CN101168539B - Method for extracting salvianolic acid B - Google Patents
Method for extracting salvianolic acid B Download PDFInfo
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- CN101168539B CN101168539B CN 200710144744 CN200710144744A CN101168539B CN 101168539 B CN101168539 B CN 101168539B CN 200710144744 CN200710144744 CN 200710144744 CN 200710144744 A CN200710144744 A CN 200710144744A CN 101168539 B CN101168539 B CN 101168539B
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Abstract
The invention discloses an extraction method of salvianolic acid B and hydroxy safflor yellow A as well as a preparation method of danhong for injection, and relates to the extraction method of the salvianolic acid and the hydroxy safflor yellow as well as the preparation method of the danhong preparation. The invention solves the problems that the extraction of the salvianolic acid B and the hydroxy safflor yellow A is not thorough, and the transportation and the storage of the prepared danhong injection are inconvenient. The salvianolic acid B of the invention is extracted according to the following steps: backflow, acidification and adsorption; thus the salvianolic acid B is obtained. The hydroxy safflor yellow A of the invention is extracted according to the following steps: boil out, acidification and adsorption; thus the hydroxy safflor yellow A is obtained. The danhong for injection is extracted according to the following steps: compounding ratio and standing, filtrating and lyophylization; thus the danhong for injection of the invention is obtained. The extraction rate of the salvianolic acid B and the hydroxy safflor yellow A of the invention is high, and the transportation and the storage of the prepared danhong injection are convenient.
Description
Technical field
The present invention relates to the extracting method of salvianolic acid.
Background technology
The DANHONG ZHUSHEYE of in " national drug standards (trying) promulgation part ", being announced be a kind of fine clinical efficacy is arranged, use existing two more than ten years history traditional Chinese patent medicine, be mainly used in the important emergent treatment of cardiovascular and cerebrovascular, common have coronary heart disease, a stenocardia etc., and the obstruction of qi in the chest that can also treat due to the extravasated blood obturation is an apoplexy etc.The method of existing salvianolic acid B that extracts and hydroxyl radical carthamin yellow carthamus A exists to be extracted not thorough to salvianolic acid B and hydroxyl radical carthamin yellow carthamus A.And adopt technique for extracting mixed in existing DANHONG ZHUSHEYE (ministerial standard WS-11220 (ZD-1220)-2002) preparation process, the active constituent content that extracts is low, and what prepare is aqueous injection, because it is oxygenous in the aqueous solution, so easy oxygenolysis of salvianolic acid B wherein and hydroxyl radical carthamin yellow carthamus A, color can deepen and produce precipitation after the standing storage, poor stability, and the transportation of aqueous injection and storage are inconvenient.
Summary of the invention
The present invention thoroughly and in the preparation DANHONG ZHUSHEYE process does not adopt technique for extracting mixed for the extracting method that solves existing salvianolic acid B and hydroxyl radical carthamin yellow carthamus A exists to salvianolic acid B and hydroxyl radical carthamin yellow carthamus A extraction, the active constituent content that extracts is low, color can deepen and produce precipitation after the standing storage, and the problem of the poor stability of the DANHONG ZHUSHEYE of preparing, transportation and storage inconvenience, and provide the extracting method of salvianolic acid B.
The extracting method of salvianolic acid B of the present invention carries out as follows: one, add 8～18 times of salvia piece quality to the red rooted salvia medicine materical crude slice earlier, mass concentration is that 35%～60% ethanol and volume are ethanol volume 1 ‰～4 ‰, concentration is the salt acid soak 1～2 hour of 1mol/L, refluxing extraction is 1.5～3 hours then, leach danshen solution, add 6～16 times of dregs of a decoction quality to the dregs of a decoction again, mass concentration is that 35%～60% ethanol and volume are ethanol volume 1 ‰～4 ‰, concentration is the hydrochloric acid of 1mol/L, refluxing extraction 1～2 hour, leach danshen solution, the danshen solution that merges extracted twice, to become relative density be 1.16～1.24 the clear cream of the red sage root to concentrating under reduced pressure again; Two, the water that adds 3～5 times of the clear cream weight of the red sage root in the clear cream of the red sage root, limit add the waterside and stir, and leave standstill under the room temperature 12～24 hours, refilter, and red sage root filtrate is 2.8～3.2 with the salt acid for adjusting pH value, obtains red sage root acidifying filtrate; Three, with macroporous adsorptive resins be 2.8～3.2 hcl acidifying with the pH value, the red sage root acidifying filtrate that step 2 is obtained is splined on the macroporous absorption post, with 5～7 times of resin volume pH values is 2.8～3.2 hydrochloric acid flushing impurity, be 35%～55% ethanol elution again with mass concentration, collect the red sage root elutriant of 1.5～2.5 times of resin volumes, the pH value of regulating red sage root elutriant with sodium hydroxide solution is 4.0～4.2, is evaporated to relative density and is 1.08～1.25 the clear cream of salvianolic acid; Promptly obtain salvianolic acid B.
