CN100358545C - Freeze drying powder and injection preparation in use for invigorating pulse-beat, and preparation method - Google Patents
Freeze drying powder and injection preparation in use for invigorating pulse-beat, and preparation method Download PDFInfo
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Abstract
The present invention relates to a blood circulation promotion freeze-drying injection for injection. The present invention is characterized in that 100 freeze-drying injections are prepared from the raw materials of 333.3g of red sage root, 333.3g of chuanxiong rhizome, 333.3g of kudzuvine root, 90g of manna and 2g of sodium bisulfite, and the volume of each freeze-drying injection is 5mL. The preparation method comprises: the red sage root, the chuanxiong rhizome and the kudzuvine root are respectively decocted, and three medicinal material extract products are obtained by the steps of decompression, concentration and alcohol deposition; the three medicinal material extract products are mixed, sodium bisulfite and manna are added, and the injection is prepared by an injection conventional preparation method of the injection. The injection is proved by pharmacodynamic tests, and the medicine of the present invention improves the erythrocyte deformability and the erythrocyte aggregation of a rat with blood stasis symptom, reduces hematocrit and the viscosity of whole human blood and plasma, inhibits the adhesiveness function of the platelet and the platelet aggregation function induced by ADP and alleviates in vivo phlebothrombosis of the rat. The present invention can be used for treating coronary heart disease angina pectoris and cerebral infarction disease.
Description
Technical field
The invention belongs to the medicinal preparation technical field of the product that contains raw material or itself and not clear structure, be specifically related to derive from the material of plant.
Background technology
The medicine that is used for the treatment of at present angina pectoris and cerebral infarction clinically has arteria coronaria to lead to sheet, Kang Er heart-soothing capsule, NAOXINTONG JIAONANG etc., the most dose of these medicines is big, onset is slow, the course of treatment is long, curative effect is definite inadequately, and some medicine patient will take the medicine of some months, even just feels onset after the half a year of taking medicine, cause very big financial burden to the patient, incured loss through delay the treatment of eqpidemic disease.Injection type, medicine can directly enter blood circulation, orally relatively can obviously improve its bioavailability, makes effect reliable rapidly, more helps the treatment to emergency cases such as angina pectoris and cerebral infarction.
Traditional method for making of Chinese medicine is that used crude drug is adopted the preparation method that decocts precipitate with ethanol, and many crude drug are placed on a decoction, has been easy to reflection between their effective ingredient, has influence on the purity of injection and the therapeutic effect of medicine.
Summary of the invention
Technical problem to be solved by this invention is to overcome the shortcoming of said medicine, the injection that provides a kind of instant effect, the treatment treatment coronary heart disease heart evident in efficacy to twist.
Another technical problem that the present invention will solve is to provide a kind of injection preparation method of lyophilized injectable powder of promoting blood circulation.
Solve the problems of the technologies described above the technical scheme that is adopted and be 100 (1.4g/ props up) lyophilized injectable powders by making with following Chinese medicine raw materials by weight proportion and adjuvant:
Radix Salviae Miltiorrhizae 333.3g
Rhizoma Chuanxiong 333.3g
Radix Puerariae 333.3g
Mannitol 90g
Sodium sulfite 2g.
Its preparation method is as follows:
1, red rooted salvia 333.3g is made only, be ground into coarse granule, add 10 times of amount concentration is that 4.3% sodium hydroxide solution decocts three times at every turn, boiled 1.5 hours at every turn, and merged decoction liquor, be evaporated to 1: 1.5 (relative density 1.10～1.15,50～60 ℃), regulate pH to 4.0, add ethanol and make determining alcohol reach 65%, stir evenly, standing over night, filter, filtrate recycling ethanol adds the injection water and is adjusted to 1: 1 to there not being the alcohol flavor; Regulate pH11～12 with aqua calcis, stir evenly, left standstill 30 minutes, regulate pH6.0～6.5, leave standstill, filter, filtrate is concentrated into 1: 3 (relative density 1.20～1.25,50～60 ℃), regulates pH to 4.0, adding ethanol makes determining alcohol reach 80%, stir evenly, left standstill 40 hours, filter, filtrate recycling ethanol is to there not being the alcohol flavor, regulate pH to 6.0, add the injection water and be adjusted to 1: 1, boiled 30 minutes, put cold, 0～5 ℃ left standstill 40 hours, filtered, and filtrate decompression is concentrated into 1: 2, measure content, get Radix Salviae Miltiorrhizae extract.
2, Rhizoma Chuanxiong medical material 333.3g is made only, be ground into coarse granule, add 7 times of water gagings at every turn and decoct three times, decocted 1.0 hours, merge decoction liquor, be evaporated to 1: 2 (relative density 1.15～1.25,50～60 ℃), adding ethanol makes determining alcohol reach 70%, stir evenly, standing over night filters, filtrate recycling ethanol adds injection water to 1: 2 to there not being the alcohol flavor; Transfer pH11～12, stir evenly, left standstill 30 minutes, transfer pH5.0～5.5, leave standstill, filter, filtrate is concentrated into 1: 4 (relative density 1.20～1.30,50～60 ℃), add ethanol and make determining alcohol reach 80%, stir evenly, standing over night filters, filtrate recycling ethanol adds the water for injection of about 6 times of amounts to there not being the alcohol flavor, boils 30 minutes, put coldly, 0～5 ℃ left standstill 40 hours, filtered, filtrate decompression is concentrated into 1: 2, measures content, gets Rhizoma Chuanxiong extract.
3,333.3g makes only with the Radix Puerariae medical material, is ground into coarse granule, adds 8 times of water gagings at every turn and decocts three times, decocted 1.5 hours, and merged decoction liquor, be evaporated to 1: 2 (relative density 1.10～1.20,50～60 ℃), add ethanol and make determining alcohol reach 50%, stir evenly, standing over night filters, the filtrate recycling ethanol and the (relative density 1.25～1.30 that is concentrated into 1: 4,50～60 ℃), add ethanol and make determining alcohol reach 80%, regulate pH to 8.0～8.2, stir evenly, left standstill 40 hours, filter, filtrate recycling ethanol adds the water for injection of 5 times of amounts to there not being the alcohol flavor, boils 30 minutes, put cold, 0～5 ℃ left standstill 40 hours, filtered, and filtrate is concentrated into 1: 2, measure content, get Radix Puerariae extract.
4, get Radix Salviae Miltiorrhizae extract, Radix Puerariae extract and Rhizoma Chuanxiong extract, mixing, adding concentration is 0.4% sodium sulfite, dissolving, mix homogeneously is regulated pH6.8～7.2, adding concentration is after 18% mannitol dissolves, it is 0.4% active carbon that the conventional proportioning for preparing injection by galenic pharmacy adds concentration, boils 20 minutes, filters, filtrate adds the injection water and is adjusted to original volume, the microporous filter membrane fine straining, in the infusion bottle of packing into filtrate branch, 115 ℃, 30 minutes pressure sterilizings.Microporous filter membrane fine straining under the aseptic condition divides in the 10ml cillin bottle of packing into, every dress 5ml, and lyophilization, Zha Gai, quality inspection promptly gets lyophilized injectable powder of the present invention.Usage and dosage: once-a-day, each 1-2 props up, and adds among 5% glucose injection 250mL or the 0.9% normal saline 250mL intravenous drip.
The lyophilized injectable powder that adopts preparation method preparation of the present invention is through the test of pesticide effectiveness, proof: medicine of the present invention can obviously improve Blood stasis rat erythrocyte deformability and aggregation thereof, reduce packed cell volume and whole blood and plasma viscosity, obviously suppress hematoblastic adhesion function and the inductive platelet aggregation of ADP, alleviate the doing of rat body angular vein thrombosis and rabbit painstaking effort external thrombus, wet weight and thrombosis length, obviously prolong hemorrhage and clotting time, have the effect of tangible blood circulation promoting and blood stasis dispelling, antithrombotic formation and antiplatelet aggregation; Medicine of the present invention is the minimizing of the dirty coronary flow of guinea-pig heart due to the antagonism pituitrin significantly, obviously dwindle the myocardial infarct size due to the dog ligation LAD, raising of ECGST section due to the alleviate myocardial ischemia, on the pallasiomy cerebral ischemic model, medicine of the present invention has Na in the cerebral tissue of anti-cerebral ischemia due to pouring into again
+, Ca
2+Retention reduces lipid peroxide metabolite (MDA) content, and encephaledema and promote the effect that brain electrical acti recovers can obviously increase the cerebral blood flow of anesthetized cat, reduces its cerebral blood flow resistance simultaneously; The death time of anoxia in mice after the present invention can significantly strengthen the ability of whole animal tolerance normobaric hypoxia and prolong isoproterenol raising myocardial oxygen consumption, external myocardial cell there is significant protective effect in the damage that anoxia lacks due to cultivating under the sugared condition, can also obviously reduces the permeability of blood capillary; Medicine energy significant prolongation clotting time of the present invention, but prothrombin time is not had influence, illustrate that its blood coagulation resisting function mainly influences endogenic blood coagulation system; Medicine of the present invention respectively can be significantly and is obviously reduced cerebral tissue ischemic region weight after the ligation of dog middle cerebral artery, can significantly reduce the effect that serum CK raises after the ligation of dog middle cerebral artery; Medicine of the present invention can obviously promote plasma in rabbit fibrin degradation product (FDP) (FDP) to form, and can remarkable and obviously shorten the plasma in rabbit euglobulin lysis time, can obviously shorten the dissolution time of rabbit whole plasm grumeleuse; Medicine of the present invention can obviously prolong the thrombotic time of rat carotid artery due to the electricity irritation, obviously suppresses the formation of rabbit painstaking effort external thrombus, obviously suppresses the thrombotic work of rat body angular vein (P＜0.001 and P＜0.05); Medicine of the present invention can obviously promote the dissolving of the rat carotid artery thrombosis that electricity irritation forms, shorten the restoration of blood flow time, after rat arteriovenous blood flow the method for bypass thrombosis, remarkable and the obvious weight in wet base (P＜0.01 and P＜0.05) that reduces thrombosis of medicine energy of the present invention, the thrombolytic rate is respectively 17.26% and 11.75%, and medicine of the present invention also has obvious dissolution to the postcava thrombosis.
The specific embodiment
The present invention is described in more detail below in conjunction with embodiment, but the invention is not restricted to these embodiment.
Embodiment 1
With production drug injection of the present invention 100 (1.4g/ props up) is that the used crude drug of example and adjuvant and weight proportion thereof are:
Radix Salviae Miltiorrhizae 333.3g
Rhizoma Chuanxiong 333.3g
Radix Puerariae 333.3g
Mannitol 90g
Sodium sulfite 2g.
Its preparation method is as follows:
1, red rooted salvia 333.3g is made only, be ground into coarse granule, add 10 times of amount concentration is that 4.3% sodium hydroxide solution decocts three times at every turn, boiled 1.5 hours at every turn, and merged decoction liquor, be evaporated to 1: 1.5 (relative density 1.10～1.15,50～60 ℃), regulate pH to 4.0, add ethanol and make determining alcohol reach 65%, stir evenly, standing over night, filter, filtrate recycling ethanol adds the injection water and is adjusted to 1: 1 to there not being the alcohol flavor; Regulate pH11～12 with aqua calcis, stir evenly, left standstill 30 minutes, regulate pH6.0～6.5, leave standstill, filter, filtrate is concentrated into 1: 3 (relative density 1.20～1.25,50～60 ℃), regulates pH to 4.0, adding ethanol makes determining alcohol reach 80%, stir evenly, left standstill 40 hours, filter, filtrate recycling ethanol is to there not being the alcohol flavor, regulate pH to 6.0, add the injection water and be adjusted to 1: 1, boiled 30 minutes, put cold, 0～5 ℃ left standstill 40 hours, filtered, and filtrate decompression is concentrated into 1: 2, measure content, get Radix Salviae Miltiorrhizae extract.
2, Rhizoma Chuanxiong medical material 333.3g is made only, be ground into coarse granule, add 7 times of water gagings at every turn and decoct three times, decocted 1.0 hours, merge decoction liquor, be evaporated to 1: 2 (relative density 1.15～1.25,50～60 ℃), adding ethanol makes determining alcohol reach 70%, stir evenly, standing over night filters, filtrate recycling ethanol adds injection water to 1: 2 to there not being the alcohol flavor; Transfer pH11～12, stir evenly, left standstill 30 minutes, transfer pH5.0～5.5, leave standstill, filter, filtrate is concentrated into 1: 4 (relative density 1.20～1.30,50～60 ℃), add ethanol and make determining alcohol reach 80%, stir evenly, standing over night filters, filtrate recycling ethanol adds the water for injection of about 6 times of amounts to there not being the alcohol flavor, boils 30 minutes, put coldly, 0～5 ℃ left standstill 40 hours, filtered, filtrate decompression is concentrated into 1: 2, measures content, gets Rhizoma Chuanxiong extract.
