous resin column of having handled well, with the water elution of 2 times of column volumes, 80% ethanol elution that reuse is 4~5 times is collected eluent earlier, reclaim ethanol and vacuum drying, promptly.
Extract yield: 4~5% content of the total saponins in radix notoginseng:>60%
Radix Salviae Miltiorrhizae extract mentioned above can be that water is carried acid precipitation extract, alcohol extract, water extract-alcohol precipitation extract or water and carried column extract.Radix Salviae Miltiorrhizae extract can utilize the preparation method of prior art to obtain, for example can utilize Chinese patent application CN1352985A, CN1247855A, CN1242364A, CN1384090A, 02117923.9, (Yunnan University of Traditional Chinese Medicine's journal, 2001,24 (4): preparation method 6) obtains Guo Ying etc.Also can grope preparation technology voluntarily obtains.Content of danshinolic acid B is 45%~70% in the Radix Salviae Miltiorrhizae extract of the present invention, and its total phenolic content is preferably in more than 80% more than 70%.No matter be or groping preparation technology voluntarily prepares Radix Salviae Miltiorrhizae extract of the present invention,, then should make with extra care, make it to meet above-mentioned content standard if do not reach above-mentioned content standard by prior art.
The present invention has carried out preferably the preparation technology of Radix Salviae Miltiorrhizae extract, and is as follows:
Get the red rooted salvia coarse powder, decoct with water secondary, add 12 times of amounts of water for the first time, add for the first time 10 times of amounts of water, each 2 hours, collecting decoction, filter, filtrate decompression is concentrated into the concentrated solution of relative density 1.08~1.12, adds ethanol to containing the alcohol amount to 85%, cold preservation 24 hours filters, and filtrate recycling ethanol is to there not being the alcohol flavor, add hydrochloric acid adjust pH to 2, extract 3 times with the ethyl acetate jolting, merge ethyl acetate liquid, evaporate to dryness.Residue adds the sour water dissolving of pH value 2, is added on the polyamide column of having handled well, with the water elution of 2 times of column volumes, discards water lotion earlier, and 95% ethanol elution of 4 times of column volumes of reuse is collected eluent, reclaims ethanol, and vacuum drying, promptly.
Radix Salviae Miltiorrhizae extract yield: 4~5% Radix Salviae Miltiorrhizae total phenolic acids content:>80% content of danshinolic acid B:>50%
Another object of the present invention is to provide a kind of pharmaceutical composition for the treatment of cardiovascular and cerebrovascular disease.Be particularly useful for treating acute myocardial infarction, coronary heart disease, angina pectoris, ischemic encephalopathy, cerebral thrombosis etc.
Said composition can add one or more pharmaceutically acceptable carriers, with oral, snuffing is gone into or the mode of parenteral is applied to the patient who needs this treatment.Be used for when oral, can be made into conventional solid preparation, as tablet, capsule, soft capsule, dispersible tablet, oral liquid, granule, chewable tablet, oral cavity disintegration tablet, drop pill, slow releasing tablet, slow releasing capsule, controlled release tablet, controlled release capsule, make liquid preparation such as water or oil-suspending agent or other liquid preparation such as syrup etc.; When being used for parenteral, can be made into solution, water or the oil-suspending agent etc. of injection, as liquid drugs injection, freeze-dried powder, aseptic powder injection, transfusion etc.The preferred form of this compositions is injection or oral formulations.
Medicine of the present invention can adopt the conventional method production in the existing pharmaceutical field, can add various pharmaceutically acceptable carriers when needing.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant of pharmaceutical field routine etc.
The present invention in order to increase its dissolubility, can add solubilizing agents such as tween 80 when making injection.Can add the isoosmotic adjusting agent that is used to regulate osmotic pressure in the transfusion, for example, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, lactic acid are received, glucose, xylitol, sorbitol and dextran etc., preferred sodium chloride or glucose.Can add excipient in the powder pin, for example, mannitol, glucose etc.
The present invention proves through pharmaceutical research and drug effect animal experiment study result, by Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and ligustrazine phosphate is the medicine that main component is made, can significantly improve the every index of hemodynamics of anesthetized dog, increase coronary flow, significantly antiplatelet aggregation significantly reduces myocardial infarct size.Experimental study result of the present invention proves that Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and ligustrazine phosphate drug combination are synergism, and drug effect obviously strengthens.
The invention has the advantages that: interaction, composition of prescription to known Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and ligustrazine phosphate are studied, with Radix Notoginseng extract and ligustrazine phosphate is that raw material directly feeds intake, preparation technology is simple, the drug loss that has caused when having avoided extracting and because the different product mass discrepancy bigger shortcoming that the crude drug mass discrepancy causes, make medicine purity higher, impurity is few, safety is higher, and mass discrepancy is little between the different batches medicine, and drug quality is more uniform and stable.Drug combination behind employing Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and the ligustrazine phosphate reasonable compatibility, prove through pharmacological experiment, drug effect is better than using separately Radix Notoginseng total arasaponins or Radix Salviae Miltiorrhizae total phenolic acids or ligustrazine phosphate, be better than also that Radix Salviae Miltiorrhizae total phenolic acids adds Radix Notoginseng extract or Radix Salviae Miltiorrhizae adds Rhizoma Chuanxiong, drug effect improves, Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and ligustrazine phosphate drug combination are synergism, and dosage reduces relatively, are with a wide range of applications.
The following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.