The extracting method of hydroxyl radical carthamin yellow carthamus A of the present invention carries out as follows: one, earlier add the water logging bubble 20～40 minutes that quality is 16～20 times of safflower quality to safflower, decocted 20～40 minutes, leach the safflower soup, adding quality to the dregs of a decoction again is the water of 14～18 times of dregs of a decoction quality, decocted 20～40 minutes, leach the safflower soup, merge the safflower soup that leaches for twice, be cooled to room temperature; Two, the safflower soup left standstill 6～24 hours with salt acid for adjusting pH value to 1.9～2.2, refiltered, and obtained safflower acidifying filtrate; Three, with macroporous adsorptive resins be 1.9～2.2 hcl acidifying with the pH value, the safflower acidifying filtrate that step 2 is obtained is splined on the macroporous absorption post, with 6～8 times of resin volume pH values is 1.9～2.2 hydrochloric acid flushing impurity, be 8%～15% ethanol elution again with mass concentration, collect the safflower elutriant of 4.5～6.0 times of resin volumes, the pH value of regulating the safflower elutriant with sodium hydroxide solution is 4.0～4.2, is evaporated to relative density and is 1.08～1.25 the clear cream of Hydroxy Carthamus yellow; Promptly get hydroxyl radical carthamin yellow carthamus A.
The method that salvianolic acid B that extracts with aforesaid method and hydroxyl radical carthamin yellow carthamus A prepare Danhong for injection is carried out as follows: be salvianolic acid B and the hydroxyl radical carthamin yellow carthamus A that 3: 1 ratio aforesaid method extracts according to red sage root and Flos Carthami medicinal material weight ratio one,, it is 75%～99% that adding water for injection makes the water content that is mixed with red red soup and makes red red soup, stirring the pH value of regulating red red soup with sodium hydroxide solution in the back is 4.0～4.5, leaves standstill under 0～10 ℃ condition 12～48 hours; Two, earlier behind the filtering with microporous membrane with 0.22 μ m or 0.45 μ m, the Millipore ultrafiltration post that passes through the molecular weight that dams again and be 30K or 50K carries out ultrafiltration, with red filtrate packing of pellet and freeze-drying; Promptly obtain Danhong for injection.
The preparation method of the extracting method of salvianolic acid B of the present invention, the extracting method of hydroxyl radical carthamin yellow carthamus A and Danhong for injection has the following advantages:
1, the red sage root adopts 35%～60% ethanol to add the hydrochloric acid reflux extraction, has prevented the decomposes of effective constituent salvianolic acid B in the aqueous solution effectively, and extracted cmpletely that extraction yield is 85%～95%, is 1.5～3 times of existing method.
2, the hydroxyl radical carthamin yellow carthamus A extraction yield is 85%～95%, is 1.1～2.4 times of existing method.
3, the refining water lotion of taking of the red sage root filters earlier, and back adjust pH passes through macroporous adsorptive resins again, has prevented that effectively effective constituent from being filtered, and makes the content of effective monomer component salvianolic acid B and the rate of transform all more than 80%.
4, when the macroporous resin of red sage root and Flos Carthami is refining, the method for sour water flushing impurity has reduced loss of active ingredients effectively when all having adopted macroporous resin to use preceding acidifying, wash-out.
5, the macroporous adsorbent resin cost that preparation technology of the present invention adopted is low, in this preparation process the adsorption rate height, elute effect is good, regeneration is easy, long service life, and is reusable at least more than 20 times.
6, the molecular weight that dams that preparation technology of the present invention adopted is that the Millipore ultrafiltration post of 30K or 50K both can thoroughly have been removed impurity, bacterium and the pyrogen in the soup effectively, can keep effective constituent effectively again.
7, preparation technology's operating process of the present invention is fairly simple, easy, efficient is high, cost is low, is particularly suitable for suitability for industrialized production.
8, salvianolic acid B and two monomer effective constituent of hydroxyl radical carthamin yellow carthamus A index are adopted in product active constituent content control of the present invention, adopt high effective liquid chromatography for measuring, measuring method is accurate, simple, stable, make that effective constituent detection and control in process of production is all highly stable, the two content is aggregated in more than 60%.
9, the present invention extracts salvianolic acid B and hydroxyl radical carthamin yellow carthamus A respectively according to their differences of physical properties, salvianolic acid B that extracts and hydroxyl radical carthamin yellow carthamus A be the extraction yield height not only, the purity of salvianolic acid B has also improved 40%～100%, and hydroxyl radical carthamin yellow carthamus A purity has also improved 30%～50%.
10, the red injection water injection of existing pellet, because oxygenous in the aqueous solution, so the easy oxygenolysis of salvianolic acid B wherein and hydroxyl radical carthamin yellow carthamus A, color can deepen and produce precipitation after the standing storage, drug failure, and also aqueous injection is not easy to transportation.