3,333.3g makes only with the Radix Puerariae medical material, is ground into coarse granule, adds 8 times of water gagings at every turn and decocts three times, decocted 1.5 hours, and merged decoction liquor, be evaporated to 1: 2 (relative density 1.10～1.20,50～60 ℃), add ethanol and make determining alcohol reach 50%, stir evenly, standing over night filters, the filtrate recycling ethanol and the (relative density 1.25～1.30 that is concentrated into 1: 4,50～60 ℃), add ethanol and make determining alcohol reach 80%, regulate pH to 8.0～8.2, stir evenly, left standstill 40 hours, filter, filtrate recycling ethanol adds the water for injection of 5 times of amounts to there not being the alcohol flavor, boils 30 minutes, put cold, 0～5 ℃ left standstill 40 hours, filtered, and filtrate is concentrated into 1: 2, measure content, get Radix Puerariae extract.
4, get Radix Salviae Miltiorrhizae extract, Radix Puerariae extract and Rhizoma Chuanxiong extract, mixing, adding concentration is 0.4% sodium sulfite, dissolving, mix homogeneously is regulated pH6.8～7.2, adding concentration is after 18% mannitol dissolves, it is 0.4% active carbon that the conventional proportioning for preparing injection by galenic pharmacy adds concentration, boils 20 minutes, filters, filtrate adds the injection water and is adjusted to original volume, the microporous filter membrane fine straining, in the infusion bottle of packing into filtrate branch, 115 ℃, 30 minutes pressure sterilizings.Microporous filter membrane fine straining under the aseptic condition divides in the 10ml cillin bottle of packing into, every dress 5ml, and lyophilization, Zha Gai, quality inspection promptly gets lyophilized injectable powder of the present invention.Usage and dosage: once-a-day, each 1～2, add among 5% glucose injection 250mL or the 0.9% normal saline 250mL intravenous drip.
In order to determine the processing step of the best of the present invention, the inventor has carried out a large amount of development tests, and various test situation are as follows:
1, extracting mode is determined experiment
Take by weighing 1/50 recipe quantity medical material, carry out comprehensive compatibility extraction experiment of three flavor medical materials respectively by table 1～table 3, with each content of effective in the extracting solution and solids yield is evaluation index, investigate of the influence of medicinal material extract mode to extraction effect, and be 100% with the single medical material, calculate the relative percent error (bracket intermediate value) of compatibility sample, the results are shown in Table 1～table 3.
Table 1 extracting mode is to the table as a result that influences of Radix Salviae Miltiorrhizae extraction effect
| Index | Radix Salviae Miltiorrhizae | Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong | Radix Salviae Miltiorrhizae, Radix Puerariae | Radix Salviae Miltiorrhizae, Radix Puerariae, Rhizoma Chuanxiong |
| Content of Danshensu and rate of change (%) solids yield and rate of change (%) | 0.5452 43.04 | 0.5114 (-6.2％) 39.59 (+7.85％) | 0.5047 (-7.4％) 34.78 (+2.11％) | 0.5934 (+8.8％) 37.38 (+13.86％) |
Table 2 extracting mode is to the table as a result that influences of Radix Puerariae extraction effect
| Index | Radix Puerariae | Radix Puerariae, Radix Salviae Miltiorrhizae | Radix Puerariae, Rhizoma Chuanxiong | Radix Puerariae, Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong |
| Puerarin content and rate of change (%) general flavone content and rate of change (%) solids yield and rate of change (%) | 3.91 12.21 25.08 | 3.78 (-3.3％) 10.38 (-15.0％) 34.78 (+2.11％) | 3.75 (-4.1％) 10.36 (-15.2％) 27.76 (+0.1％) | 4.12 (+5.4％) 11.07 (-9.3％) 37.38 (+13.86％) |
Table 3 extracting mode is to the table as a result that influences of Rhizoma Chuanxiong extraction effect
| Index | Rhizoma Chuanxiong | Rhizoma Chuanxiong, Radix Salviae Miltiorrhizae | Rhizoma Chuanxiong, Radix Puerariae | Radix Salviae Miltiorrhizae, Radix Puerariae, Rhizoma Chuanxiong |
| Ferulaic acid content and rate of change (%) solids yield and rate of change (%) | 0.0427 30.38 | 0.0270 (-36.77％) 39.59 (+7.85％) | 0.0501 (+17.3％) 27.76 (+0.1％) | 0.0350 (-18.0％) 37.38 (+13.86％) |
By above experimental result as can be seen, the solids yield is difficult to estimate the substantial effect of extracting mode to extraction effect as index.Single component quantitative assessment index shows that three medicines mix fries in shallow oil ferulaic acid content reduction by 18.0%; Radix Salviae Miltiorrhizae and Rhizoma Chuanxiong compatibility, danshensu and ferulaic acid content reduce by 6.2% and 36.77% respectively; Radix Puerariae and Radix Salviae Miltiorrhizae or Rhizoma Chuanxiong compatibility, puerarin content reduces by 3.3% and 4.1% respectively, and Radix Puerariae total flavones reduces by 15.0% and 15.2% respectively.As seen, raw material of Chinese medicine mixed extraction of the present invention is difficult to take into account the extraction efficiency of different effective ingredient, and the medical material compatibility is unfavorable for the extraction of effective ingredient; For satisfying the requirement of injection to refining purification, the medical material branch is proposed selection and the application that helps refining purification process, determines that raw material of Chinese medicine extracting mode of the present invention is that three flavor medical materials extract respectively.
(1) Radix Salviae Miltiorrhizae extraction process
Character at aqueous soluble active constituent polyhydric phenols in the Radix Salviae Miltiorrhizae will reach rational leaching, and after solvent was determined, extracting temperature B, solvent load A, extraction time D and extraction time C was the key factor that influence is leached.Plan according to uniform Design factor, number of levels design principle, is used U with the preferred reasonably extract technology condition of uniform Design
9(9
8) uniform designs table, set up factor level table the 1,2,3,5 row.
Experiment arrangement such as table 4.
Experimental technique and result: take by weighing Radix Salviae Miltiorrhizae 50 gram respectively, press table 6 experiment arrangement, behind the extracting solution, mix, filtration measures cumulative volume, gets an amount of mensuration solids yield, extracting solution uv absorption and content of Danshensu.Each experiment repeats 3 times, gets the experiment averages three times.Result of the test sees Table 5.
Table 4 Radix Salviae Miltiorrhizae leaches factor experimental result table
| Level | Solvent load A (doubly) | Extraction temperature B (℃) | Extraction time C (hour) | Extraction time D (inferior) |
| 1 2 3 4 5 6 7 8 9 | 3 5 7 8 9 10 11 12 12 | 50 60 70 75 80 85 90 97 (boiling) 97 (boiling) | 0.5 1.0 1.5 2.0 2.0 2.5 2.5 3.0 3.0 | 1 2 2 2 2 2 3 3 4 |
Table 5 Radix Salviae Miltiorrhizae leaching experiment is table as a result
| The experiment number | Experiment condition | Solids yield (%) | Uv absorption A283.8 | Content of Danshensu (%) | |||
| A | B | C | D | ||||
| 1 2 3 4 5 6 7 8 9 | 3 5 7 8 9 10 11 12 12 | 60 75 85 97 50 70 80 90 97 | 2.0 3.0 1.5 2.5 1.0 2.5 0.5 2.0 3.0 | 3 2 2 1 3 2 2 2 4 | 28.92(+2.58) 32.90(+0.93) 31.53(-2.35) 31.19(-2.15) 37.87(-1.37) 40.11(-1.41) 36.33(-0.32) 54.23(+4.28) 62.69(-0.77) | 0.380(+3.72) 0.443(-1.53) 0.418(-1.47) 0.463(-0.61) 0.465(+0.13) 0.556(-2.09) 0.451(-3.65) 0.687(+5.51) 0.990(-0.89) | 0.352(+33.58) 0.298(-35.13) 0.473(-6.57) 0.644(+3.76) 0.160(+10.61) 0.364(-15.06) 0.325(-40.56) 1.060(+23.03) 1.290(-4.08) |
The level value of each factor is used the multiple linear regression routine processes on computers after normalization is handled, its regression equation is as follows, and through the F check, regression equation is all set up.Regression equation calculation value and measured value and relative percent error thereof (see Table value in 5 brackets,
With the solids yield is index, and regression equation is:
y
1＝10.4230+18.7192A+4.6797B+10.9444C+19.2018D
S=1.2902; R=0.9962; F=132.488 F＞F
0.01,4.4=16.0, regression equation is set up.
With the extracting solution ultraviolet absorption value is index, and regression equation is:
y
2＝0.0082+0.3084A+0.1255B+0.2414C+0.3154D
S=0.0235; R=0.9962; F=132.1763 F＞F
0.01,4.4=16.0; Regression equation is set up.
With the content of Danshensu is index, and regression equation is:
y
3＝-3.8734+2.3230A+7.3517B+2.1404C+5.0966D
S=1.5640; R=0.9515; F=9.5668 F＞F
0.05,4.4=6.39, regression equation is set up.
Interpretation of result and conclusion: by above-mentioned regression equation as seen, the coefficient of each factor just is all, and shows that level value is big more in the horizontal span of factor, and each desired value is also big more; With solids yield and uv absorption is in two regression equations of index, and the coefficient order of magnitude sequence consensus of each factor is all D＞A＞C＞B, extraction time is described to the having the greatest impact of index, and extracts the Temperature Influence minimum.Is that index is calculated regression equation with the content of Danshensu, relative error is bigger, and the regression equation credibility reduces, but can find out from experimental data, during content of Danshensu high temperature apparently higher than low temperature.Therefore, determining and extracting temperature is 97 ℃, and extraction time is 3 times, and solvent load is 10 times, and extraction time is 1.5 hours.
(2) Radix Puerariae extraction process
Radix Puerariae is a stem class medical material, has the problem of whether soaking in leaching process.On prerun and relevant documents and materials basis, set up factor level table such as table 9, select even experimental design table U for use
9(9
8) the 1st, 2,3,5 row experiment arrangement such as tables 6.
Table 6 Radix Puerariae extraction factor experimental result table
| Level | Soak time A (hour) | Solvent load B (doubly) | Extraction time C (hour) | Extraction time D (inferior) |
| 1 2 3 4 5 6 7 8 9 | 0 2 4 6 8 10 12 14 16 | 4 5 6 7 8 9 10 11 12 | 0.5 0.5 1.0 1.0 1.0 1.5 1.5 2.0 2.0 | 1 1 2 2 2 3 3 3 4 |
Experimental technique and result: take by weighing Radix Puerariae 50 gram respectively, press table 6 experiment arrangement, behind the extracting solution, mix, filtration measures cumulative volume, gets an amount of mensuration solids yield, general flavone content and puerarin content.Each experiment repeats 3 times, gets the experiment averages three times, and result of the test sees Table 7.
Table 7 Radix Puerariae extracts the experimental result table
| The experiment number | Experiment condition | Solids yield (%) | General flavone content (%) | Puerarin content (%) | |||
| A | B | C | D | ||||
| 1 2 3 4 5 6 7 8 9 | 0 2 4 6 8 10 12 14 16 | 5 7 9 11 4 6 8 10 12 | 1.0 2.0 1.0 1.5 0.5 1.5 0.5 1.0 2.0 | 3 2 2 1 3 3 2 1 4 | 21.89(-10.53) 28.15(+5.26) 24.00(-6.93) 18.90(-6.93) 22.05(-3.13) 28.17(+2.28) 24.41(+9.47) 18.66(-9.09) 31.71(-1.19) | 9.94(-7.67) 11.48(+6.51) 10.32(+6.91) 7.44(-8.53) 8.99(-1.41) 11.34(-1.28) 10.00(+8.13) 6.81(-5.93) 14.97(-0.24) | 3.18(-11.51) 4.07(+5.04) 3.44(+7.39) 3.03(-3.08) 3.21(-0.98) 4.18(+1.81) 3.21(+10.25) 2.49(-13.07) 5.26(-0.91) |
The level value of each factor is used the multiple linear regression routine processes on computers after normalization is handled, its regression equation is as follows, and through the F check, regression equation is all set up.The relative percent error with measured value of regression equation calculation value sees Table value in 7 brackets.