[specific embodiment]
The preparation selection process of experimental example 1 Radix Notoginseng extract
Get pseudo-ginseng, be ground into coarse powder, add alcohol reflux 3 times, each 3 hours, filter, filtrate recycling ethanol is to there not being the alcohol flavor, add water to an amount of (every 1ml is equivalent to the 2g crude drug), cold preservation 24 hours filters, filtrate is added on the low pole macroporous resin column of having handled well, with the water elution of 2 times of column volumes, 80% ethanol elution that reuse is 4~5 times is collected eluent earlier, reclaim ethanol and vacuum drying, promptly.
Experimental example 2: content of the total saponins in radix notoginseng determination test in the Radix Notoginseng extract
Radix Notoginseng total arasaponins reference substance 10mg is got in the preparation of reference substance solution, in 60 ℃ of vacuum dryings 2 hours, accurately claim surely, put in the 25ml measuring bottle, adding methanol makes dissolving and is diluted to scale, shake up, filter with dry filter paper, precision is measured subsequent filtrate 5ml, put in the 25ml measuring bottle, add methanol and be diluted to scale, shake up, promptly.
This product 15mg is got in the preparation of need testing solution, and accurate the title decides, and puts in the 25ml measuring bottle, adds methanol and makes dissolving and be diluted to scale, shakes up, and filters with dry filter paper, and precision is measured subsequent filtrate 5ml, puts in the 25ml measuring bottle, adds methanol and is diluted to scale, shakes up, promptly.
The algoscopy precision is measured need testing solution and each 1ml of reference substance solution, puts in the 10ml tool plug test tube evaporate to dryness respectively, put coldly, add 5% vanillin glacial acetic acid solution 0.2ml, add perchloric acid 0.8ml again, 60 ℃ of insulations 15 minutes, be cooled to room temperature, add glacial acetic acid 5ml, shake up, do blank simultaneously,, measure absorbance at 560nm wavelength place according to ultraviolet visible spectrophotometry, calculate, promptly.
Method according to experimental example 1 obtains three batches of Radix Notoginseng extracts, and wherein the assay of Radix Notoginseng total arasaponins the results are shown in Table 1:
The assay result and the yield of table 1 Radix Notoginseng extract
Batch | Total saponin content (%) | Panax Notoginseng saponin R
1Content (%)
| The ginsenoside Rg
1Content (%)
| Ginsenoside Rb
1Content (%)
|
1 | 65.15 | 3.76 | 31.74 | 29.65 |
2 | 68.17 | 3.89 | 33.54 | 30.74 |
3 | 70.18 | 3.92 | 34.84 | 31.42 |
Experimental example 3 Radix Salviae Miltiorrhizae extraction process screening tests
1. extraction process by water screening test
Amount of water, extraction time and extraction time are bigger to the influence of extracting the result, are index with Radix Salviae Miltiorrhizae total phenolic acids content and yield of extract therefore, investigate three kinds of factors to extracting result's influence with orthogonal experiment.Factor and level design see Table 2.
Table 2 Radix Salviae Miltiorrhizae alcohol extraction process is investigated the factor level table
Level/factor | A amount of water (doubly) | B extraction time (hour) | C extraction time (inferior) |
1 | 18 | 3 | 1 |
2 | 20 | 4 | 2 |
3 | 22 | 5 | 3 |
Test method takes by weighing Radix Salviae Miltiorrhizae 100g, and totally 9 parts, extract by the requirement of each row of table 2 number, merge extractive liquid, filters, and filtrate concentrates and is settled to 500ml, and is standby.
The assay of red total phenolic acid
The preparation precision of reference substance solution takes by weighing exsiccant protocatechualdehyde reference substance 1mg, is dissolved in water, and is settled to 100ml.
The preparation precision of need testing solution is measured sample solution 25ml, put in the 50ml volumetric flask, thin up to scale precision is measured 0.1ml and is put and add ethanol 5ml in the 25ml volumetric flask, adds 0.3% sodium lauryl sulphate 2ml, 0.6% potassium ferricyanide, one 0.9% ferric oxide (face and use preceding mixed in equal amounts) 1ml, so 5min is placed in the dark place, add the 0.1mol/L hydrochloric acid solution to scale, shake up, after 20min is placed in the dark place, put in the lena colorimetric pool, the 720nm place measures.
The algoscopy colorimetry
The mensuration precision of yield of extract is measured each sample solution 25ml, puts in the evaporating dish of constant weight, and behind water bath method, 105 ℃ of dryings 3 hours, cooling was 30 minutes in the dislocation exsiccator, accurately rapidly claims to decide weight.Calculate yield of extract.The results are shown in Table 3.