Embodiment
Embodiment one: the extracting method of the salvianolic acid B of present embodiment carries out as follows: one, add 8～18 times of salvia piece quality to the red rooted salvia medicine materical crude slice earlier, mass concentration is that 35%～60% ethanol and volume are ethanol volume 1 ‰～4 ‰, concentration is the salt acid soak 1～2 hour of 1mol/L, refluxing extraction is 1.5～3 hours then, leach danshen solution, add 6～16 times of dregs of a decoction quality to the dregs of a decoction again, mass concentration is that 35%～60% ethanol and volume are ethanol volume 1 ‰～4 ‰, concentration is the hydrochloric acid of 1mol/L, refluxing extraction 1～2 hour, leach danshen solution, the danshen solution that merges extracted twice, to become relative density be 1.16～1.24 the clear cream of the red sage root to concentrating under reduced pressure again; Two, the water that adds 3～5 times of the clear cream weight of the red sage root in the clear cream of the red sage root, limit add the waterside and stir, and leave standstill under the room temperature 12～24 hours, refilter, and red sage root filtrate is 2.8～3.2 with the salt acid for adjusting pH value, obtains red sage root acidifying filtrate; Three, with macroporous adsorptive resins be 2.8～3.2 hcl acidifying with the pH value, the red sage root acidifying filtrate that step 2 is obtained is splined on the macroporous absorption post, with 5～7 times of resin volume pH values is 2.8～3.2 hydrochloric acid flushing impurity, be 35%～55% ethanol elution again with mass concentration, collect the red sage root elutriant of 1.5～2.5 times of resin volumes, the pH value of regulating red sage root elutriant with sodium hydroxide solution is 4.0～4.2, is evaporated to relative density and is 1.08～1.25 the clear cream of salvianolic acid; Promptly obtain salvianolic acid B.
In the present embodiment relative density of the clear cream of the red sage root of step 1 be under 40～60 ℃ the condition the clear cream of the red sage root with respect to the density of water; The relative density of the clear cream of salvianolic acid of step 3 be under 40～60 ℃ the condition the clear cream of salvianolic acid with respect to the density of water.
Embodiment two: the difference of present embodiment and embodiment one is: earlier adding 12～14 times of salvia piece quality, mass concentration to the red rooted salvia medicine materical crude slice in the step 1 is that 45%～55% ethanol and volume are that ethanol volume 2 ‰～3 ‰, concentration are the salt acid soak 1.2～1.8 hours of 1mol/L, and refluxing extraction is 2～2.5 hours then.Other step and parameter are identical with embodiment one.
Embodiment three: the difference of present embodiment and embodiment one is: earlier adding 13 times of salvia piece quality, mass concentration to the red rooted salvia medicine materical crude slice in the step 1 is that 50% ethanol and volume are that ethanol volume 2.5 ‰, concentration are the salt acid soak 1.5 hours of 1mol/L, and refluxing extraction is 2.2 hours then.Other step and parameter are identical with embodiment one.
Embodiment four: the difference of present embodiment and embodiment one is: adding 10～12 times of dregs of a decoction quality, mass concentration to the dregs of a decoction again in the step 1 is that 48%～52% ethanol and volume are that ethanol volume 1.5 ‰～3.5 ‰, concentration are the hydrochloric acid of 1mol/L, refluxing extraction 1.2～1.8 hours.Other step and parameter are identical with embodiment one.
Embodiment five: the difference of present embodiment and embodiment one is: adding 11 times of dregs of a decoction quality, mass concentration to the dregs of a decoction again in the step 1 is that 51% ethanol and volume are that ethanol volume 2 ‰, concentration are the hydrochloric acid of 1mol/L, refluxing extraction 1.5 hours.Other step and parameter are identical with embodiment one.
Embodiment six: present embodiment with the difference of embodiment one is: to become relative density be 1.1 8～1.22 the clear cream of the red sage root to the danshen solution concentrating under reduced pressure in the step 1.Other step and parameter are identical with embodiment one.
Embodiment seven: present embodiment with the difference of embodiment one is: to become relative density be 1.20 the clear cream of the red sage root to the danshen solution concentrating under reduced pressure in the step 1.Other step and parameter are identical with embodiment one.
Embodiment eight: the difference of present embodiment and embodiment one is: the water that adds 4 times of the clear cream weight of the red sage root in the step 2 in the clear cream of the red sage root.Other step and parameter are identical with embodiment one.
Embodiment nine: the difference of present embodiment and embodiment one is: the clear cream of the red sage root left standstill 15～21 hours after adding water in the step 2.Other step and parameter are identical with embodiment one.
Embodiment ten: the difference of present embodiment and embodiment one is: the clear cream of the red sage root left standstill 17～19 hours after adding water in the step 2.Other step and parameter are identical with embodiment one.
Embodiment 11: the difference of present embodiment and embodiment one is: the clear cream of the red sage root left standstill 18 hours after adding water in the step 2.Other step and parameter are identical with embodiment one.
Embodiment 12: the difference of present embodiment and embodiment one is: red sage root filtrate is 3.0 with the salt acid for adjusting pH value in the step 2.Other step and parameter are identical with embodiment one.
The mass concentration of hydrochloric acid is 3%～10% in the present embodiment.
Embodiment 13: the difference of present embodiment and embodiment one is: in the step 3 be 3.0 hcl acidifying with the pH value with macroporous adsorptive resins.Other step and parameter are identical with embodiment one.
Embodiment 14: the difference of present embodiment and embodiment one is: the hydrochloric acid with 6 times of column volume pH values 3.0 in the step 3 washes impurity.Other step and parameter are identical with embodiment one.
Embodiment 15: the difference of present embodiment and embodiment one is: be 40%～50% ethanol elution again with mass concentration in the step 3.Other step and parameter are identical with embodiment one.
Embodiment 16: the difference of present embodiment and embodiment one is: be 55% ethanol elution again with mass concentration in the step 3.Other step and parameter are identical with embodiment one.