With the solids yield is index, and regression equation is:
y
1＝17.2791+1.6669A-1.0686B+7.2704C+6.9382D
S=2.3979; R=0.9180; F=5.3952 F＞F
0.10,4.4=11, regression equation is set up.
With the general flavone content is index, and regression equation is:
y
2＝0.3043-0.0562A+0.1108B+0.1169C+0.2960D
S=0.0493; R=0.9609; F=13.8984 F＞F
0.05,4.4=6.39, regression equation is set up.
With the puerarin content is index, and regression equation is:
y
3＝2.0424+0.2243A+0.2557B+1.1559C+1.6295D
S=0.3437; R=0.9550; F=10.3575 F＞F
0.05,4.4=6.39, regression equation is set up.
Interpretation of result and conclusion: as seen by above-mentioned regression equation, with total flavones and puerarin is index, each factor affecting rule unanimity, be all D＞C＞B＞A, the solids yield is basically identical also, factor D is more remarkable to the influence of each index, is all positive correlation, should select the maximum horizontal value D except that level 9 for use
8Factor C, B become positive correlation to the influence of effective ingredient, should select big level value C for use
7, B
5Factor A is to the minimum that influences of effective ingredient, also is time minimum to the influence of solids, the optional level value A that gets
1In view of the above, and according to producing actual time consumption and energy consumption situation, drafting Radix Puerariae, to leach optimal conditions be A
1B
5C-D
8
(3) Rhizoma Chuanxiong extraction process
Except that containing volatilization class oils composition, also contain alkaloids and organic acid effective ingredient in the Rhizoma Chuanxiong medical material, all kinds of constitutive properties are different, should adopt corresponding method and solvent.Set up factor level table such as table 13, select even experimental design table U for use
9(9
8) the 1st, 2,3,5 row experiment arrangement, experimental result sees Table 8.
Table 8 Rhizoma Chuanxiong extraction factor experimental result table
| Level | Soak time A (hour) | Solvent load B (doubly) | Extraction time C (hour) | Extraction time D (inferior) |
| 1 2 3 4 5 6 7 8 9 | 0 0 0 0.5 0.5 0.5 1 1 1 | 4 4 5 5 6 6 7 7 8 | 0.5 0.5 0.5 1.0 1.0 1.0 1.5 1.5 1.5 | 1 1 1 2 2 2 3 3 3 |
Experimental technique and result: take by weighing Rhizoma Chuanxiong 50 gram respectively, press table 9 experiment arrangement, behind the extracting solution, mix, filtration measures cumulative volume, gets an amount of mensuration solids yield, extracting solution uv absorption and ferulaic acid content.Each experiment repeats 3 times, gets the experiment averages three times, the results are shown in Table 9.
Table 9 Rhizoma Chuanxiong extracts the experimental result table
| The experiment number | Experiment condition | Solids yield (%) | Uv absorption A280.2 | Ferulaic acid content (%) | |||
| A | B | C | D | ||||
| 1 2 3 4 5 6 7 8 9 | 0 0 0 0.5 0.5 0.5 1.0 1.0 1.0 | 4 5 6 7 4 5 6 7 8 | 1.0 1.5 0.5 1.5 0.5 1.0 0.5 1.0 1.5 | 3 2 1 1 3 2 2 1 3 | 31.304(-2.71) 31.078(+2.75) 18.021(+2.24) 24.997(-4.85) 29.659(-3.14) 29.988(+4.42) 27.155(+2.12) 24.082(-2.36) 38.544(+1.05) | 0.403(-8.84) 0.406(+3.09) 0.230(+6.56) 0.324(-6.83) 0.416(+0.48) 0.405(+8.92) 0.355(-3.26) 0.310(-3.71) 0.605(+2.49) | 0.0752(-10.05) 0.0735(+5.03) 0.0530(+5.93) 0.0602(-9.24) 0.0734(+2.34) 0.0640(+8.25) 0.0640(-5.59) 0.0544(-0.52) 0.1306(+2.41) |
The level value of each factor is used the multiple linear regression routine processes on computers after normalization is handled, its regression equation is as follows, and through the F check, regression equation is all set up, and the relative percent error with measured value of regression equation calculation value sees Table 9 (values in the bracket).
With the solids yield is index, and regression equation is:
y
1＝18.6614+3.9969A-1.5674B+7.1176C+9.9319D
S=1.2774; R=0.9875; F=39.1508 F＞F
0.01,4.4=16.0, regression equation is set up.
With the extracting solution ultraviolet absorption value is index, and regression equation is:
y
2＝0.1702+0.0397A+0.0671B+0.0889C+0.2239D
S=0.0314; R=0.9764; F=20.4775 F＞F
0.01,4.4=16.0, regression equation is set up.
With the ferulaic acid content is index, and regression equation is:
y
3＝0.0998-0.1750A+0.5987B+0.0466C+0.7045D
S=0.0643; R=0.9810; F=25.5509 F＞F
0.01,4.4=16.0, regression equation is set up.
Interpretation of result and conclusion: by above-mentioned regression equation as seen, each factor to three indexs to influence rule complete consistent, but factor D is all maximum to the influence of each index, and all is positive correlation, should choose maximum " 3 times "; Factor C is a time principal element to solids yield, uv absorption index, and all is positive correlation also, should select higher value " 1.0 hours " for use; Factor B is a positive correlation to the effective ingredient index, be negative correlation to solids yield index, but influence degree is less, can select 7 times of higher values for use; Factor A influences minimum, from producing actual consideration, chooses " not soaking ".Through taking all factors into consideration, drafting Rhizoma Chuanxiong extraction optimal conditions is A
1B
7C
6D
7
2, extract purification process
(1) Radix Salviae Miltiorrhizae extract purification process
Water alcohol method: Radix Salviae Miltiorrhizae extract is concentrated into 1: 1,60% precipitate with ethanol; Reclaim ethanol, be concentrated into 1: 2,80% precipitate with ethanol reclaims ethanol, is adjusted to 1: 2, gets the water alcohol method refined solution.
The stone mercaptans: Radix Salviae Miltiorrhizae extract is concentrated into 8: 1, adds aqua calcis and regulates pH12, adds sulfuric acid solution and transfers pH6.0～6.5, leaves standstill, and filters, and is concentrated into 1: 1; Method is handled once for another example, is concentrated into 1: 2; 80% precipitate with ethanol reclaims ethanol, is adjusted to 1: 2, gets stone mercaptans refined solution.
The clarifier method of purification: 101 clarifier solution of adding 15% in the Radix Salviae Miltiorrhizae extract, 80 ℃ of liquid temperature leave standstill, and filter, and filtrate is concentrated into 1: 1, and method is handled once for another example, is concentrated into 1: 2, gets 101 clarifier method refined solutions; Radix Salviae Miltiorrhizae extract is concentrated into 1: 10, adds 10% 1+1 clarifier solution, and 80 ℃ are incubated 20 minutes, filter, and filtrate is concentrated into 2: 1, and method is handled once for another example, is concentrated into 1: 2, promptly gets 1+1 clarifier method refined solution.
Resin adsorption method: get the macroporous adsorbent resin of having handled well, wet method dress post, behind the eliminating bubble, the Radix Salviae Miltiorrhizae extract upper prop is collected effluent simultaneously, stops upper prop during to the absorption terminal point, water elution, reuse 50% ethanol elution is to eluent FeCl
3Test solution is checked negative, collects 50% ethanol elution, is concentrated into 1: 2, gets the resin adsorption method refined solution.
Get above-mentioned each refined solution, measure its solids yield and content of Danshensu respectively, be 100% calculating retention rate (value in the bracket) with each index before the extracting solution purification simultaneously, and do the paired observation of clarity, appearance character, the results are shown in Table 10
Different purification process index components content of table 10 Radix Salviae Miltiorrhizae and appearance character comparison sheet
| Solids yield % | Content of Danshensu % | Purity % | Appearance character | |
| Extracting solution water alcohol method stone mercaptans clarifier A method clarifier B method resin adsorption method | 52.97 9.15 (17.3％) 15.66 (29.6％) 35.60 (67.2％) 33.62 (63.5％) 5.57 (10.5％) | 2.476 1.300 (52.5％) 1.353 (54.6％) 1.417 (57.2％) 1.508 (60.9％) 0.466 (18.8％) | 4.67 14.21 8.64 3.98 4.49 8.37 | Muddy, be placed with the precipitation clarification, place and after two days precipitation is arranged, not sticking wall clarification, place the back and do not have precipitation, dark-brown is not clarified, brownish black, thickness is not clarified, brownish black, thickness is not clarified, brownish black, thickness |
Interpretation of result and conclusion: the clarifier method can not significantly reduce the solids yield, and the refined solution appearance character is poor; Resin adsorption method is too big to the loss rate of danshensu; The stone mercaptans is similar to the water alcohol method purification effect, but water alcohol method refined solution less stable if sample solids yield is higher, is not removed part in advance, then adopts stone mercaptans complex operation.As seen, single purification process can not reach the requirement of injection purification.Therefore, select water alcohol method and the bonded method of stone mercaptans to carry out purification, promptly first precipitate with ethanol, remove partial impurities after, reuse stone mercaptans purification.
The screening of alcohol precipitation concentration first: it is an amount of to get red rooted salvia, by optimal conditions extract extracting solution, be concentrated into 1: 1.5, be divided into 6 parts then, adding 95% ethanol respectively, to make determining alcohol be 50%, 60%, 70%, 80%, 85%, fully stirs the refrigerator standing over night, filter, filtrate recycling ethanol adds water and is adjusted to 1: 1, gets and measures the wherein content of danshensu in right amount respectively, content * 100% before content/precipitate with ethanol the results are shown in Table 11 behind calculating retention rate=precipitate with ethanol.
As seen, alcohol precipitation concentration is to the appreciable impact that remains with of effective component in red sage, and along with the increase of alcohol precipitation concentration, retention rate reduces, and considers the difficulty or ease of solid-liquid separation behind practical operation situation and the precipitate with ethanol, selects 65% concentration to make for the first time precipitate with ethanol and handles.
The determining of pH value in the precipitate with ethanol first: pH value is remarkable to the influence of effective ingredient in the precipitate with ethanol operation.The material of getting it filled extracts concentrated solution, and surveying pH value is stock solution pH, regulates pH, and adding 95% ethanol, to make determining alcohol be 65%, leave standstill to make and precipitate fully, filter, filtrate recycling ethanol adds water and is adjusted to finite concentration, measure the content of danshensu, and be 100% to calculate retention rate, the results are shown in Table 12 with content before the precipitate with ethanol.
Table 11 alcohol precipitation concentration is to the table that influences of effective component in red sage
| Index | Before the precipitate with ethanol | 50％ | 60％ | 65％ | 70％ | 80％ | 85％ |
| Uv absorption retention rate % content of Danshensu retention rate % appearance character | 0.483 100 1.295 100 muddinesses | 0.367 76.08 // not stratified | 0.301 62.32 1.047 80.83 are difficult for layering | 0.279 57.76 0.999 77.20 standing demix | 0.215 44.51 0.965 76.95 layerings | 0.170 35.20 0.984 75.98 layerings | 0.157 32.51 0.965 74.49 layerings are fast |
PH value is to the table that influences of danshensu in the table 12 Radix Salviae Miltiorrhizae precipitate with ethanol
| Index | Stock solution pH5.7 | Uncomfortable (pH5.7) | Transfer pH4.0 | Transfer pH7.0 |
| Content of Danshensu retention rate % | 1.295 100 | 0.9998 77.20 | 1.1216 86.61 | 0.9547 73.72 |
As seen, turn down the reservation that pH value helps danshensu in the precipitate with ethanol, heightening pH value then retention rate reduces, and therefore determines to regulate pH4.0 before the precipitate with ethanol.
Stone mercaptans craft screening: contain effective ingredient phenolic acid compound and impurity such as tannin, resin in the red sage root water soluble ingredient, wherein phenolic acid compound and tannin structural similarity, be difficult to separate with conventional method, by optimizing stone mercaptans process conditions, can reach the purpose that keeps effective ingredient as far as possible, removes impurity, wherein sulphuric acid readjustment pH value and secondary alcohol precipitation concentration are the principal elements that influences stone mercaptans purification effect, so give experiment screening.