Table 3 Radix Salviae Miltiorrhizae extraction process by water is investigated orthogonal test table
Sequence number | Factor | Total phenolic acid (%) | Yield of extract (%) |
A | B | C | D |
1 | 1 | 1 | 1 | 1 | 0.572 | 15.12 |
2 | 1 | 2 | 2 | 2 | 0.631 | 16.23 |
3 | 1 | 3 | 3 | 3 | 0.569 | 16.78 |
4 | 2 | 1 | 2 | 3 | 0.763 | 18.32 |
5 | 2 | 2 | 3 | 1 | 0.656 | 18.09 |
6 | 2 | 3 | 1 | 2 | 0.737 | 17.67 |
7 | 3 | 1 | 3 | 2 | 0.761 | 19.11 |
8 | 3 | 2 | 1 | 3 | 0.802 | 18.42 |
9 | 3 | 3 | 2 | 1 | 0.823 | 19.84 |
K1 | 1.772 | 2.096 | 2.111 | 2.051 | | |
K2 | 2.156 | 2.089 | 2.217 | 2.129 | | |
K3 | 2.386 | 2.129 | 1.986 | 2.134 | | |
R | 0.614 | 0.043 | 0.231 | 0.083 | | |
Table 4 Radix Salviae Miltiorrhizae extraction process analysis of variance table
Factor | Sum of sguares of deviation from mean | Degree of freedom | Inequality | The F value | Significance |
A | 0.06415 | 2 | 0.032 | 44.549 | p<0.05 |
B | 0.0003 | 2 | 0.000 | 0.208 | |
C | 0.00891 | 2 | 0.00446 | 6.188 | |
D (error term) | 0.00144 | 2 | 0.00024 | | |
F
0.05(2,2)=19.00 F
0.05(2,2)=99.00
By table 3, table 4 result as can be seen, amount of water is to influence the principal element that Radix Salviae Miltiorrhizae extracts, and extraction time and extraction time are little to its influence, in conjunction with commercial production, with reference to yield of extract, determine that the Radix Salviae Miltiorrhizae extraction process is A
3B
3C
2, that is: get Radix Salviae Miltiorrhizae, decocting with water twice, twice all is 2 hours, adds 12 times of amounts of water for the first time, adds 10 times of amounts of water for the second time.
2. the investigation of alcohol precipitation process
The investigation of alcohol precipitation concentration:
Test method takes by weighing Radix Salviae Miltiorrhizae 200g, totally 3 parts, decocts with water respectively twice, 1.5 hours for the first time, add 12 times of amounts of water, 1 hour for the second time, add 10 times of amounts of water, merge extractive liquid, filters, it is 1.17~1.20 (80 ℃) that filtrate is concentrated into relative density, uses Ethanol Treatment 2 times, and containing amount of alcohol in the solution for the first time is 75%, for the second time be respectively 80%, 85%, 90%, each all cold preservation is placed, and filters, reclaim ethanol, concentrate and be settled to 1000ml.Precision is measured 50ml, according to 1 following assay and yield of extract assay method, measures the content and the yield of extract of total phenolic acid.Result of the test sees Table 5.
Table 5 alcohol precipitation concentration is investigated result of the test
Alcohol precipitation concentration (%) | Total phenolic content (%) | Yield of extract (%) |
80 | 0.737% | 9.49 |
85 | 0.779% | 6.55 |
90 | 0.798% | 4.97 |
By table 5 result as can be seen, when alcohol precipitation concentration is 85%, can guarantee the content of total phenolic acid, can reach the purpose of remove impurity again, therefore selecting ethanol precipitation twice concentration is 85%.
Experimental example 4 Radix Salviae Miltiorrhizae extract discrimination tests
Get this product 0.2g, porphyrize adds 70% methanol 25ml, and reflux 1 hour filters, and filtrate evaporate to dryness, residue add 70% methanol 1ml makes dissolving, as need testing solution.Other gets the salvianolic acid B reference substance, adds 70% methanol and makes the solution that every 1ml contains 2mg, in contrast product solution.Use tlc determination, draw above-mentioned two kinds of each 5ul of solution, put in same silica gel G F respectively
254On the lamellae, be developing solvent, launch, take out, dry, put under the ultra-violet lamp (254nm) and inspect with toluene-chloroform-ethyl acetate-methanol-formic acid (2: 3: 4: 0.5: 2).
In the test sample chromatograph, with reference substance chromatograph relevant position on, show the speckle of same color.
The test of experimental example 5 Radix Salviae Miltiorrhizae extract assays
Total phenolic content is measured colorimetry
The preparation precision of reference substance solution takes by weighing the protocatechualdehyde reference substance 1mg that is dried to constant weight in 105 ℃, is dissolved in water, and is settled to 100ml.
The preparation precision of need testing solution takes by weighing sample 50mg, put in the 50ml volumetric flask, thin up to scale precision is measured 0.1ml and is put and add ethanol 5ml in the 25ml volumetric flask, adds 0.3% sodium lauryl sulphate 2ml, 0.6% potassium ferricyanide, one 0.9% ferric oxide (face and use preceding mixed in equal amounts) 1ml, so 5min is placed in the dark place, add the 0.1mol/L hydrochloric acid solution to scale, shake up, after 20min is placed in the dark place, put in the lena colorimetric pool, the 720nm place measures.
The assay high performance liquid chromatography of salvianolic acid B
Chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica; With methanol-acetonitrile-formic acid-water (30: 10: 1: 59) be mobile phase; The detection wavelength is 286nm.Theoretical cam curve is calculated by the danshensu peak should be not less than 1500.
It is an amount of that the preparation precision of reference substance solution takes by weighing the salvianolic acid B reference substance, adds 75% methanol and make the solution that every 1ml contains 0.14mg, promptly.
The about 0.2g of this product is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, and the accurate 75% methanol 50ml that adds claims to decide weight, reflux 1h takes out, and Fang Leng claims to decide weight again, supplies with 75% methanol to subtract weight loss, shake up, filter, get subsequent filtrate, promptly.
Accurate respectively reference substance solution and each 20ul of need testing solution of drawing of algoscopy injects chromatograph of liquid, measures, promptly.
Method according to experimental example 3 obtains three batches of water-soluble extract of red sage root, and wherein total phenolic acid and content of danshinolic acid B measurement result see Table 6:
The assay result and the yield of table 6 Radix Salviae Miltiorrhizae extract
Batch | Total phenolic content (%) | Content of danshinolic acid B (%) |
1 | 75.11 | 51 |
2 | 77.85 | 54 |
3 | 70.27 | 48 |
By the drug effect concertedness and the proportioning screening of the following pharmacological evaluation proof present composition, the present composition is hereinafter to be referred as compositions.