Embodiment 17: the difference of present embodiment and embodiment one is: the pH value with sodium hydroxide solution adjusting red sage root elutriant in the step 3 is 4.1.Other step and parameter are identical with embodiment one.
The mass concentration of sodium hydroxide solution is 3%～10% in the present embodiment.
Embodiment 18: the difference of present embodiment and embodiment one is: be evaporated to relative density in the step 3 and be 1.16～1.20 the clear cream of salvianolic acid.Other step and parameter are identical with embodiment one.
Embodiment 19: the difference of present embodiment and embodiment one is: be evaporated to relative density in the step 3 and be 1.18 the clear cream of salvianolic acid.Other step and parameter are identical with embodiment one.
Embodiment 20: the difference of present embodiment and embodiment one is: the model of macroporous adsorbent resin is HZ801, AB-8 or D101 in the step 3.Other step and parameter are identical with embodiment one.
Embodiment 21: the difference of present embodiment and embodiment one is: go up the sample flow velocity in the step 3 and be 1 resin volume/hour～1.5 resin volumes/hour.Other step and parameter are identical with embodiment one.
Embodiment 22: the difference of present embodiment and embodiment one is: in the step 3 flow velocity of flushing impurity be 1 resin volume/hour～1.5 resin volumes/hour.Other step and parameter are identical with embodiment one.
Embodiment 23: the difference of present embodiment and embodiment one is: in the step 3 elution flow rate be 1 resin volume/hour～1.5 resin volumes/hour.Other step and parameter are identical with embodiment one.
Embodiment 24: the difference of present embodiment and embodiment one is: the red sage root elutriant of collecting 2 times of resin volumes in the step 3.Other step and parameter are identical with embodiment one.
Embodiment 25: the difference of present embodiment and embodiment one is: the weight of macroporous adsorbent resin is 2 times of red sage root weight in the step 3.Other step and parameter are identical with embodiment one.
Embodiment 26: the extracting method of the hydroxyl radical carthamin yellow carthamus A of present embodiment carries out as follows: one, earlier add the water logging bubble 20～40 minutes that quality is 16～20 times of safflower quality to safflower, decocted 20～40 minutes, leach the safflower soup, adding quality to the dregs of a decoction again is the water of 14～18 times of dregs of a decoction quality, decocted 20～40 minutes, leach the safflower soup, merge the safflower soup that leaches for twice, be cooled to room temperature; Two, the safflower soup left standstill 6～24 hours with salt acid for adjusting pH value to 1.9～2.2, refiltered, and obtained safflower acidifying filtrate; Three, with macroporous adsorptive resins be 1.9～2.2 hcl acidifying with the pH value, the safflower acidifying filtrate that step 2 is obtained is splined on the macroporous absorption post, with 6～8 times of resin volume pH values is 1.9～2.2 hydrochloric acid flushing impurity, be 8%～15% ethanol elution again with mass concentration, collect the safflower elutriant of 4.5～6.0 times of resin volumes, the pH value of regulating the safflower elutriant with sodium hydroxide solution is 4.0～4.2, is evaporated to relative density and is 1.08～1.25 the clear cream of Hydroxy Carthamus yellow; Promptly get hydroxyl radical carthamin yellow carthamus A.
In the present embodiment relative density of the clear cream of Hydroxy Carthamus yellow of step 3 be under 40～60 ℃ the condition the clear cream of Hydroxy Carthamus yellow with respect to the density of water.
Embodiment 27: the difference of present embodiment and embodiment 26 is: earlier add the water logging bubble 25～35 minutes that quality is 17～19 times of safflower quality to safflower in the step 1, decocted 25～35 minutes.Other step and parameter are identical with embodiment 26.
Embodiment 28: the difference of present embodiment and embodiment 26 is: earlier add the water logging bubble 30 minutes that quality is 18 times of safflower quality to safflower in the step 1, decocted 30 minutes.
Other step and parameter are identical with embodiment 26.
Embodiment 29: the difference of present embodiment and embodiment 26 is: adding quality to the dregs of a decoction again in the step 1 is the water of 15～17 times of dregs of a decoction quality, decocts 25～30 minutes.Other step and parameter are identical with embodiment 26.
Embodiment 30: the difference of present embodiment and embodiment 26 is: adding quality to the dregs of a decoction again in the step 1 is 16 times of dregs of a decoction quality, decocts 28 minutes.Other step and parameter are identical with embodiment 26.
The embodiment hentriaconta-: the difference of present embodiment and embodiment 26 is: the safflower soup left standstill 10～20 hours with salt acid for adjusting pH value to 2.0～2.1 in the step 2.Other step and parameter are identical with embodiment 26.
Embodiment 32: the difference of present embodiment and embodiment 26 is: the safflower soup left standstill 15 hours with salt acid for adjusting pH value to 2.0 in the step 2.Other step and parameter are identical with embodiment 26.
The hydrochloric acid mass concentration of present embodiment is 3%～10%.
Embodiment 33: the difference of present embodiment and embodiment 26 is: in the step 3 be 2.1 hcl acidifying with the pH value with macroporous adsorptive resins.Other step and parameter are identical with embodiment 26.
Embodiment 34: the difference of present embodiment and embodiment 26 is: the hydrochloric acid with 7 times of resin volume pH values 2.0 in the step 3 washes impurity.Other step and parameter are identical with embodiment 26.