Sulphuric acid readjustment pH value is selected: the precipitate with ethanol for the first time and to be concentrated into 1: 2 Radix Salviae Miltiorrhizae extract an amount of of learning from else's experience, and add lime cream and regulate pH12, stir evenly, leave standstill, continue and regulate pH value with sulfuric acid solution, centrifugal, getting supernatant and measure uv absorption, is 100% calculating retention rate with the extracting solution effective ingredient.The results are shown in Table 13.
Table 13 Radix Salviae Miltiorrhizae stone mercaptans readjustment pH value is to the table that influences of effective ingredient
| Index | Extracting solution | pH7.0 | pH6.5 | pH6.0 | pH4.9 | pH4.4 | pH3.8 |
| Ultraviolet absorption value rate of change % | 0.452 100 | 0.324 71.7 | 0.358 79.2 | 0.363 80.3 | 0.336 74.7 | 0.338 74.8 | 0.326 72.1 |
As seen, the readjustment pH value is 6.0～6.5 o'clock, and the retention rate of effective component in red sage is better, determines that therefore the readjustment pH value is 6.0～6.5.
The selection of secondary alcohol precipitation concentration: medicinal liquid is behind lime sulphur treatment, and residual have polysaccharide, starch and a calcium salt, needs further precipitate with ethanol to remove.Get medicinal liquid behind the lime sulphur treatment, be concentrated into 1: 3, add ethanol respectively and make determining alcohol reach 70%, 80%, 85%, fully stir, leave standstill, filter, reclaim ethanol, add entry liquor strength to the precipitate with ethanol, measure before and after the precipitate with ethanol calcium salt residual quantity in the medicinal liquid respectively.The results are shown in Table 14.
Table 14 alcohol precipitation concentration calcium salt is removed influences table
| Before the precipitate with ethanol | 70％ | 80％ | 85％ | |
| Calcium residual quantity (%) | 0.1200 | 0.0375 | 0.0075 | 0.0075 |
As seen precipitate with ethanol is handled and can be reduced calcium salt residual quantity in the medicinal liquid, acts on more remarkable with the alcohol precipitation concentration rising.Take all factors into consideration the influence of alcohol precipitation concentration, determine that the secondary alcohol precipitation concentration is 80% the danshensu retention rate.
Conclusion: through above process conditions screening, the Radix Salviae Miltiorrhizae purification process is defined as: Radix Salviae Miltiorrhizae extract is concentrated into 1: 1.5, regulates pH to 4.0,65% precipitate with ethanol, and standing over night filters, and filtrate recycling ethanol adds the injection water and is adjusted to 1: 1; Regulate pH11～12 with aqua calcis, left standstill 30 minutes, the reuse sulfuric acid solution is regulated pH6.0～6.5, leaves standstill, filter, filtrate is concentrated into 1: 3 (relative density 1.20～1.25,50～60 ℃), regulates pH to 4.0,80% precipitate with ethanol left standstill 40 hours, filtered, filtrate recycling ethanol is regulated pH to 6.0, adds the injection water and is adjusted to (1: 1), boiled 30 minutes, and put coldly, 0～5 ℃ left standstill 40 hours, filter, filtrate decompression is concentrated into 1: 2, promptly gets the Radix Salviae Miltiorrhizae refined solution.
Prepare the Radix Salviae Miltiorrhizae refined solution by this purifying process, 4 batches of statistics that feed intake wherein, content of Danshensu 4.54 ± 1.64mg/ml, pH5～6 do not detect tannin, calcium ion, sulfate ion passed examination, the solution brownish red is clear and bright, illustrates that this extraction and purification process is feasible.
(2) Radix Puerariae extract purification process
Water alcohol method: Radix Puerariae extracting solution is concentrated into 1: 1,50% precipitate with ethanol; Reclaim ethanol, be concentrated into 1: 2,80% precipitate with ethanol is regulated pH7.5～8.0 with 10% sodium hydroxide solution; Reclaim ethanol, be adjusted to 1: 2, get the water alcohol method refined solution.
The lead acetate sedimentation method: Radix Puerariae extracting solution is concentrated into 1: 1,50% precipitate with ethanol; Reclaim ethanol, thin up to 1: 1, add the lead acetate precipitation, filtrate adds the Lead monosubacetate precipitation again, and precipitation is suspended in 70% ethanol, deleading, supernatant is standby; Be deposited in 70% ethanol and refluxed 2 hours, backflow and supernatant merge, and reclaim ethanol, are adjusted to 1: 2, get lead acetate sedimentation method refined solution.
The stone mercaptans: Radix Puerariae extracting solution is concentrated into 1: 1, adds aqua calcis and regulates pH12, and supernatant is standby; Precipitation is suspended in 70% ethanol, adds sulfuric acid solution and regulates pH4.0, and centrifugal, supernatant is standby; Be deposited in 70% ethanol and refluxed 2 hours; Backflow and preceding two kinds of supernatant merge, and regulate pH7, reclaim ethanol, are concentrated into 1: 2; 80% precipitate with ethanol reclaims ethanol, is adjusted to 1: 2, gets stone mercaptans refined solution.
The clarifier method of purification: 101 clarifier solution of adding 20% in the Radix Puerariae extracting solution, 80 ℃ of liquid temperature leave standstill, and filter, and filtrate is concentrated into 1: 1, and method is handled once for another example, is concentrated into 1: 2, gets 101 clarifier method refined solutions; Radix Puerariae extracting solution is concentrated into 12: 1, adds 8% 1+1 clarifier solution, and 80 ℃ are incubated 20 minutes, filter, and filtrate is concentrated into 2: 1, and method is handled once for another example, is concentrated into 1: 2, promptly gets 1+1 clarifier method refined solution.
Resin adsorption method: get the macroporous adsorbent resin of having handled well, wet method dress post, after getting rid of bubble, the Radix Puerariae extracting solution upper prop is collected effluent simultaneously, stop upper prop during to the absorption terminal point, water elution, reuse 50% ethanol elution to eluent color shoals, and collects 50% ethanol elution, be concentrated into 1: 2, get the resin adsorption method refined solution.
Get above-mentioned each refined solution, measure its solids yield, general flavone content and puerarin content respectively, be 100% calculating retention rate (value in the bracket) with each index before the extracting solution purification simultaneously, and do the paired observation of appearance character, the results are shown in Table 15
Different purification process index components content of table 15 Radix Puerariae and appearance character comparison sheet
| Solids yield % | Puerarin content % | General flavone content % | Total flavones purity % | Appearance character | |
| Extract water alcohol method stone mercaptans lead acetate precipitation method fining agent A method fining agent B method resin adsorption method | 31.21 13.69 (43.9％) 21.00 (67.4％) 18.60 (59.6％) 19.28 (61.8％) 18.14 (58.1％) 14.60 (46.8％) | 4.64 2.83 (61.0％) 2.61 (56.2％) 1.08 (23.3％) 3.27 (70.5％) 3.18 (68.5％) 3.87 (83.4％) | 12.81 8.76 (68.4％) 7.56 (59.1％) 3.41 (26.60％) 8.75 (68.4％) 11.25 (87.8％) 11.05 (91.3％) | 41.04 63.99 36.00 18.34 45.38 62.02 75.68 | Muddy, be placed with precipitation and turbidity, clarification is clarified after transferring pH, the precipitation clarification is arranged after the placement, postpone adularescent precipitation is not clarified for a long time, color depth, stiff is not clarified, color depth, stiff is not clarified, color depth, stiff |
Interpretation of result and conclusion: stone mercaptans, the lead acetate sedimentation method and clarifier method all can not significantly reduce the solids yield; The lead acetate sedimentation method also make the total flavones loss rate increase; The clarifier method is also not obvious to the improvement effect of Radix Puerariae medicinal liquid clarification situation; Though the retention rate height of resin adsorption method total flavones, purity also improves, and is considerable method, and the medicinal liquid appearance character is poor, and in actual production, this method is used for injection and also has some problems to be difficult to for the moment solve; And water alcohol method is all better to the each side index.Therefore, select water alcohol method purification Radix Puerariae medicinal liquid through taking all factors into consideration.
Alcohol precipitation concentration is to the influence of effective ingredient: get Radix Puerariae extracting solution, be concentrated into 1: 2, it is 50%, 60%, 65%, 70%, 75%, 80%, 85% that adding ethanol makes determining alcohol respectively, standing over night, centrifugal, supernatant reclaims ethanol, add water and be made into 1: 2 concentration, measure wherein Radix Puerariae total flavones content, and make outward appearance and observe, the results are shown in Table 16
Table 16 alcohol precipitation concentration is to the table that influences of kudzu vine root
| Index | Before the precipitate with ethanol | 50％ | 60％ | 65％ | 70％ | 80％ | 85％ |
| The general flavone content appearance character | 12.45 it is muddy | 12.42 standing demix | 11.71 layering | 11.69 layering | 12.39 layering | 11.24 layering is fast | 10.25 layering is fast |
Alcohol precipitation concentration selects 50% alcohol precipitation concentration just to be enough to impurity such as sedimentation starch below 80% Radix Puerariae total flavones being kept influence not quite first, effectively keeps effective ingredient.
The screening of Radix Puerariae secondary precipitate with ethanol pH value: behind 50% precipitate with ethanol, impurity major parts such as starch are removed, but not exclusively, are necessary to carry out one time the high concentration precipitate with ethanol again
[21]Get Radix Puerariae extracting solution, behind 50% precipitate with ethanol, reclaim ethanol, be concentrated into 1: 4,80% precipitate with ethanol is regulated pH7.6,8.2,9.0 respectively, standing over night, measure wherein puerarin content, general flavone content and solid contents, and be 100% to calculate retention rate, the results are shown in Table 17 with uncomfortable pH precipitate with ethanol result.
Table 17 Radix Puerariae precipitate with ethanol (80%) back adjust pH is to the table that influences of effective ingredient
| Index | Uncomfortable (pH6.2) | Transfer pH7.6 | Transfer pH8.2 | Transfer pH9.0 |
| Puerarin content and retention rate % general flavone content and retention rate % solid contents % retention rate % appearance character | 2.908 100 7.108 100 7.96 100 looks shallow | 2.919 100.38 7.286 102.50 7.80 97.99 looks shallow | 2.871 98.73 7.069 99.45 7.62 95.48 looks slightly dark | 2.734 94.02 6.971 98.07 7.11 89.45 looks are deepened |
It is little to the kudzu vine root influence to regulate pH value in the Radix Puerariae secondary precipitate with ethanol, heightening pH value, and the medicinal liquid color burn, but can further reduce solid contents, remove impurity.Therefore, determine to regulate in the secondary precipitate with ethanol pH value 8.0～8.2.
Conclusion: through above process conditions screening, the Radix Puerariae purification process is defined as: Radix Puerariae extracting solution is concentrated into 1: 2,50% precipitate with ethanol, and standing over night filters, and filtrate is concentrated into 1: 4; 80% precipitate with ethanol is regulated pH8.0～8.2 with 10% sodium hydroxide solution, leaves standstill 40 hours, filters, filtrate recycling ethanol adds the water for injection of about 5 times of amounts, boils 30 minutes, puts cold, 0～5 ℃ left standstill 40 hours, filtered, and filtrate is concentrated into 1: 2, got the Radix Puerariae refined solution.
Prepare the Radix Puerariae refined solution by this purifying process, 4 batches of statistics that feed intake wherein, puerarin content 46.6 ± 9.32mg/ml, general flavone content 117.66 ± 23.54mg/ml, pH7～8, the solution brownish red is clear and bright, illustrates that this extraction and purification process is feasible.
(3) Rhizoma Chuanxiong extract purification process
Water alcohol method: the Rhizoma Chuanxiong extracting solution is concentrated into 1: 1,60% precipitate with ethanol; Reclaim ethanol, be concentrated into 1: 2,80% precipitate with ethanol; Reclaim ethanol, be adjusted to 1: 2, get the water alcohol method refined solution.
The stone mercaptans: the Rhizoma Chuanxiong extracting solution is concentrated into 10: 1, adds aqua calcis and regulates pH12, adds sulfuric acid solution again and regulates pH4.5～5.0, leaves standstill, and filters, and is concentrated into 1: 1; Method is handled once for another example, is concentrated into 1: 2; 70% precipitate with ethanol reclaims ethanol, is adjusted to 1: 2, gets stone mercaptans refined solution.