Experimental example 5: the research of present composition drug combination drug effect
Animal subject: the Wister rat, male, body weight 200~220g, 10 every group, is divided into 33 groups at random by 330.
Test sample: normal saline matched group
Radix Notoginseng extract group: Radix Notoginseng extract injection, self-control
Radix Salviae Miltiorrhizae extract group: Radix Salviae Miltiorrhizae total phenolic acids injection, self-control
Ligustrazine phosphate group: ligustrazine phosphate injection, self-control
Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group, self-control
Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group, self-control
Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract+ligustrazine phosphate (various dose proportioning), 27 groups, self-control
Experimental technique: rat is divided into 33 groups immediately: the normal saline matched group; The Radix Notoginseng extract group; The Radix Salviae Miltiorrhizae extract group; The ligustrazine phosphate group; Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group; Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group; Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group (is organized 1: three+pellet+river=30mg+10mg+10mg; Organize 2: three+pellet+river=30mg+10mg+50mg; Organize 3: three+pellet+river=30mg+10mg+400mg; Organize 4: three+pellet+river=30mg+50mg+10mg; Organize 5: three+pellet+river=30mg+50mg+50mg; Organize 6: three+pellet+river=30mg+50mg+400mg; Organize 7: three+pellet+river=30mg+400mg+10mg; Organize 8: three+pellet+river=30mg+400mg+50mg; Organize 9: three+pellet+river=30mg+400mg+400mg; Organize 10: three+pellet+river=150mg+10mg+10mg; Organize 11: three+pellet+river=150mg+10mg+50mg; Organize 12: three+pellet+river=150mg+10mg+400mg; Organize 13: three+pellet+river=150mg+50mg+10mg; Organize 14: three+pellet+river=150mg+50mg+50mg; Organize 15: three+pellet+river=150mg+50mg+400mg; Organize 16: three+pellet+river=150mg+400mg+10mg; Organize 17: three+pellet+river=150mg+400mg+50mg; Organize 18: three+pellet+river=150mg+400mg+400mg; Organize 19: three+pellet+river=400mg+10mg+10mg; Organize 20: three+pellet+river=400mg+10mg+50mg; Organize 21: three+pellet+river=400mg+10mg+400mg; Organize 22: three+pellet+river=400mg+50mg+10mg; Organize 23: three+pellet+river=400mg+50mg+50mg; Organize 24: three+pellet+river=400mg+50mg+400mg; Organize 25: three+pellet+river=400mg+400mg+10mg; Organize 26: three+pellet+river=400mg+400mg+50mg; Organize 27: three+pellet+river=400mg+400mg+400mg).Each medicine all is diluted to desired concn with normal saline, and dosage is 15mg/kg, the tail vein injection administration.
The rat experiment myocardial infarction model: it is fixing that animal pentobarbital intraperitoneal injection of anesthesia (45mg/kg) is faced upward the position.Tracheal intubation is made the longitudinal incision of 2cm in breastbone left side, nearly breastbone side is cut off the 3rd, the 4th costicartilage, open the thoracic cavity after, connect artificial respirator (ventilation 2ml/100g, 50 times/min).Cut off pericardium, expose heart, left anterior descending coronary artery root threading is in order to ligation, and record standard II lead electrocardiogram was stablized 10 minutes, and the ligation left anterior descending coronary artery is closed the thoracic cavity.With syringe sucking-off animal throat secretions, make animal recover autonomous respiration.Behind the ligation coronary artery 15min, intravenously administrable.Behind the ligation coronary artery 4 hours, win heart, 5 of the following crosscuts of ligature, carry out chlorination nitro blue tetrazolium (N-BT) dyeing, calculating myocardium infarcted region area accounts for the percentage ratio of ventricle and heart area, and carries out statistical procedures (t check).The results are shown in Table 7.