Embodiment 35: the difference of present embodiment and embodiment 26 is: be 10%～12% ethanol elution again with mass concentration in the step 3.Other step and parameter are identical with embodiment 26.
Embodiment 36: the difference of present embodiment and embodiment 26 is: be 11% ethanol elution again with mass concentration in the step 3.Other step and parameter are identical with embodiment 26.
Embodiment 37: the difference of present embodiment and embodiment 26 is: the pH value with sodium hydroxide solution adjusting safflower elutriant in the step 3 is 4.1.Other step and parameter are identical with embodiment 26.
The mass concentration of the sodium hydroxide solution of present embodiment is 3%～10%.
Embodiment 38: the difference of present embodiment and embodiment 26 is: be evaporated to relative density in the step 3 and be 1.12～1.20 the clear cream of Hydroxy Carthamus yellow.Other step and parameter are identical with embodiment 26.
Embodiment 39: the difference of present embodiment and embodiment 26 is: be evaporated to relative density in the step 3 and be 1.16 the clear cream of Hydroxy Carthamus yellow.Other step and parameter are identical with embodiment 26.
Embodiment 40: the difference of present embodiment and embodiment 26 is: the model of macroporous adsorbent resin is X-5 or D4020 in the step 3.Other step and parameter are identical with embodiment 26.
Embodiment 41: the difference of present embodiment and embodiment 26 is: go up the sample flow velocity in the step 3 and be 1 resin volume/hour～1.5 resin volumes/hour.Other step and parameter are identical with embodiment 26.
Embodiment 42: the difference of present embodiment and embodiment 26 is: in the step 3 flow velocity of flushing impurity be 1 resin volume/hour～1.5 resin volumes/hour.Other step and parameter are identical with embodiment 26.
Embodiment 43: the difference of present embodiment and embodiment 26 is: in the step 3 elution flow rate be 1 resin volume/hour～1.5 resin volumes/hour.Other step and parameter are identical with embodiment 26.
Embodiment 44: the difference of present embodiment and embodiment 26 is: the safflower elutriant of collecting 5 times of resin volumes in the step 3.Other step and parameter are identical with embodiment 26.
Embodiment 45: the difference of present embodiment and embodiment 26 is: the weight of macroporous adsorbent resin is 4 times of safflower weight in the step 3.Other step and parameter are identical with embodiment 26.
Embodiment 46: the hydroxyl radical carthamin yellow carthamus A that present embodiment is extracted with the salvianolic acid B and the embodiment 26 of embodiment one extraction prepares the method for Danhong for injection to carry out as follows: one, according to red sage root and Flos Carthami medicinal material weight ratio is that 3: 1 ratio is got the hydroxyl radical carthamin yellow carthamus A that salvianolic acid B that embodiment one extracts and embodiment 26 are extracted, it is 75%～99% that adding water for injection makes the water content that is mixed with red red soup and makes red red soup, stirring the pH value of regulating red red soup with sodium hydroxide solution in the back is 4.0～4.5, leaves standstill under 0～10 ℃ condition 12～48 hours; Two, earlier behind the filtering with microporous membrane with 0.22 μ m or 0.45 μ m, the Millipore ultrafiltration post that passes through the molecular weight that dams again and be 30K or 50K carries out ultrafiltration, with red filtrate packing of pellet and freeze-drying; Promptly obtain Danhong for injection.
Embodiment 47: the difference of present embodiment and embodiment 46 is: the water content that makes red red soup in the step 1 is 80%～95%.Other step and parameter are identical with embodiment 46.
Embodiment 48: the difference of present embodiment and embodiment 46 is: the water content that makes red red soup in the step 1 is 85%～90%.Other step and parameter are identical with embodiment 46.
Embodiment 49: the difference of present embodiment and embodiment 46 is: the water content that makes red red soup in the step 1 is 88%.Other step and parameter are identical with embodiment 46.
Embodiment 50: the difference of present embodiment and embodiment 46 is: the pH value of regulating red red soup with sodium hydroxide solution in the step 1 is 4.2～4.4.Other step and parameter are identical with embodiment 46.
The mass concentration of the sodium hydroxide solution of present embodiment is 3%～10%.
Embodiment 51: the difference of present embodiment and embodiment 46 is: the pH value of regulating red red soup with sodium hydroxide solution in the step 1 is 4.3.Other step and parameter are identical with embodiment 46.
Embodiment 52: the difference of present embodiment and embodiment 46 is: left standstill under 2～8 ℃ condition 18～30 hours in the step 1.Other step and parameter are identical with embodiment 46.
Embodiment 53: the difference of present embodiment and embodiment 46 is: left standstill under 4～6 ℃ condition 25～28 hours in the step 1.Other step and parameter are identical with embodiment 46.
Embodiment 54: the difference of present embodiment and embodiment 46 is: left standstill under 5 ℃ condition 27 hours in the step 1.Other step and parameter are identical with embodiment 46.