The clarifier method of purification: the Rhizoma Chuanxiong extracting solution adds 10% 101 clarifier solution, and 80 ℃ of liquid temperature leave standstill, and filters, and filtrate is concentrated into 1: 1, and method is handled once for another example, is concentrated into 1: 2, gets 101 clarifier method refined solutions; The Rhizoma Chuanxiong extracting solution is concentrated into 12: 1, adds 8% 1+1 clarifier solution, and 80 ℃ are incubated 20 minutes, filter, and filtrate is concentrated into 1: 2, gets 1+1 clarifier method refined solution.
Resin adsorption method: get the macroporous adsorbent resin of having handled well, wet method dress post, after getting rid of bubble, Rhizoma Chuanxiong extracting solution upper prop is collected effluent simultaneously, stop upper prop during to the absorption terminal point, water elution, reuse 50% ethanol elution to eluent color shoals, and collects 50% ethanol elution, be concentrated into 1: 2, get the resin adsorption method refined solution.
Get above-mentioned each refined solution, measure its solids yield and ferulaic acid content respectively, be 100% calculating retention rate with content before the purification simultaneously, and do the paired observation of appearance character, the results are shown in Table 18.
Different purification process index components content of table 18 Rhizoma Chuanxiong and appearance character comparison sheet
| Solids yield % | Ferulaic acid content % | Purity % | Appearance character | |
| Extracting solution water alcohol method stone mercaptans clarifier A method clarifier B method resin adsorption method | 36.11 13.64 (37.8％) 14.99 (41.5％) 30.50 (87.2％) 28.83 (71.5％) 6.59 (18.3％) | 0.0465 0.0295 (63.4％) 0.0311 (66.9％) 0.0399 (85.8％) 0.0316 (68.0％) 0.0415 (89.3％) | 0.129 0.216 0.207 0.131 0.122 0.630 | Muddy, be placed with the precipitation clarification, it is muddy to place all backs, the sticking wall of precipitation is arranged, color is clarified more deeply, precipitation is arranged but not sticking wall after placing for two weeks, color is more shallow not to be clarified, color depth is not clarified, the color depth brownish black is muddy opaque |
Interpretation of result and conclusion: to reduction solids yield, clarifier method weak effect, and resin adsorption method is effective ways; For the reservation of ferulic acid, clarifier method and resin adsorption method are all better, but the appearance character of its refined solution is too poor, can not reach the requirement of injection.The water alcohol method easy operating, but have precipitation to produce after placing; Stone mercaptans Treatment Stability is better, though place the back precipitation is arranged, not sticking wall.So each method pros and cons of balance are actual in conjunction with producing, and select water alcohol method and the bonded method purification of stone mercaptans Rhizoma Chuanxiong extract.
Alcohol precipitation concentration is to the influence of effective ingredient: get the Rhizoma Chuanxiong extracting solution, be concentrated into 1: 2, it is 50%, 60%, 70%, 80%, 85% that adding ethanol makes determining alcohol respectively, standing over night, centrifugal, supernatant reclaims ethanol, add water and be made into 1: 2 medicinal liquid, measure the refined solution uv absorption, and make outward appearance and observe, the results are shown in Table 19.
Table 19 alcohol precipitation concentration is to the table that influences of Rhizoma Chuanxiong effective ingredient
| Index | Before the precipitate with ethanol | 50％ | 60％ | 65％ | 70％ | 80％ | 85％ |
| Uv absorption retention rate % appearance character | 0.707 100 muddinesses | 0.666 94.20 is not stratified | 0.664 93.92 is not stratified | 0.631 89.25 are difficult for layering | 0.592 83.73 standing demix | 0.568 80.34 layerings | 0.536 75.81 layerings are fast |
Alcohol precipitation concentration has in various degree influence to the Rhizoma Chuanxiong effective ingredient, and the concentration below 80% can keep effective ingredient more than 80%, considers from practical operation, selects 70% alcohol precipitation concentration.
Sulphuric acid readjustment pH value is selected: get above-mentioned alcohol deposit fluid and reclaim ethanol, be concentrated into 1: 2, add aqua calcis and regulate pH12, continue and regulate pH7.0, pH6.5, pH6.0, pH5.4, pH4.9 with sulfuric acid solution, measure the supernatant uv absorption respectively, the results are shown in Table 20.
Table 20 Rhizoma Chuanxiong stone mercaptans readjustment pH value is to the table that influences of effective ingredient
| Index | Extracting solution | pH7.0 | pH6.5 | pH6.0 | pH5.4 | pH4.9 |
| Uv absorption and rate of change % | 0.624 100 | 0.438 70.2 | 0.484 77.6 | 0.493 79.0 | 0.503 80.6 | 0.490 78.5 |
Select sulphuric acid readjustment pH5～5.5.
Secondary precipitate with ethanol standing time is to the influence of effective ingredient: the purpose of precipitate with ethanol is to remove residual impurity such as calcium salt in the stone mercaptans, as preceding 80% concentration better effects is arranged, but precipitate with ethanol has or not influence to the effective ingredient ferulaic acid content standing time, remains to be investigated.Get the Rhizoma Chuanxiong concentrated solution, add ethanol and make and reach 80% concentration, placed 12,24,48 hours, the 50ml that takes a sample respectively reclaims ethanol, adds water to 1: 1, measures content, the results are shown in Table 21.
Table 21 Rhizoma Chuanxiong precipitate with ethanol standing time is to the influence of effective ingredient
| Index | Former concentrated solution | Placed 12 hours | Placed 24 hours | Placed 48 hours |
| Ferulaic acid content (mg/ml) retention rate (%) | 0.0567 100 | 0.0557 98.24 | 0.0540 95.24 | 0.0522 92.06 |
Alcoholic solution increases standing time, and the effective ingredient ferulic acid has reduction trend.Actual in conjunction with producing, precipitate with ethanol was advisable in 24 hours standing time.
Conclusion: through above process conditions screening, the Rhizoma Chuanxiong purification process is defined as: the Rhizoma Chuanxiong extracting solution is concentrated into 1: 2,70% precipitate with ethanol, and standing over night filters filtrate recycling ethanol, water for injection to 1: 2; Regulate pH11～12 with aqua calcis, left standstill 30 minutes, continue and regulate pH5.0～5.5 with sulfuric acid solution, leave standstill filtration, filtrate is concentrated into 1: 4; 80% precipitate with ethanol, standing over night filters, and filtrate recycling ethanol adds the injection water to normal concentration, boils 30 minutes, puts coldly, and 0～5 ℃ left standstill 40 hours, filtered, and filtrate decompression is concentrated into 1: 2, promptly gets the Rhizoma Chuanxiong refined solution.
Prepare the Rhizoma Chuanxiong refined solution by this purifying process, 4 batches of statistics that feed intake wherein, ferulaic acid content 0.494 ± 0.16mg/ml, pH4.5～5.5, calcium ion, sulfate ion passed examination, the solution yellow is clear and bright, illustrates that this extraction and purification process is feasible.
In order to verify beneficial effect of the present invention, the injection of the present invention (name is called blood circulation promoting injecta during test) that adopts preparation method of the present invention preparation has carried out Pharmacodynamic test of active extract through Sichuan University's West China basic medical and legal medical expert institute and pharmacology teaching and research room of department of pharmacy of unming Medical College, and various test situation are as follows:
1, the anti-toy cerebral ischemia test of medicine of the present invention
Experiment material: medicine of the present invention, Pharmaceutical College, Huaxi Medical Univ preparation research chamber provides lot number: 971106, specification 10ml/ props up, 1g crude drug/ml; FUFANG DANSHEN ZHUSHEYE, southern pharmaceutical factory produces, lot number 970411, specification 10ml/ props up, 2g crude drug/ml; LPO measures test kit, and Nanjing is built up bio-engineering research and produced, lot number 971102.
Experimental animal: domestic cat, body weight 2.5～3.5kg, male and female are regardless of, and (female person does not have pregnant) provided by animal section of unming Medical College; Pallasiomy, body weight 60～90g, male and female have concurrently, are provided by Yunnan Province natural drug pharmacology key lab, and the quality certification number is defended pipe A12 number for cloud.
Test apparatus: RM-6000 type polygraph, Japanese photoelectricity company product; Photoelectricity VC-II type memory oscilloscope, RF-5000 type spectrofluorophotometer, photoelectricity QC-IIIJ stack Nogata analyser, Japan produces.
(1) medicine of the present invention is to the protective effect of pallasiomy cerebral ischemia and reperfusion injury thereof
Select healthy adult pallasiomy bilateral carotid arteries folder to close after 10 minutes and poured into again 5 days, cause the cerebral ischemia reperfusion injury model.The intravenous injection every day listed drug dose of table 22 once during irritating again.Observe the following index of mensuration then respectively and come of the influence of overall merit medicine of the present invention cerebral ischemia and reperfusion injury.
1. medicine of the present invention to the pallasiomy cerebral ischemia reperfusion injury after the influence of electroencephalogram (EEG)
It is subcutaneous to insert the pallasiomy forebrain with needle electrode, reference electrode places occipitalia subcutaneous, another termination Japanese photoelectricity VC-II type memory oscilloscope, before the record ischemia, iv5 minute, ischemia 10 minutes, irritate the EEG of 10 minutes, 30 minutes and 60 minutes again, and EEG signals is imported Japanese photoelectricity QC-IIIJ stack Nogata analyser, write down its rectangular histogram, with the uv number of X-axis as EEG current potential amplitude, by EEG before the ischemia is 100%, calculates EEG current potential amplitude % with following formula, the results are shown in Table 22.
EEG current potential amplitude %=ischemia or irritate back amplitude ÷ ischemia again before amplitude * 100%
By table 22 as seen, closing the pallasiomy bilateral common carotid arteries 10 minutes (being cerebral ischemia 10 minutes), the EEG amplitude produces immediately and significantly continues low flat change, and medicine of the present invention and FUFANG DANSHEN ZHUSHEYE all can not the above-mentioned variations of antagonism.But irritating phase 60 minutes EEG current potentials amplitude more slowly recovers near level after the administration, NS group only reaches 41.3% of level after the administration, illustrate that its influence to EEG due to the reperfusion injury is little, otherwise medicine of the present invention increases the recovery that promotes reperfusion injury EEG current potential amplitude with dosage, and more all there were significant differences with time point for medicine high dose group of the present invention 10,30,60 minutes and NS group.Illustrate that it not only acts on significantly, and promote reperfusion injury EEG to change the speed of recovery also obviously prior to matched group.The recovery rate that medicine high and low dose of the present invention and FUFANG DANSHEN ZHUSHEYE promote the brain cell anoxia to annotate damage EEG current potential amplitude again is respectively 96.4%, 47.4% and 84.7%.
Table 22 medicine of the present invention to the pallasiomy cerebral ischemia reperfusion injury after the influence of electroencephalogram current potential amplitude
| Group | Animal (only) | Dosage (the g crude drug/kg) | EEG current potential amplitude (%) | |||||
| Contrast 5min behind the medicine | Ischemia 10min | Irritate again | Recovery rate (%) | |||||
| 10min | 30min | 60min | ||||||
| NS medicine of the present invention medicine of the present invention | 8 9 8 8 | 10ml/kg 2.0g/kg iv 1.0g/kg iv 2.0g/kg iv | 116.6± 28.05 104.6± 14.94 104.1± 14.94 113.5± 22.32 | 22.3± 10.57☆ 33.7± 17.16☆ 33.7± 17.16☆ 34.0± 15.42☆ | 30.0±10.57 57.2± 36.05* 35.2±88.76 54.0±34.42 | 41.7± 20.32 84.9± 64.20*△ 39.6± 97.75 79.1± 33.50* | 48.2± 25.64 100.8± 63.84**△ 49.3± 97.34 96.1± 39.03* | 41.3 96.4 47.4 84.7 |
Annotate: ☆: check ☆ P＜0.05 * and NS group contrast T check * P＜0.05, * * P＜0.01 with 5 minutes own control T after the administration;
△: high and low dose medicine significance test of the present invention △ P＜0.05
2. medicine of the present invention to cerebral ischemia and reperfusion injury after the Ca of brain cortical tissue
2+, Na
+Influence with water content
Get cortex of temporal lobe with reference to the Young method, inhale the mark of dehematizing with filter paper, put in the quartz beaker of weighing in advance, after accurately taking by weighing weight in wet base, put 100 ℃ of baking box bakings 4 hours, take by weighing dry weight again, calculate the water content of cerebral tissue by weight in wet base one dry weight/weight in wet base * 100%, through digestion, ashing, dilution process, use atomic absorption spectrophotometer Ca then
2+, Na
+Content the results are shown in Table 23.