Table 7 compositions is to the influence (x of rat experiment myocardial inyaretion scope
On average± s)
Group | Number of animals | Infarcted region/ventricle (%) | Infarcted region/heart (%) |
Model group | 10 | 33.67±7.85 | 26.48±5.10 |
The Radix Notoginseng extract group | 10 | 25.98±4.58
* | 21.03±3.25
* |
The Radix Salviae Miltiorrhizae extract group | 10 | 23.58±7.85
* | 19.68±5.45
* |
The ligustrazine phosphate group | 10 | 26.95±5.62
* | 23.45±4.23
* |
Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group | 10 | 20.58±6.25
**# | 16.49±5.45
**# |
Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group | 10 | 21.61±6.84
**# | 17.62±6.21
**# |
Group 1 | 10 | 17.91±6.25
**##& | 14.86±4.82
**##& |
Group 2 | 10 | 17.02±5.54
**##& | 14.03±5.23
**##& |
Group 3 | 10 | 17.23±5.41
**##& | 14.21±4.86
**##& |
Group 4 | 10 | 14.25±6.14
**##& | 12.51±5.61
**##& |
Group 5 | 10 | 13.05±2.48
**##& | 11.52±2.53
**##& |
Group 6 | 10 | 13.52±5.41
**##& | 11.63±4.52
**##& |
Group 7 | 10 | 14.06±4.23
**##& | 12.21±4.56
**##& |
Group 8 | 10 | 14.52±3.14
**##& | 12.52±2.52
**##& |
Group 9 | 10 | 14.25±5.16
**##& | 12.54±5.42
**##& |
Group 10 | 10 | 13.25±4.56
**##& | 11.23±4.82
**##& |
Group 11 | 10 | 13.02±3.56
**##& | 10.98±3.56
**##& |
Group 12 | 10 | 12.89±3.45
**##& | 10.53±3.12
**##& |
Group 13 | 10 | 12.01±2.87
**##& | 10.13±2.97
**##& |
Group 14 | 10 | 11.68±4.52
**##& | 9.89±4.23
**##& |
Group 15 | 10 | 11.89±3.52
**##& | 10.05±2.58
**##& |
Group 16 | 10 | 12.42±6.23
**##& | 10.58±5.26
**##& |
Group 17 | 10 | 12.35±3.56
**##& | 10.45±3.75
**##& |
Group 18 | 10 | 12.64±3.54
**##& | 10.58±3.98
**##& |
Group 19 | 10 | 13.59±4.36
**##& | 11.25±5.36
**##& |
Group 20 | 10 | 13.24±2.52
**##& | 10.98±3.24
**##& |
Group 21 | 10 | 13.87±3.45
**##& | 11.35±4.12
**##& |
Group 22 | 10 | 13.54±3.25
**##& | 11.11±3.52
**##& |
Group 23 | 10 | 12.52±4.25
**##& | 10.85±3.85
**##& |
Group 24 | 10 | 13.85±3.58
**##& | 11.32±3.51
**##& |
Group 25 | 10 | 14.25±4.35
**##& | 12.36±3.58
**##& |
Group 26 | 10 | 15.24±2.58
**##& | 12.59±3.51
**##& |
Group 27 | 10 | 15.64±4.25
**##& | 12.98±3.76
**##& |
Annotate:
*P<0.05,
*P<0.01 is compared with model group;
#P<0.05,
##P<0.01 is compared with Radix Notoginseng extract group or Radix Salviae Miltiorrhizae extract group or ligustrazine phosphate group;
﹠amp;P<0.05 is compared with Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group or Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group.
Conclusion: each administration group all has tangible function of resisting myocardial ischemia, the effect of Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group or Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group be better than single with Radix Notoginseng total arasaponins or Radix Salviae Miltiorrhizae extract or ligustrazine phosphate (
#P<0.05), but be weaker than Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group effect (
﹠amp;P<0.05).The effect of Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group is the strongest, and relevant with the consumption proportion of each composition components, and effect is optimum during three+pellet+river=150mg+50mg+50mg.
Experimental example 6: compositions antiplatelet aggregative activity
Laboratory animal: the Wister rat, male, body weight 200~220g, 10 every group, is divided into 9 groups at random by 90.
Test sample: normal saline matched group
Radix Notoginseng extract group: Radix Notoginseng extract injection, self-control
Radix Salviae Miltiorrhizae extract group: Radix Salviae Miltiorrhizae total phenolic acids injection, self-control
Ligustrazine phosphate group: ligustrazine phosphate injection, self-control
Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group, self-control
Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group, self-control
Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract+ligustrazine phosphate=150mg+50mg+50mg is divided into basic, normal, high 3 dosage groups, self-control
Test method: rat is divided into 9 groups at random, 10 every group, is respectively the normal saline matched group; The Radix Notoginseng extract group; The Radix Salviae Miltiorrhizae extract group; The ligustrazine phosphate group; Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group; Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group; Three+pellet+river=150mg+50mg+50mg group is divided into basic, normal, high 3 dosage groups.Each treated animal administration, once a day, successive administration 7 days, after the last administration 1 hour, from abdominal aortic blood, anticoagulant adopted 3.28% sodium citrate after the Animal Anesthesia, with blood with 1: 9 mixed.With anticoagulated whole blood 1500r.min under 20 ℃ of conditions
-1Centrifugal 5min obtains platelet rich plasma (PPR).After leaving and taking quantitative PPR, will remain PPR once more with 3000r.min
-1Centrifugal 10min obtains the rich or poor platelet blood plasma of own control (PPP).Regulate PPR concentration with PPP, make each PPR concentration identical.In 37 ℃ constant temperature hole after the preheating, (final concentration is 3 μ mol.L to add ADP with PPR
-1) cause and write down maximum agglutination rate by platelet aggregation.The results are shown in Table 8.
Table 8 antiplatelet aggregative activity (X ± SD)
Group | Dosage (mg/kg) | Maximum agglutination rate | The P value |
The normal saline matched group | 15 | 86.65±18.46 | - |
The Radix Notoginseng extract group | 15 | 70.37±16.28 | <0.05 |
The Radix Salviae Miltiorrhizae extract group | 15 | 71.58±16.27 | <0.05 |
The ligustrazine phosphate group | 15 | 68.47±16.78 | <0.05 |
Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group | 15 | 63.15±15.28
* | <0.01 |
Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group | 15 | 62.87±17.25
* | <0.01 |
Three+pellet+river (low dosage) | 10 | 54.05±16.25
** | <0.001 |
Three+pellet+river (middle dosage) | 15 | 54.13±15.85
** | <0.001 |
Three+pellet+river (high dose) | 20 | 53.54±16.52
** | <0.001 |
Annotate:
*P<0.05 is compared with Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group or Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group
*P<0.01 is compared with Radix Notoginseng extract group or Radix Salviae Miltiorrhizae extract group or ligustrazine phosphate group
Conclusion: each administration group is anticoagulant obviously, the wherein effect of Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group the strongest (P<0.001), and relevant with the dosage of compositions, effect is optimum during dosage 20mg/kg.The effect of Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group or Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group is better than single with Radix Notoginseng extract or Radix Salviae Miltiorrhizae extract or ligustrazine phosphate, but is weaker than the effect of Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group.