Embodiment 55: the preparation method of the Danhong for injection of present embodiment carries out as follows: one, extract salvianolic acid B; Two, extract hydroxyl radical carthamin yellow carthamus A; Three, be that 3: 1 ratio is got the hydroxyl radical carthamin yellow carthamus A that salvianolic acid B that step 1 extracts and step 2 are extracted according to red sage root and Flos Carthami medicinal material weight ratio, it is 85% that adding water for injection makes the water content that is mixed with red red soup and makes red red soup, stirring the pH value of regulating red red soup with sodium hydroxide solution in the back is 4.2, leaves standstill under 5 ℃ condition 40 hours; Four, earlier behind the filtering with microporous membrane with 0.22 μ m, the Millipore ultrafiltration post that passes through the molecular weight that dams again and be 30K carries out ultrafiltration, with red filtrate packing of pellet and freeze-drying; Promptly obtain Danhong for injection.
The method that the present embodiment step 1 is extracted salvianolic acid B is: one, add 16 times of salvia piece quality to the red rooted salvia medicine materical crude slice earlier, mass concentration is that 50% ethanol and volume are ethanol volume 2 ‰, concentration is the salt acid soak 1.5 hours of lmol/L, refluxing extraction is 2 hours then, leach danshen solution, add 12 times of dregs of a decoction quality to the dregs of a decoction again, mass concentration is that 55% ethanol and volume are ethanol volume 2 ‰, concentration is the hydrochloric acid of lmol/L, refluxing extraction 1.5 hours, leach danshen solution, the danshen solution that merges extracted twice, to become relative density be 1.18 the clear cream of the red sage root to concentrating under reduced pressure again; Two, the water that adds 4 times of the clear cream weight of the red sage root in the clear cream of the red sage root, limit add the waterside and stir, and leave standstill under the room temperature 20 hours, refilter, and red sage root filtrate is 2.9 with the salt acid for adjusting pH value, obtains red sage root acidifying filtrate; Three, 's 2.9 hcl acidifying with macroporous adsorptive resins with the pH value, the red sage root acidifying filtrate that step 2 is obtained is splined on the macroporous absorption post, last sample flow velocity be 1 resin volume/hour～1.5 resin volumes/hour, hydrochloric acid flushing impurity with 6 times of resin volume pH values 2.9, elution flow rate be 1 resin volume/hour～1.5 resin volumes/hour, use 2 times of column volumes again, mass concentration is 50% ethanol elution, elution flow rate be 1 resin volume/hour～1.5 resin volumes/hour, collect the red sage root elutriant of 2.0 times of resin volumes, the pH value of regulating red sage root elutriant with sodium hydroxide solution is 4.1, is evaporated to relative density and is 1.16 the clear cream of salvianolic acid; Promptly obtain salvianolic acid B.
The method that the present embodiment step 2 is extracted hydroxyl radical carthamin yellow carthamus A is: one, add the water logging bubble 30 minutes that quality is 18 times of safflower quality to safflower earlier, decocted 30 minutes, leach the safflower soup, add the water logging bubble 35 minutes that quality is 16 times of a dregs of a decoction quality to the dregs of a decoction again, decocted 35 minutes, leach the safflower soup, merge the safflower soup that leaches for twice, be cooled to room temperature; Two, the safflower soup left standstill 18 hours with salt acid for adjusting pH value to 2.0, refiltered, and obtained safflower acidifying filtrate; Three, 's 2.0 hcl acidifying with macroporous adsorptive resins with the pH value, the safflower acidifying filtrate that step 2 is obtained be splined on go up on the macroporous absorption post sample flow velocity be 1 resin volume/hour～1.5 resin volumes/hour, hydrochloric acid flushing impurity with 7 times of column volume pH values 2.0, elution flow rate be 1 resin volume/hour～1.5 resin volumes/hour, be 12% ethanol elution again with mass concentration, elution flow rate be 1 resin volume/hour～1.5 resin volumes/hour, collect the safflower elutriant of 5.0 times of resin volumes, the pH value of regulating the safflower elutriant with sodium hydroxide solution is 4.1, is evaporated to relative density and is 1.16 the clear cream of Hydroxy Carthamus yellow; Promptly get hydroxyl radical carthamin yellow carthamus A.
The Danhong for injection that present embodiment is made carries out following pharmacodynamics test research:
1, Danhong for injection is to dog myocardial infarction model and hemodynamic influence due to the coronary ligation
Purpose: observe the Danhong for injection administration to dog myocardial infarction and hemodynamic influence due to the coronary ligation.
Method: get 30 of healthy adult domesticated dogs, male and female half and half, body weight 13.6 ± 1.25kg is divided into 5 groups at random by body weight, is respectively model group, the basic, normal, high dosage group of Danhong for injection and DANHONG ZHUSHEYE positive controls.During test with dog with vetanarcol 30mgkg
-1Anesthesia, trachea cannula connects the breathing apparatus, and is subcutaneous with needle electrode insertion four limbs and front.Open chest along left border of sternum 3-4 intercostal, fully expose heart.Cut off pericardium and make the pericardium hammock.With 1/3 place on No. 0 silk thread ligation ramus descendens anterior arteriae coronariae sinistrae, each group administration respectively behind the ligation 15min, the basic, normal, high dosage group of Danhong for injection dosage is respectively 3mgkg
-1, 6mgkg
-1, and 12mgkg
-1, positive controls gives DANHONG ZHUSHEYE, and dosage is 4mlkg
-1, model group gives 0.9% sodium chloride injection of respective volume.With 15min, 30min, 60min, 90min, 120min, 180min epicardial electrogram EKG before the ligation of polygraph record, behind the stable back of ligation, administration, and calculate ∑-ST and NST.With 15min, 30min, 60min, 90min, 120min, 180min haemodynamics physiological parameter before polygraph, the ligation of doppler flowmeter detection record, after the stable back of ligation and the administration.Separate carotid artery, extract arterial blood, get coronary vein blood through external jugular vein intubate to coronary sinus vein and survey the oxygen content of blood with oximeter.The 2ml that draws blood simultaneously measures the content of serum lactic dehydrogenase (SLDH) (LDH), creatine phosphokinase (CK) with automatic biochemistry analyzer.3h behind the medicine, dirty the weighing of coring, myocardial infarction area is measured in TTC dyeing.