Table 23 medicine of the present invention to the pallasiomy ischemia at perfusion injury cerebral cortex water, Ca
2+, Na
+The influence of content
| Group | Dosage | The Mus number | H 2O | Na + | Ca 2+ |
| NS matched group medicine of the present invention medicine of the present invention medicine of the present invention | 10ml/kg 2.0g/kg iv 1.0g/kg iv 2.0g/kg iv | 15 8 9 8 | 78.4±1.42 74.5±3.15 **★ 77.1±1.24 * 76.2±1.39* | 216.5±16.8 185.2±26.09 **▲ 200.24±13.92 *▲ 234.0±39.3 | 64.46±2.303 1.57±1.026 **▲ 3.50±1.247 44.80±2.06 |
Annotate: 1. each administration group is compared the T check with the NS matched group: * P＜0.05, * * P＜0.01;
2. each administration group is compared with Radix Salviae Miltiorrhizae ▲ P＜0.05, compares ★ P＜0.05 with medicine low dosage of the present invention
By table 23 as seen, medicine low dose group of the present invention can obviously reduce pallasiomy cerebral ischemia re-pouring cerebral cortex Na
+And water content (P＜0.05), the more remarkable reduction of high dose group Na
+And water content (P＜0.01), be dose-effect relationship, and can make Ca
2+Amount obviously descends, and the compound Salviae Miltiorrhizae group only shows that the water yield descends, and can not make Na
+And Ca
2+Amount descends, and with medicine high dose group of the present invention significant difference (P＜0.05) is arranged relatively, illustrates alleviating cerebral edema due to the cerebral ischemia re-pouring, antagonism Na
+And Ca
2+Rising aspect medicine of the present invention be better than FUFANG DANSHEN ZHUSHEYE.
3. medicine of the present invention to the pallasiomy cerebral ischemia reperfusion injury after the influence of brain lipid peroxide (LPO)
Lipid peroxide (LPO) is measured: the disconnected neck of animal is put to death, and gets the left side forebrain, and employing thiobarbituricacid (TBA) method is measured the LPO in the cerebral tissue.TBA product RF-5000 type fluorescent spectrophotometer assay, 1,1,3,3, a tetramethoxy propane is as outer standard, and the LPO level is represented (seeing Table 24) with its metabolite malonaldehyde (MDA) weight in wet base.
Table 24 medicine of the present invention is to the table that influences of pallasiomy cerebral ischemia reperfusion injury brain lipid peroxide
| Group | Dosage (the g crude drug/kg) | Number of animals (only) | MDA (n mol/g weight in wet base) |
| NS matched group medicine of the present invention medicine Radix Salviae Miltiorrhizae Injection of the present invention | 10ml/kg 2.0g/kg iv 1.0g/kg iv 2.0g/kg iv | 15 8 9 8 | 228.0±55.25 179.5±32.06 253.9±38.38 133.2±30.82 |
Annotate: 1. each administration group is compared with matched group: * P＜0.05, * * P＜0.01; 2. ▲: each administration group compares with Radix Salviae Miltiorrhizae＜and 0.05.
By table 24 as seen, FUFANG DANSHEN ZHUSHEYE and isodose medicine of the present invention all can significantly reduce cerebral cortex lipid peroxide contents behind the ischemical reperfusion injury, help the recovery of function of brain cell.FUFANG DANSHEN ZHUSHEYE obviously is better than medicine of the present invention on the level that reduces MDA.
(2) medicine of the present invention is to the influence of anesthetized cat cerebral blood flow and cerebral vascular resistance (vascular resistance increases due to the NA)
Healthy domestic cat, body weight 2.5～3.5kg, male and female are regardless of, 3% pentobarbital sodium 30mg/kg ip. anesthesia, lie on the back and be fixed in operating-table, the medisection skin of neck separates the outer branch of left carotid and its cranium of ligation, put the electromagnetic flowmeter probe of diameter 2mm, trace cerebral blood flow (CBF) with this.Separate left carotid, insert the intubate of filling, connect pressure transducer and measure arteriotony (Bp) with heparin (500u/ml) normal saline.Adopt analog multiplier-divider that Bp is promptly got the index cerebral vascular resistance (R) of deriving divided by CBF, trace bilateral brain body surface EEG and ECG simultaneously, more than the equal synchronous recording of every index in RM-6000 type polygraph.
Separate a side femoral vein and do administration fully, etc. every index stable after, trace one section normal control curve, then divide 4 groups of constant speed (1ml/min) iv NS 2ml/kg (matched group) respectively at random, medicine 1.0g/kg of the present invention, medicine 2g/kg of the present invention and FUFANG DANSHEN ZHUSHEYE 2g/kg (Radix Salviae Miltiorrhizae group).Retouch the every index of meter behind the 5min, constant speed iv norepinephrine (NA) 1.0ug/kg/min again observes each index and changes situation, and after giving NA 0,5,10,25,30min respectively traces one section curve, and own control adopts pairing T check before and after the data at different levels; Adopt relatively between the data at different levels in groups that T check (significance level a=0.05) the results are shown in Table 25, table 26.
1. medicine of the present invention is to the influence (seeing Table 25) of anesthetized cat blood pressure
By table 25 as seen, medicine of the present invention and FUFANG DANSHEN ZHUSHEYE illustrate that to the equal no significant difference of systolic pressure and diastolic pressure reading administration front and back of anesthetized cat it does not make significant difference to normal arterial pressure.Under this experimental condition, two medicines all can not resist the hypertension due to the NA.
2. medicine of the present invention is to the influence of anesthetized cat cerebral blood flow and cerebral blood flow resistance
Result of the test sees Table 26.
By table 26 as seen, comparison from administration front and back cerebral blood flow and cerebral blood flow resistance own control, medicine of the present invention and FUFANG DANSHEN ZHUSHEYE high and low dose all have significant cerebral blood flow increasing amount and reduce the effect of cerebral blood flow resistance synchronously, and effect intensity there was no significant difference between three groups.To increasing of the cerebral blood flow resistance due to the NA, medicine low dose group of the present invention and FUFANG DANSHEN ZHUSHEYE all do not show antagonism, but medicine high dose group cerebral blood flow resistance of the present invention is less because of the increasing degree due to the NA, with the same period NS group significance meaning is relatively arranged.
2, medicine of the present invention is to the influence of experimental dog ischemia
Medicine: medicine of the present invention, Pharmaceutical College, Huaxi Medical Univ preparation research chamber provides; Lot number: 000501, specification: 10g crude drug/10ml/ props up; Usage and dosage: each 10ml that is grown up medicine of the present invention adds normal saline 100ml intravenous drip, and every day 1 time, promptly consumption is Coming-of-Age Day: 10g crude drug/10ml * 10ml/l time/day, it is standby to be mixed with the desired concn medicinal liquid with 0.9% sodium chloride injection during test.
Reagent: pentobarbital sodium, by the import packing of chemical experimental factory, Foshan City, 25g dress, lot number: 990901; Gentian Violet: reagent three factories in Shenyang produce, lot number: 980501; Alkali phosphatase (ALP) test kit (enzyme performance rate method): give birth to company's product, lot number: 000315 in Beijing.Creatine phosphokinase (CK) test kit (enzyme performance rate method): give birth to company's product, lot number: 00301 in Beijing.
Instrument: BeckMan700 type automatic clinical chemistry analyzer, Beckman Instruments Inc.(U.S.A.) makes; JA-5003 type electronic balance, balance equipment factory in Shanghai makes; Model-820 type histotome, U.S. AO company produces; The OLYMPUSPM-10AD photomicroscope, Japan produces.
Test method: 48 of careless dogs, be divided into 6 groups at random, every group 8, lumbar injection 2.5% pentobarbital sodium 1.0ml/kg (25mg/kg) anesthesia, fixedly dog is on operating-table, separating the left side femoral vein uses for getting blood, then in the right tail of the eye of dog and auris dextra root 1/2 place (mid point), with electric knife percutaneous incision skin, separating muscle, open skull with the special-purpose cranial drill brill of operation, enlarge the skull hole with rongeur, cut cerebral dura mater, find middle cerebral artery after, extract enzymatic determination serum levels of ALP and CK before medicine 3.0ml blood is done the middle cerebral artery ligation by femoral vein earlier.Get and be about to the middle cerebral artery ligation behind the blood and make and cause cerebral ischemia, this moment, by forelimb intravenous injection drug administration 1 time, dosage and grouping saw Table 27 immediately.
Table 27 medicine dog of the present invention focal ischemia test dosage regimen
| Group | Tried thing | Drug level (g/ml) | Administration volume (ml/kg) | Dosage (g/kg) | Administration number of times (inferior) | Route of administration | Clinical multiple |
| Dosage group medicine small dose group of the present invention in the 0.9% sodium chloride injection group matrix liquid group compound injection of red sage root group medicine high dose group of the present invention medicine of the present invention | 0.9 sodium chloride injection matrix liquid matrix liquid medicine of the present invention medicine of the present invention medicine of the present invention | 0.9％ -- 1 1 0.5 0.25 | 2 2 2 2 2 2 | -- -- -- 2 1 0.5 | 1 1 1 1 1 1 | 1.v 1.v 1.v 1.v 1.v 1.v | -- ≈12 ≈6 ≈3 |
The middle cerebral artery ligation is promptly tried thing after about 6 hours, get blood 3.0ml by femoral vein once more and make enzymatic determination, and separate bilateral common carotid arteries and press from both sides and close it, at once the centrifugal end perfusion 20ml Gentian Violet saturated solution that closes from the right carotid folder dyes to cerebral tissue, put to death animal then, open cranium and take out cerebral tissue, claim full brain heavy, downcut undyed cerebral tissue (being the ischemic region cerebral tissue) then and weigh, obtain the percentage rate that it accounts for full brain weight, and carry out histopathologic examination and (get cerebral tissue routinely, 1 week of 10% formaldehyde fixed, H.E dyeing, paraffin embedding section, optical microscope is observed down), judge whether it belongs to cerebral ischemic injury (seeing Table 28).The serum zymetology is determined on the Beckman 700 type automatic clinical chemistry analyzers carries out.The results are shown in Table 28～table 30.
Table 28 medicine of the present invention is to the influence of experimental dog cerebral ischemia
| Group | Dosage * number of times (g/kg * c) | Dog number (only) | Full brain heavy (g) | Cerebral ischemia district heavy (g) | Ischemic region weighs/full brain heavy (%) |
| 0.9% sodium chloride injection group matrix liquid group compound injection of red sage root group medicine liquid of the present invention group | Equal-volume * 1 equal-volume * 12 * 12 * 11 * 1 0.5 * 1 | 8 8 8 8 8 8 | 63.99 ±13.33 63.10 ±8.53 64.26 ±6.78 65.76 ±5.53 66.76 ±5.75 62.31 ±5.44 | 20.48 ±8.19 17.92 ±3.91 14.14* ±3.37 9.77** ±3.89 12.72 ±2.24 15.95 ±3.31 | 28.76 ±4.99 28.47 ±7.61 22.26* ±5.90 15.05** ±6.30 18.68* ±2.94 25.60 ±4.80 |
Annotate: 1. 2. each administration group and the comparison of substrate liquid group of substrate liquid group and 0.9% sodium chloride injection group comparison P＞0.05
*P＜0.05
*P＜0.01
Table 28 shows, 2 and 1g/kg dosage medicine of the present invention respectively can be significantly and obviously reduce cerebral tissue ischemic region weight (P＜0.01 and P＜0.05) after the ligation of dog middle cerebral artery.
Table 29 medicine of the present invention is to the active influence of experimental cerebral ischemia dog serum ALP
Annotate: 1. relatively 2. each administration group and relatively P＞0.05 table 8 demonstration of substrate liquid group of P＞0.05 of substrate liquid group and 0.9% sodium chloride injection group, 2-0.5g/kg having, dosage medicine of the present invention reduces serum levels of ALP trend of rising after the ligation of dog middle cerebral artery, but not statistically significant (P＞0.05).
Table 30 medicine of the present invention is to the active influence of experimental cerebral ischemia dog serum CK
Annotate: 1. substrate liquid group and 0.9% sodium chloride injection group compare P＞0.05 2. each administration group and substrate liquid group comparison * P＜0.01 * * P＜0.01
Table 30 shows, 2g/kg dosage medicine of the present invention can significantly reduce the effect (P＜0.01) that serum CK raises after the ligation of dog middle cerebral artery.