Experimental example 7: the composite injection intravenously administrable is to the hemodynamic influence of anesthetized open-chest dog
Laboratory animal: the hybrid dog, 45, body weight is at 11.0~13.0 kilograms, 5 every group.
Test sample: normal saline matched group
Radix Notoginseng extract group: Radix Notoginseng extract injection, self-control
Radix Salviae Miltiorrhizae extract group: Radix Salviae Miltiorrhizae total phenolic acids injection, self-control
Ligustrazine phosphate group: ligustrazine phosphate injection, self-control
Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group, self-control
Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group, self-control
Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract+ligustrazine phosphate=150mg+50mg+50mg is divided into basic, normal, high 3 dosage groups, self-control
Test method: get 45 hybrid dogs, body weight is at 11.0~13.0 kilograms, and 5 every group, the male and female dual-purpose is divided into 9 groups at random, is respectively the normal saline matched group; The Radix Notoginseng extract group; The Radix Salviae Miltiorrhizae extract group; The ligustrazine phosphate group; Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group; Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group; Three+pellet+river=150mg+50mg+50mg group is divided into basic, normal, high 3 dosage groups.With 0.9% normal saline preparation desired concn medicinal liquid, each organizes the administration volume and is the 20mg/kg body weight each administration group before administration.
Animal subject is taked under the right arm reclining malleation artificial respiration after pentobarbital sodium (30mg/kg) intravenous injection anesthesia, opens breast between 4~5 sides of body of a left side, opens pericardium in distance vagus nerve 2cm place, and parietal layer is made outstanding bed it is sutured in thoracic wall, and heart is fully exposed.Separate aorta, be inserted in electromagnetic flowmeter probe (10-12mm), measure cardiac output (CO) at aortic root; At the left anterior descending coronary artery root, separate visceral pericardium, isolate about 1cm coronary artery, be inserted in the electromagnetic flowmeter probe (2~3mm), the measurement coronary flow, (CBF); This two probe is connected on the LMTC-621 type electromagnetic flowmeter, separates a bilateral common carotid artery, intubate connects pressure transducer, record arteriotony (AP) and mean arterial pressure (MAP); With internal diameter is that the cardiac catheter of 1.5mm is inserted left ventricle from the apex of the heart, amplify left indoor pressure (LVP) by YZ-1 type pressure transducer through carrier wave, the LVP electric signal amplifies 10 times through direct current amplifier, write down left chamber EDP (LVEDP), with the LVP electric signal again through BMI type differentiator derivative recording left indoor pressure rate of change (dp/dt
Max), it is subcutaneous to insert the animal subject extremity with pin type electrode, and record mark II lead electrocardiogram (EGG-II) is to measure heart rate (HR).These parameters changes equal synchronous recording and leads instrument in RM-6300 physiology more.
Separate femoral artery and take out arterial blood, through External Carotid Artery for Intubation to the coronary sinus vein venous blood samples, according to CY-2 oxygen analyser operation instructions, with freshly prepared anaerobic solution (crystallization of 0.01M borax soln adding anhydrous sodium sulfite is mixed with 2% sodium sulfite solution) zeroing, use with the distilled water of air balance temperature constant and make sensitivity adjusting.Treat to measure oxygen content with oxygen analyser behind the instrument stabilizer.Calculating myocardium oxygen consumption after the off-test.
After operation finishes, observe above-mentioned every index, after stable with the index of record value before as administration, behind the intravenously administrable in 1 ', 3 ', 5 ', 10 ', 20 ', 30 ' gathers above-mentioned every index, and with every index rate of change (%) of each time point after the administration, the t-test that does significance pairing data between group with every index rate of change (%) of each corresponding time point of matched group handles.
Result and conclusion: result of the test shows: the basic, normal, high dosage group of Radix Notoginseng extract group, Radix Salviae Miltiorrhizae extract group, ligustrazine phosphate group, Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group, Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group, Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract+ligustrazine phosphate=150mg+50mg+50mg, compare significant difference with matched group; Wherein the basic, normal, high dosage group effect of Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract+ligustrazine phosphate=150mg+50mg+50mg is the strongest, and relatively there were significant differences (P<0.01) with Radix Notoginseng extract group, Radix Salviae Miltiorrhizae extract group, ligustrazine phosphate group, Radix Notoginseng extract+Radix Salviae Miltiorrhizae extract group, Radix Salviae Miltiorrhizae extract+ligustrazine phosphate group.
Experimental example 8: composite injection stability test
Sample: composite injection (self-control, three+pellet+river=150mg+50mg+50mg)
Investigation project: character, pH value, clarity
Long-time stability experimental technique and result: this product is put under the condition of 25 ℃ ± 2 ℃ of temperature, relative humidity 60% ± 10% and placed 6 months, 12 months, every index has no significant change, result of the test show composite injection long-term place basicly stable.
Embodiment 1: the preparation of Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and ligustrazine phosphate powder
Prescription:
Radix Notoginseng extract 150g
Radix Salviae Miltiorrhizae extract 50g
Ligustrazine phosphate 50g
Mannitol 300g
Sterile water for injection adds to 3000ml
Prepare 1000 altogether
Preparation technology:
1) vessel of at first dosing being used and antibiotic glass bottle, plug etc. carry out aseptic process.
2) take by weighing supplementary material according to recipe quantity.