The result: (∑-ST) and myocardial ischemia scope (NST): behind the coronary ligation, each organizes dog ST section and obviously raises (1) degree of myocardial ischemia.With the same period model group compare, ∑-ST and NST all have reduction behind each dosage group of Danhong for injection and the positive control drug DANHONG ZHUSHEYE medicine, behind the high dose group medicine behind ∑-ST of 30～90min, the medicine NST of 60～120min significantly reduce, with model group significant difference (P＜0.05) is arranged relatively, though other times point no difference of science of statistics all has reduction trend; In behind the dosage group medicine behind the ∑-ST, medicine of 60min the NST of 60～90min significantly reduce, with model group significant difference (P＜0.05) is arranged relatively; Low dose group ∑-ST and NST all have reduction trend, but no difference of science of statistics; 30min, 60～90min have obvious restraining effect to the increase of raising with NST of ∑-ST respectively behind the DANHONG ZHUSHEYE positive controls medicine, with model group significant difference (P＜0.05) are arranged relatively.Compare with positive controls, each time point ∑-ST and NST there was no significant difference behind the basic, normal, high dosage group of the Danhong for injection medicine, but the Danhong for injection high dosage has further reduction ∑-ST and NST trend, and concrete experimental data is as shown in Table 1 and Table 2.
Table 1 Danhong for injection to acute myocardial ischemia epicardial electrogram ∑-ST (
, influence mv)
Annotate: 1. n=6; 2.
*P＜0.05,
*Compare with model group P＜0.01.
Table 2 Danhong for injection is to coronary artery ligation dog myocardial ischemia scope
Influence
Annotate: 1. n=6; 2.
*P＜0.05,
*Compare with model group P＜0.01; 3.
ΔP＜0.05,
The Δ ΔCompare with the preceding value of medicine P＜0.01.
(2) myocardial infarct size: 195min respectively organizes dog and tangible myocardial infarction all occurs behind the coronary ligation, compares with model group, and each dosage of positive control drug and Danhong for injection all obviously dwindles myocardial infarct size.Dosage group myocardial infarction district area accounts for left ventricle and whole-heartedly ratio is low by 2.68% and 6.36% than model group respectively in the Danhong for injection, and high dose group reduces by 3.06% and 6.55% respectively, and obvious statistical significance (P＜0.05) is all arranged; The Danhong for injection low dose group reduces by 1.29% and 5.15% respectively, and reduction trend is arranged, but does not have tangible statistical significance; Positive controls myocardial infarction district area accounts for left ventricle and ratio whole-heartedly is low by 2.18% and 5.75% than model group respectively, and obvious statistical significance (P＜0.05) is arranged.Compare with positive controls, the basic, normal, high dosage group of Danhong for injection myocardial infarction district area there was no significant difference, but the Danhong for injection high dosage has further reduction myocardial infarction district area trend, and concrete experimental data is as shown in table 3.
Table 3 Danhong for injection is to coronary artery ligation dog myocardial infarct size
Influence
Annotate: 1. n=6; 2.
*P＜0.05,
*Compare with model group P＜0.01.
(3) creatine kinase (CK) and serum lactic dehydrogenase (LDH): each treated animal serum CK and LDH obviously raise before than ligation after the coronary artery ligation.Though the 180min seroenzyme also has rising behind each dosage group of Danhong for injection and the positive controls medicine, the amplitude that raises all has reduction in various degree.Compare Danhong for injection 3mgkg with model group
-1180min has certain inhibition trend to the rising of seroenzyme behind the medicine, but there was no significant difference; 6mgkg
-1And 12mgkg
-1Administration can obviously suppress the activity rising (P＜0.05 or P＜0.01) of Serum LDH, CK.Also can significantly suppress the rising of Serum LDH, CK behind the DANHONG ZHUSHEYE medicine, significant difference (P＜0.05) relatively be arranged with model group.Compare with positive controls, the basic, normal, high dosage group of Danhong for injection suppresses the activity rising effect there was no significant difference of Serum LDH, CK, but the Danhong for injection high dose group can further suppress the activity rising degree of LDH, CK, and concrete experimental data is shown in table 4 and table 5.
Table 4 Danhong for injection to coronary artery ligation dog serum serum lactic dehydrogenase (
, influence U/L)
Annotate: 1. n=6; 2.
*P＜0.05,
*Compare with model group P＜0.01.
Table 5 Danhong for injection to coronary artery ligation dog serum creatine creatase (
, influence U/L)
Annotate: 1. n=6; 2.