3, the medicine of the present invention test that resists myocardial ischemia
Medicine: medicine of the present invention, Pharmaceutical College, Huaxi Medical Univ preparation research chamber provides.Lot number 971106, specification 10ml/ props up, 1g crude drug/ml; FUFANG DANSHEN ZHUSHEYE, southern pharmaceutical factory produces, lot number 970411, specification 10ml/ props up, 2g crude drug/ml; SHENGMAI ZHUSHEYE: Pharmacuetical Plant of Huaxi Medical Univ. produces, lot number: 980202,980601, and specification 10ml/ props up, 0.568g crude drug/ml; The timolol eye drop: Tianjin pharmaceutical factory of central authorities produces, lot number 971108, and 5ml/ props up, 5mg/ml; The moulding medicine: the pituitrin injection, the biochemical-pharmaceutical factory, Shanghai produces, lot number 971102,1ml/ props up, 10u/ml.
Reagent: LDH and CPK test kit: Beijing Zhongsheng Biological Engineering High Technology Company's product, lot number 980201; Calf serum, the biological study of West China, Chengdu is produced, lot number 98.0606; The M199 culture medium, GIBCO, lot number 21200-076; Dulbeccos PBS:GIBCO, lot number 21600-010; Trypsin Tripsin 1: 250), GIBCO, lot number 27250-042; Smart nitrogen: specification 99.9% is provided by the attached First Academy of West China medical university oxygen group.
Animal: Chinese grass seeds dog (hybrid dog): body weight 8.5～15kg, male and female have (female person does not have pregnant) concurrently and are provided by animal section of unming Medical College; Cavia porcellus: body weight 300～400g, male and female have concurrently, are carried by animal section of unming Medical College; Kunming mouse: body weight 18～20g, the one-level animal, male and female half and half are provided by Huaxi Medical Univ's Experimental Animal Center, one-level laboratory animal, the quality certification number: No. the 67th, the real moving pipe in river; SD kind rat: male and female have concurrently, and body weight 160～200g is provided by magnificent medical university Experimental Animal Center, one-level laboratory animal, each lattice card number: No. the 70th, the real moving pipe word in river.
Instrument: RM-6000 type polygraph: Japanese photoelectricity company product; Gould 3400S/DASA 4600 type polygraphs, the U.S. produces; Tianjin, island U-135 balance recorder; Beckman C * 7 type full automatic biochemical apparatus, the U.S. produces.
(1) medicine of the present invention is to anesthetized dog myocardial ischemia test (ligation anterior descending coronary method)
28 of healthy dogs, the male and female dual-purpose, tracheal intubation after with sodium pentobarbital 30mg/kg intravenous anesthesia (artificial respiration uses when opening breast fully), separate left side femoral artery and carotid artery, the polyethylene pipe (in be full of heparin-saline) that inserts suitable bore respectively is to femoral artery and left ventricle, connect pressure transducer and measure periphery femoral blood pressure (AP) and left indoor pressure (LVP), the phase is not pressed (LVEDP) and the maximum climbing speed (LVdp/dt) of left ventricular pressure in the left ventricle diastole; Open breast and separate dark aortic root, put suitable in diameter probe through MF-27 electromagnetic flowmeter determination blood flow as cardiac output (C0), measure electrocardiogram＜ECG (II) simultaneously 〉, the visceral pericardium electrocardiogram.Except that directly measuring the above-mentioned parameter, friendship is tried to achieve a minute tension-time index by literature method, and (TTIXHRO is with indirect determination myocardial oxygen consumption index.More than eight equal synchronous recordings of index on RM-6000 type polygraph, etc. every index stable after, give normal saline (NS), FUFANG DANSHEN ZHUSHEYE and medicine high and low dose of the present invention respectively through the vein constant speed, injection capacity is 2ml/kg, annotated in 10 minutes, before the injection, inject and has partly measured and 3 time points of full dose, all write down above-mentioned parameters once.Then in the dual ligation of ramus descendens anterior arteriae coronariae sinistrae (LAD1/2 place, the first order vessel branch initial part of supply ventricle antetheca and apex), 5,10,15,60 and 120 minutes above-mentioned parameters of start record respectively after the ligation.And before ligation LAD and after the ligation 3 hours, extracting vein blood is measured lactic acid dehydrogenase (LDH) and creatine phosphokinase (CPK) content; Heart is won in last blood-letting rapidly, and the NBT staining is weighed and measured the left ventricle infarction size, with the T check significance is carried out relatively in ligation LAD front and back and each test group differences, the results are shown in Table 31.
1. medicine of the present invention is to the influence of myocardial ischemia zymetology index and left chamber infarction size
Table 31 medicine of the present invention to the influence of anesthetized dog ligation LAD myocardial ischemia zymetology index and left ventricle infarction size (
N=7)
| Group | Dosage g crude drug/kg | LDH(U/L) | CPK(U/L) | Left side chamber infarction size (%) | ||
| Before the ligation | After the ligation | Before the ligation | After the ligation | |||
| Normal saline group compound recipe side Radix Salviae Miltiorrhizae group high dose group of the present invention low dose group of the present invention | - 2.0 2.0 1.0 | 261±167 143±157 171±85 285±202 | 340±313 202±157 400±113 360±207 | 254±176 175±118 207±142 171±143 | 692±176 345±176 651±235 472±217 | 16.8±6.2 7.4±2.4 ※ ※ 9.2±1.0 ※ ※ 8.1±2.4 ※ ※ |
Annotate: ※ ※: with NS matched group T significance test P＜0.01.
By table 31 as seen, behind medicine of the present invention and the isodose FUFANG DANSHEN ZHUSHEYE intravenously administrable, the left ventricle infarction size due to the dog ligation anterior descending coronary ischemia is significantly dwindled, illustrate that it all has the tangible myocardial ischemia effect that improves.But for the increase of myocardial cell owing to caused LDH of ischemia and CPK burst size, under this experimental condition, medicine of the present invention and FUFANG DANSHEN ZHUSHEYE are all failed to show and are suppressed or the improvement effect.
2. medicine of the present invention sees Table 32 to the result of the test that influences of the dirty heart rate of guinea-pig heart, myocardial contraction.
Table 32 medicine of the present invention to the influence of the dirty heart rate of guinea-pig heart, myocardial contraction (
N=10)
| Group | Final concentration (mg/ml) | Heart rate (inferior/min) | Myocardial contraction (cm) | ||
| Before the medicine | Behind the medicine | Before the medicine | Behind the medicine | ||
| NS group medicine of the present invention medicaments compound Radix Salviae Miltiorrhizae Injection of the present invention | Equal-volume 3.8 1.9 3.8 | 108±17 109±15 118±13 123±14 | 107±10 99±20 108±12 108±10 | 2.31±0.74 2.83±1.82 3.10±1.50 3.36±1.27 | 2.34±0.80 2.90±1.75 4.05±1.34 4.55±1.15 |
By table 32 as seen, medicine of the present invention and FUFANG DANSHEN ZHUSHEYE self relatively there is no notable difference to guinea-pig heart dirty heart rate and two index administrations front and back of myocardial contraction, illustrate that its basic activity to the normal heart that exsomatizes does not have directly significantly influence.
(2) medicine of the present invention is to the influence of the myocardial oxygen consumption equilibrium of supply and demand
1. medicine of the present invention is to the exponential influence of anesthetized dog myocardial oxygen consumption
After directly recording parameters such as HR, ECG, MAP, CO, LVP, LVdp/dt, LVEDP by anesthetized dog myocardial ischemia test with RM-6000 type polygraph, press document
[1]Method can be tried to achieve myocardial oxygen consumption index (TT1 * HR), the results are shown in Table 33.
Table 33 medicine of the present invention is to (the influence of TT1 * HR) of anesthetized dog myocardial oxygen consumption index
| Group | Dosage (g/kg) | Number of animals (only) | TT1×HR(mmHg·s/min) | |
| Before the administration | After the administration | |||
| Normal saline group high dose group of the present invention low dose group compound Salviae Miltiorrhizae of the present invention matched group | - 2.0 1.0 2.0 | 7 7 7 7 | 227±17.2 213±12.7 212±18.0 218±12.8 | 233±3.4 205±23.9 215±16.2 209±11.4 |
Annotate: each administration group and normal saline group be P＞0.05 relatively
By table 33 as seen, each test group myocardial oxygen consumption index there are no significant difference before and after the administration, thus point out medicine of the present invention that the oxygen consumption of anesthetized dog cardiac muscle is not had obvious influence indirectly.
2. medicine of the present invention brings out the influence of rat heart muscle ischemia to pituitrin
The healthy SD rat, body weight 200 ± 20g, male and female half and half are divided into 5 groups, 12 every group by the principle of sex equalization.Press the listed drug dose of table 29, every day, tail vein injection was respectively organized medicine once (volumetric injection is 0.5ml/100g), continuous 6 days, after the last administration 1 hour, anaesthetize with 10% urethane 0.5g/kg ip, one section normal II ECG that leads is write down with Gould3400s/DASA4600 type polygraph in fixing back, back of the body position, iv pituitrin 1u/kg then, 15s after administration, 30s, 1min, 2min, 3min, 4min, 5min, 10min, 15min, 20min writes down the II ECG that leads respectively, and with the T ripple of any time wherein or ST section rising 0.1mv or decline 0.05mv as the positive number of animals of myocardial ischemia, experimental result x
2Check is organized a significant difference relatively.The results are shown in Table 34.
Table 34 medicine of the present invention is brought out the influence of acute myocardial ischemia by pituitrin to rat
| Group | Drug dose (the g crude drug/kg/d) | Number of animals (only) | The myocardial ischemia number of animals | P (comparing) with the NS group | |
| Number positive | Negative number | ||||
| Dosage group low dose group of the present invention among the high dose group the present invention of the present invention of normal saline group SHENGMAI ZHUSHEYE | 5ml×6d 2.5g×6d 3.4g×6d 1.7g×6d 3.4×6d | 12 12 12 12 12 | 12 0 0 2 5 | 0 12 12 △ 10 7 | ＜0.001 ＜0.001 ＜0.001 ＜0.01 |
Annotate: △: compare p＜0.05 with low dose group
By table 34 as seen, the negative animal number average of each dosage group of medicine of the present invention is significantly higher than the normal saline group, illustrate that it has tangible antagonism to the acute myocardial ischemia that is caused by the coronary vasospasm due to the pituitrin, exists tangible dose-effect regularity in medicine high and low dose group of the present invention.Results suggest medicine of the present invention promotes the balance of myocardial cell to the oxygen demand by the supply that improves ischemic myocardium blood oxygen.
3. medicine of the present invention is to the influence of anoxia in mice tolerance
A. medicine of the present invention is to the influence of mice normobaric hypoxia test
80 of the Kunming mouses of body weight 18-20g, after being divided into 4 groups by the principle of body weight equalization, tabular medicine and dosage under the difference lumbar injection, continuous 5 days, behind the last injectable drug 30 minutes, place in 1 500ml ground glass bottle (every bottle of end fills the 10g sodica calx) airtight each mice, observe its hypoxia death time, significance between organizing relatively the results are shown in Table 35.
Table 35 medicine of the present invention is to the influence of mice normobaric hypoxia death time
Annotate: ※ ※ ※: organize significance test p＜0.001 △ with NS: medicine high and low dose group significance test p of the present invention＜0.05
By table 35 as seen, medicine of the present invention and positive control drug chlorpromazine be the death time of energy significant prolongation mice normal pressure resistant anoxia all, particularly brain, heart organ are to anoxybiotic tolerance to point out it that enhancing body is arranged, and there is tangible dose-effect rule in its potentiation between the high and low dose group.
B. medicine of the present invention brings out the influence of mice normal pressure resistant anoxia test to isoproterenol
80 of the Kunming mouses of body weight 18-20g, after being divided into 4 groups by the principle of body weight equalization, tabular medicine and dosage under the difference lumbar injection, continuous 5 days, behind the last injectable drug 30 minutes, give every mouse subcutaneous injection isoproterenol, after 20 minutes mice is placed in the 500ml ground glass bottle (the bottle end fills the 10g sodica calx), its hypoxia death time of observed and recorded, carry out the T check of group difference and compare, the results are shown in Table 36.
By table 36 as seen; the equal energy of beta receptor blocker timolol and medicine of the present invention significant prolongation injection isoproterenol improves the mice normobaric hypoxia death time after the myocardial oxygen consumption level; point out it like reducing myocardium keto consumption, myocardial ischemia is had the certain protection effect.