3) heating for dissolving in ligustrazine phosphate, Radix Notoginseng extract and Radix Salviae Miltiorrhizae extract adding dosing amount 50% sterile water for injection is complete.It is complete that mannitol adds the sterile water for injection heated and stirred dissolving of dosing amount 40%, merges above-mentioned solution, adds sterile water for injection to full dose.
4) needle-use activated carbon of adding dosing amount 0.1%, heated and stirred 15 minutes.
5) through sand filtration rod filtering decarbonization.Measure the also pH value of regulator solution.
6) through the microporous filter membrane fine straining of 0.22um.
7) clarity of inspection solution, the semi-finished product chemical examination.
8) be sub-packed in the antibiotic glass bottle half tamponade.Sample is put into the freeze dryer lyophilization.Pre-freeze-45 ℃ 5 hours, low-temperature vacuum drying-45 ℃~0 ℃ 20 hours was warming up to 25 ℃ of vacuum dryings 3 hours then.
9) lyophilizing finishes, and lid is rolled in tamponade.
10) finished product is examined entirely, the packing warehouse-in.
Embodiment 2: the preparation of Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and the little pin of ligustrazine phosphate
Prescription:
Radix Notoginseng extract 150g
Radix Salviae Miltiorrhizae extract 50g
Ligustrazine phosphate 50g
Mannitol 300g
Sterile water for injection adds to 3000ml
Prepare 1000 altogether
Preparation technology:
3) vessel of at first dosing being used and antibiotic glass bottle, plug etc. carry out aseptic process.
4) take by weighing supplementary material according to recipe quantity.
3) heating for dissolving in ligustrazine phosphate, Radix Notoginseng extract and Radix Salviae Miltiorrhizae extract adding dosing amount 50% sterile water for injection is complete.It is complete that mannitol adds the sterile water for injection heated and stirred dissolving of dosing amount 40%, merges above-mentioned solution, adds sterile water for injection to full dose.
4) needle-use activated carbon of adding dosing amount 0.1%, heated and stirred 15 minutes.
5) through sand filtration rod filtering decarbonization.Measure the also pH value of regulator solution.
6) through the microporous filter membrane fine straining of 0.22um.
7) clarity of inspection solution, the semi-finished product chemical examination.
8) be sub-packed in the antibiotic glass bottle half tamponade.Sample is put into the freeze dryer lyophilization.Pre-freeze-45 ℃ 5 hours, low-temperature vacuum drying-45 ℃~0 ℃ 20 hours was warming up to 25 ℃ of vacuum dryings 3 hours then.
9) lyophilizing finishes, and lid is rolled in tamponade.
10) finished product is examined entirely, the packing warehouse-in.
Embodiment 3: the preparation of Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and ligustrazine phosphate transfusion
The sodium chloride transfusion:
Prescription:
Radix Notoginseng extract 150g
Radix Salviae Miltiorrhizae extract 50g
Ligustrazine phosphate 50g
Sodium chloride 900g
Water for injection adds to 100000ml
Prepare 1000 bottles altogether
Preparation technology:
1) handles the previous day such as pipeline that dosing uses and container etc., face with the fresh water for injection flushing of preceding reuse.
2) ligustrazine phosphate, Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract are added the dissolving of dosing amount 50% water for injection heated and stirred fully, sodium chloride is complete with the water for injection dissolving of dosing amount 40%.
3) merge above-mentioned solution, benefit adds to the full amount of water for injection.
4) needle-use activated carbon of adding dosing amount 0.1%, heated and stirred 15 minutes.
5) through sand filtration rod filtering decarbonization.Measure the also pH value of regulator solution.
6) through the microporous filter membrane fine straining of 0.45um.
7) clarity of inspection solution, the semi-finished product chemical examination.
8) fill is in the infusion bottle of 100ml.
9) 115 ℃ of pressure sterilizings are 30 minutes.
10) lamp inspection, finished product is examined entirely, the packing warehouse-in.
Glucose infusion liquid:
Prescription:
Radix Notoginseng extract 150g
Radix Salviae Miltiorrhizae extract 50g
Ligustrazine phosphate 50g
Glucose 5000g
Water for injection adds to 100000ml
Prepare 1000 bottles altogether
Preparation technology:
1) carries and handle the previous day such as pipeline that dosing uses and container etc., face with the fresh water for injection flushing of preceding reuse.
2) with heated and stirred dissolving in ligustrazine phosphate, Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract adding dosing amount 50% water for injection fully, that glucose is complete with the water for injection dissolving of dosing amount 40%, heated and boiled 15 minutes.
3) merge above-mentioned solution, benefit adds to the full amount of water for injection.
4) needle-use activated carbon of adding dosing amount 0.1%, heated and stirred 15 minutes.
5) through sand filtration rod filtering decarbonization.Measure the also pH value of regulator solution.
6) through the microporous filter membrane fine straining of 0.45um.
7) clarity of inspection solution, the semi-finished product chemical examination.
8) fill is in the infusion bottle of 100ml.
9) 115 ℃ of pressure sterilizings are 30 minutes.
10) lamp inspection, finished product is examined entirely, the packing warehouse-in.
Embodiment 4: the preparation of Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and Ligustrazine Phosphate pill
Prescription:
Radix Notoginseng extract 150g
Radix Salviae Miltiorrhizae extract 50g
Ligustrazine phosphate 50g
Starch 40.0g
Microcrystalline Cellulose 40.0g
The 2%HPMC aqueous solution is an amount of
Magnesium stearate 6.0g
Carboxymethylstach sodium 12.0g
Prepare 1000 altogether
Preparation technology:
1) it is standby ligustrazine phosphate, Radix Notoginseng extract and Radix Salviae Miltiorrhizae extract to be pulverized 100 mesh sieves.