Table 36 medicine of the present invention brings out the influence of mice normobaric hypoxia death time to isoproterenol
Annotate: ※ ※ ※: organize significance test p＜0.001 △ △ △ with NS: medicine high and low dose group significance test p of the present invention＜0.001
4. medicine of the present invention is cultivated the protection test of anoxia sugar deficiency injury to external myocardial cell
Select Wistar rat neonatal rat for use, under the sterile working, get ventricular muscles and shred to 1mm
3, Hanks liquid flushing back with trypsin repeatedly digest, centrifugal, precipitation with 15% calf serum suspend, counting, adjustment concentration is 2 * 106/ culture bottles, 37 ℃ of 5%CO
2Incubator was cultivated 4-6 days, was paved with to be standby behind the cell monolayer.Test is divided into seven groups: 1. DPBS buffer normal control group; 2. fill the nitrogen anoxia and lack sugared model control group; 3. SHENGMAI ZHUSHEYE positive drug control group; 4. receptor blocking agent---timolol matched group; 5.-7. be respectively high, medium and low three the dosage experiments groups of medicine of the present invention.Except that 1., surplus respectively the group adds 3ml and fills the nitrogen medicinal liquid and filled nitrogen 30 seconds in each Tissue Culture Flask, screw bottle cap, sealing was cultivated 6 hours, get supernatant with Beckman C * 7 type full automatic biochemical apparatus at the 340nm wavelength through spectrophotometry lactic acid dehydrogenase (LDH) content wherein, and with T check comparable group differences.
The results are shown in Table 37.
Table 37 medicine of the present invention is to cultivating the influence of myocardial cell anoxia sugar deficiency injury
| Group | Dosage or concentration (g/l or M) | Cell bottle number | LDH (unit) |
| Dosage group low dose group of the present invention among the high dose group the present invention of the present invention of Normal group Anoxia model group shengmai injection group Timolol group | - - 1g/L 1×10 -5 2g/L 1g/L 2g/L | 6 6 6 6 5 6 6 | 17.50±6.06 63.67±25.23 △△△ 29.66±15.20 ※※ 23.50±3.51 ※※※ 20.40±6.31 ※※※ 25.17±12.03 ※※※ 40.33±19.71 ※ |
Annotate: ※: lack relatively ※ P＞0.05 of sugared model group with anoxia; ※ ※ P＜0.05; ※ ※ ※ P＜0.01
△: anoxia lacks sugared model group and the normal control group compares △ △ △ P＜0.01
By table 37 as seen; LDH content very significantly is higher than normal myocardium cell culture group in the scarce sugared model myocardial cell culture fluid of anoxia; be released into LDH content in the culture fluid and SHENGMAI ZHUSHEYE and timolol and medicine height of the present invention, middle dosage group all can obviously reduce myocardial cell injury, show that medicine of the present invention has tangible direct protective action to external myocardial cell anoxia sugar deficiency injury.
Function of the present invention: blood circulation and channel invigorating.
Of the present invention curing mainly: be used for the treatment of angina pectoris.
Specification of the present invention: 1.4g/ props up.
Usage and dosage of the present invention: once-a-day, each 1～2, add among 5% glucose injection 250mL or the 0.9% normal saline 250mL intravenous drip.
The influence that table 25 medicine of the present invention changes anesthesia domestic cat blood pressure (mmHg)
| Medicine and dosage group | Cat number (only) | Before the administration | After the administration (min) | To (min) behind the NA | ||||||
| 0 | 5 | 10 | 15 | 20 | 30 | |||||
| NS (3g/kg) low dose group (1.0g/kg) high dose group (2g/kg) the danshen injections group (2g/kg) of promoting blood circulation of promoting blood circulation | 6 7 8 9 | Systolic pressure diastolic pressure systolic pressure diastolic pressure systolic pressure diastolic pressure systolic pressure diastolic pressure | 200.41±6.15 150.1±11.18 198.0±9.65 157.0±13.19 194.9±16.31 146.8±14.62 198.3±13.7 148.7±7.86 | 202.0±17.54 148±12.60 195.3±8.92 155.2±29.28 193.3±20.34 142.5±27.66 200.6±9.49 148.8±6.22 | 225±17.24* 165.0±15.17* 246.0±33.27* 166.8±18.35* 226.5±26.92* 164.8±16.48* 228.0±18.78* 171.5±12.95* | 202.1±19.24 152.2±16.48 192.2±21.34 153.8±16.57 191.5±26.56 145.0±29.58 195.1±23.41 149.2±9.85 | 206.2±18.61 151.6±16.64 193.2±231 155.2±6.4 190.2±23.19 142.5±20.53 194.6±22.56 149.0±6.59 | 201.1±18.82 148.4±9.63 193.2±18.95 154.0±5.65 189.4±16.45 141.6±14.98 196.4±20.21 146.3±6.54 | 199.2±19.77 151.2±10.87 195.2±21.30 153.3±14.10 190.6±20.36 143.0±16.67 195.6±24.22 145.8±5.86 | 200±15.81 151.6±11.24 195.5±20.23 153.4±11.51 189.9±18.00 145.3±14.86 194.6±24.22 145.7±6.21 |
Annotate: contrast P＜0.05 before * and the administration contrasts there was no significant difference between each group
Table 26 medicine of the present invention is to the influence of anesthesia domestic cat cerebral blood flow (ml/min) and cerebral blood flow resistance (ml/min/mmHg) variation
| Medicine and dosage group | Number of animals | Before the administration | (min) 5 after the administration | To (min) behind the NA | ||||||
| 0 | 5 | 10 | 15 | 20 | 30 | |||||
| NS (2g/kg) low dose group (1.0g/kg) high dose group (2g/kg) the danshen injections group (2g/kg) of promoting blood circulation of promoting blood circulation | 6 7 8 9 | The resistance of blood flow of CBF resistance of blood flow CBF resistance of blood flow CBF resistance of blood flow CBF | 27.0±69.78 5.9±1.03 25.8±3.71 6.1±0.80 26.0±6.39 5.9±1.01 26.0±4.60 5.9±1.11 | 27.6±8.67 5.8±1.20 28.9±3.19* 5.2±0.48* 29.5±7.76* 4.3±1.10*# 28.8±4.86* 4.8±0.95* | 24.4±6.34* 7.5±1.96* 27.8±3.49 7.0±0.97* 25.6±5.40 6.5±1.26*# 25.1±4.52* 6.9±1.07* | 24.4±8.32 6.7±4.36 25.0±2.53 6.3±0.58 24.5±4.57 5.7±1.18 24.9±5.25 6.2±1.41 | 23.8±9.70 7.1±2.42 24.6±3.01 6.3±0.78 24.4±5.59 6.0±1.44 24.8±5.78 6.3±1.43 | 23.8±9.67 7.0±2.32 25.0±3.46 6.2±0.34 24.4±5.78 6.2±1.72 25.0±5.40 6.3±1.30 | 24.0±10.4 7.0±2.15 25.8±4.49 6.0±0.51 24.2±6.08 6.4±1.90 24.2±5.56 6.4±1.42 | 24.4±9.8 7.2±2.62 26.0±4.98 6.0±0.65 24.3±6.08 6.1±1.43 24.3±6.02 6.3±1.50 |
Annotate: contrast P＜0.05 before * and the administration; # and NS group contrast P＜0.05
Claims (1)
1, a kind of injection preparation method of lyophilized injectable powder of promoting blood circulation, 100 lyophilized injectable powders at every packing 1.4g comprise raw material Radix Salviae Miltiorrhizae 333.3g, Rhizoma Chuanxiong 333.3g, Radix Puerariae 333.3g, mannitol 90g, sodium sulfite 2g, it is characterized in that this preparation method comprises the steps:
(1) Radix Salviae Miltiorrhizae 333.3g is made only, be ground into coarse granule, add 10 times of amount concentration is that 4.3% sodium hydroxide solution decocts three times at every turn, boiled 1.5 hours at every turn, and merged decoction liquor, be evaporated to 1: 1.5, relative density 1.10～1.15, is regulated pH to 4.0 by 50～60 ℃, adding ethanol makes determining alcohol reach 65%, stir evenly, standing over night filters, filtrate recycling ethanol adds the injection water and is adjusted to 1: 1 to there not being the alcohol flavor; Regulate pH11～12 with aqua calcis, stir evenly, left standstill 30 minutes, regulate pH6.0～6.5, leave standstill, filter, filtrate is concentrated into 1: 3, relative density 1.20～1.25,50～60 ℃, regulate pH to 4.0, add ethanol and make determining alcohol reach 80%, stir evenly, left standstill 40 hours, and filtered, filtrate recycling ethanol is regulated pH to 6.0 to there not being the alcohol flavor, add the injection water and be adjusted to 1: 1, boiled 30 minutes, put coldly, 0～5 ℃ left standstill 40 hours, filter, filtrate decompression is concentrated into 1: 2, measures content, gets Radix Salviae Miltiorrhizae extract;
(2) Rhizoma Chuanxiong medical material 333.3g is made only, be ground into coarse granule, add 7 times of water gagings at every turn and decoct three times, decocted 1.0 hours, and merged decoction liquor, be evaporated to 1: 2, relative density 1.15～1.25,50～60 ℃, add ethanol and make determining alcohol reach 70%, stir evenly, standing over night, filter, filtrate recycling ethanol adds injection water to 1: 2 to there not being the alcohol flavor; Transfer pH11～12, stir evenly, left standstill 30 minutes, transfer pH5.0～5.5, leave standstill, filter, filtrate is concentrated into 1: 4, relative density 1.20～1.30,50～60 ℃, add ethanol and make determining alcohol reach 80%, stir evenly standing over night, filter, filtrate recycling ethanol adds the water for injection of about 6 times of amounts to there not being the alcohol flavor, boils 30 minutes, put coldly, 0～5 ℃ left standstill 40 hours, filtered, filtrate decompression is concentrated into 1: 2, measures content, gets Rhizoma Chuanxiong extract;
(3) Radix Puerariae medical material 333.3g is made only, be ground into coarse granule, add 8 times of water gagings at every turn and decoct three times, decocted 1.5 hours, and merged decoction liquor, be evaporated to 1: 2, relative density 1.10～1.20, adds ethanol and makes determining alcohol reach 50% by 50～60 ℃, stir evenly, standing over night filters, filtrate recycling ethanol also is concentrated into 1: 4, relative density 1.25～1.30,50～60 ℃, add ethanol and make determining alcohol reach 80%, regulate pH to 8.0～8.2, stir evenly, left standstill 40 hours, and filtered, filtrate recycling ethanol is to there not being the alcohol flavor, add the water for injection of 5 times of amounts, boiled 30 minutes, put cold, 0～5 ℃ left standstill 40 hours, filtered, and filtrate is concentrated into 1: 2, measure content, get Radix Puerariae extract;
(4) get Radix Salviae Miltiorrhizae extract, Radix Puerariae extract and Rhizoma Chuanxiong extract, mixing, adding concentration is 0.4% sodium sulfite, dissolving, mix homogeneously is regulated pH6.8～7.2, and adding concentration is after 18% mannitol dissolves, it is 0.4% active carbon that the conventional proportioning for preparing injection by galenic pharmacy adds concentration, boiled 20 minutes, and filtered, filtrate adds the injection water and is adjusted to original volume, the microporous filter membrane fine straining, filtrate is divided in the infusion bottle of packing into, 115 ℃, 30 minutes pressure sterilizings, microporous filter membrane fine straining under the aseptic condition, divide in the 10ml cillin bottle of packing into, every dress 5ml, lyophilization, Zha Gai, quality inspection gets lyophilized injectable powder of the present invention.
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| CN103948783A (en) * | 2014-04-30 | 2014-07-30 | 王莉 | Pharmaceutical composition for preventing and/or treating cerebral infarction |
| CN115919929A (en) * | 2022-12-01 | 2023-04-07 | 金华寿仙谷药业有限公司 | Antioxidant traditional Chinese medicine composition and application thereof |
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| CN1583041A (en) * | 2004-06-02 | 2005-02-23 | 曹恒信 | Tongmai drops and their preparation |
| CN1611247A (en) * | 2003-10-28 | 2005-05-04 | 吴晓枫 | Chinese medicine injection liquid for treating cerebro-cardio vascular disease and diabetes |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1611247A (en) * | 2003-10-28 | 2005-05-04 | 吴晓枫 | Chinese medicine injection liquid for treating cerebro-cardio vascular disease and diabetes |
| CN1583041A (en) * | 2004-06-02 | 2005-02-23 | 曹恒信 | Tongmai drops and their preparation |
Non-Patent Citations (1)
| Title |
|---|
| 卫生部药品标准中药成方制剂第四册. 卫生部，169，卫生部. 1991 * |
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