2) take by weighing supplementary material according to recipe quantity.
3) hypromellose 2% the aqueous solution made soluble in water is standby.
4) with Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract, ligustrazine phosphate, starch, microcrystalline Cellulose mix homogeneously, adding 2%HPMC aqueous solution is an amount of, stirs, and makes suitable soft material.
5) cross 20 mesh sieve system granules.
6) granule is dried under 60 ℃ condition.
7) dry good granule adds magnesium stearate and carboxymethylstach sodium, crosses 18 mesh sieve granulate, mix homogeneously.
8) sampling, the semi-finished product chemical examination.
9) the sheet weight sheet of determining according to chemical examination.
10) finished product is examined entirely, the packing warehouse-in.
Embodiment 5: the capsular preparation of Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and ligustrazine phosphate
Prescription:
Radix Notoginseng extract 150g
Radix Salviae Miltiorrhizae extract 50g
Ligustrazine phosphate 50g
Starch 20.0g
Microcrystalline Cellulose 60.0g
The 2%HPMC aqueous solution is an amount of
Magnesium stearate 6.0g
Prepare 1000 altogether
Preparation technology:
1) it is standby ligustrazine phosphate, Radix Notoginseng extract and Radix Salviae Miltiorrhizae extract to be pulverized 100 mesh sieves.
2) take by weighing supplementary material according to recipe quantity.
3) hypromellose 2% the aqueous solution made soluble in water is standby.
4) with Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract, ligustrazine phosphate, starch, microcrystalline Cellulose mix homogeneously, adding 2%HPMC aqueous solution is an amount of, stirs, and makes suitable soft material.
5) cross 20 mesh sieve system granules.
6) granule is dried under 60 ℃ condition.
7) dry good granule adds magnesium stearate, crosses 18 mesh sieve granulate, mix homogeneously.
8) sampling, the semi-finished product chemical examination.
9) loading amount of determining according to chemical examination incapsulates.
10) finished product is examined entirely, the packing warehouse-in.
Embodiment 6: the particulate preparation of Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and ligustrazine phosphate
Prescription:
Radix Notoginseng extract 150g
Radix Salviae Miltiorrhizae extract 50g
Ligustrazine phosphate 50g
Icing Sugar 2000.0g
The 2%HPMC50% alcoholic solution is an amount of
Prepare 1000 bags altogether
Preparation technology:
1) it is standby sucrose to be pulverized 100 mesh sieves.It is standby that ligustrazine phosphate, Radix Notoginseng extract and Radix Salviae Miltiorrhizae extract were pulverized 100 mesh sieves.
2) take by weighing supplementary material according to recipe quantity.
3) the method mix homogeneously that ligustrazine phosphate, Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and Icing Sugar are progressively increased with equivalent, adding 2%HPMC50% alcoholic solution is an amount of, stirs, and makes suitable soft material,
4) cross 20 mesh sieve system granules.
5) granule is dried under 60 ℃ condition.
6) dried granule is crossed 18 mesh sieve granulate.
7) sampling, the content of principal agent is determined loading amount in the semi-finished product chemical examination granule.
8) packing, finished product is examined entirely, the packing warehouse-in.
Embodiment 7: the preparation of Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and phosphoric ligustrazine drop agent
Prescription:
Radix Notoginseng extract 150g
Radix Salviae Miltiorrhizae extract 50g
Ligustrazine phosphate 50g
Polyethylene glycol 6000 1000g
Preparation technology:
Pulverized behind 100 mesh sieves ligustrazine phosphate, Radix Notoginseng extract and Radix Salviae Miltiorrhizae extract standby.With polyethylene glycol 6000 heating and melting in water-bath, treat to add ligustrazine phosphate, Radix Notoginseng extract and Radix Salviae Miltiorrhizae extract after whole fusions, stirring and dissolving, 60 mesh sieves filter, and keep 60 ℃ to splash in the liquid paraffin that is chilled to below 10 ℃ and make ball.
Embodiment 8: the preparation of Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and Lingustrazine Phosphate soft capsule
Prescription:
Radix Notoginseng extract 150g
Radix Salviae Miltiorrhizae extract 50g
Ligustrazine phosphate 50g
Soybean oil 1000.0g
Soybean phospholipid 500g
Cera Flava 500g
Prepare 1000 altogether
Preparation technology:
With the soybean oil of recipe quantity and soybean phospholipid, Cera Flava heating and melting, mixing is put coldly, adds Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract, ligustrazine phosphate and grinds well, and is pressed into soft capsule and gets final product.
Embodiment 9: the preparation of Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract and ligustrazine phosphate oral liquid
Prescription:
Radix Notoginseng extract 150g
Radix Salviae Miltiorrhizae extract 50g
Ligustrazine phosphate 50g
Sodium benzoate 15g
Stevioside 10g
Purified water adds to 10000ml
Prepare 1000 altogether
Preparation technology:
1) with heated and stirred dissolving in the purified water of Radix Notoginseng extract, Radix Salviae Miltiorrhizae extract, ligustrazine phosphate adding dosing amount 50% fully.
2) sodium benzoate and stevioside is complete with the water dissolution of dosing amount 20%.
3) merge above-mentioned two solution, mend and add water to full dose.
4) filtering with microporous membrane of mistake 0.8um.
5) semi-finished product chemical examination.
6) fill.Finished product is examined entirely, the packing warehouse-in.