*P＜0.05,
*Compare with model group P＜0.01.
(4) left ventricular pressure (LVP), left ventricular end diastolic are pressed (LVEDP) and the maximum rate of change (dp/dt of left ventricular pressure
Max): compare with model group, 15min administration after each dosage of Danhong for injection and the positive controls ligation, LVP does not see variation; For LVEDP, middle and high dosage group of Danhong for injection and the DANHONG ZHUSHEYE positive controls left ventricular diastolic end voltage rise height that decline causes to myocardial contractility has significant reduction effect, 90～180min and model group relatively have significant difference (P＜0.05 or P＜0.01) behind the medicine, after the administration of Danhong for injection low dosage left ventricular diastolic end voltage rise height there is certain inhibition trend, but there was no significant difference, concrete experimental data is shown in table 6, table 7 and table 8.
Table 6 Danhong for injection is to dog dp/dt
MaxInfluence
Annotate:
*P＜0.05,
*Compare with model group P＜0.01.
Table 7 Danhong for injection is to the influence of dog LVEDP
Annotate:
*P＜0.05,
*Compare with model group P＜0.01.
Table 8 Danhong for injection is to the influence of dog LVP
Annotate:
*P＜0.05,
*Compare with model group P＜0.01.
For dp/dt
Max, with model group relatively, middle and high dosage group of Danhong for injection and DANHONG ZHUSHEYE positive controls all have the increase effect, 90～180min and model group relatively have significant difference (P＜0.05) behind the medicine, after the administration of Danhong for injection low dosage to dp/dt
MaxIncrease trend is arranged, but there was no significant difference.
Compare the basic, normal, high dosage group of Danhong for injection LVP, LVEDP and dp/dt with positive controls
MaxDifference is not obvious, but Danhong for injection high dose group LVEDP, dp/dt
MaxFurther reduction and increase trend are arranged respectively.
(5) aorta flow (CO) and coronary flow (CAF): compare with model group, behind the middle and high dosed administration of Danhong for injection, velocity of variation behind anesthetized dog CO and the CAF medicine there is the increase effect, respectively at 60～120min behind the medicine and 60～180min significant difference (P＜0.05) is arranged, though other times point no difference of science of statistics all has increase trend; Behind the Danhong for injection low dosage medicine behind CO and the CAF medicine velocity of variation increase trend is also arranged, but no difference of science of statistics.Behind the DANHONG ZHUSHEYE positive controls medicine 90～120min to CO and CAF medicine after velocity of variation be significantly increased effect, with model group significant difference (P＜0.05) is arranged relatively.Compare with positive controls, the basic, normal, high dosage group of Danhong for injection CO, CAF there was no significant difference, but the Danhong for injection high dosage has further increase CO, CAF trend, and concrete experimental data is shown in table 9 and table 10.
Table 9 Danhong for injection is to the influence of dog CO
Annotate: 1. n=6; 2.
*P＜0.05,
*Compare with model group P＜0.01.
Table 10 Danhong for injection is to the influence of dog CAF
Annotate: 1. n=6; 2.
*P＜0.05,
*Compare with model group P＜0.01.
(6) arteriovenous blood oxygen content: compare with model group, the middle and high dosage group of Danhong for injection and DANHONG ZHUSHEYE positive controls 60min after administration begin, the atrerial venous oxygen content difference value obviously rises (P＜0.05 or P＜0.01), and wherein the Danhong for injection high dose group can reach 180min behind the medicine action time.The Danhong for injection low dose group has increase trend, but there was no significant difference.With positive controls relatively, each time point of the basic, normal, high dosage group of Danhong for injection atrerial venous oxygen content difference value there are no significant difference, but the Danhong for injection high dose group can further increase the atrerial venous oxygen content difference value, concrete experimental data is as shown in table 11.
Table 11 Danhong for injection is to the influence of dog atrerial venous oxygen content difference value
Annotate: 1. n=6; 2.
*P＜0.05,
*Compare with model group P＜0.01.
Conclusion: ∑-ST and NST raise behind (1) this trial model dog coronary ligation, and Serum LDH and CK are active to be increased, and the 195min myocardial infarction area is obvious after the ligation, illustrates that model prepares successfully.
(2) Danhong for injection has significant protective effect to the dog myocardial ischemia, has the cardiovascular function of improvement, stimulates circulation, increases effects such as atrerial venous oxygen content difference value, and its onset dosage is 3～6mgkg
-1Under this test conditions, positive control drug DANHONG ZHUSHEYE and Danhong for injection 6mgkg
-1∑-ST, NST that dog myocardial infarction is caused raise, and Serum LDH, the active increase of CK and LVEDP raise tangible reduction effect is arranged, to dp/dt
Max, CO, CAF and atrerial venous oxygen content difference value have the increase effect, with model group significant difference (P＜0.05) arranged more all respectively; Danhong for injection 3mgkg
-1Above index all there is certain effect trend, but not obvious with the model group comparing difference; Danhong for injection 12mgkg
-1Can further improve dog myocardial ischemia index and dp/dt
Max, CO, CAF and arteriovenous blood oxygen content.Increase Danhong for injection effect with dosage presents certain dose-effect relationship, and 30min begins onset behind the medicine, and effective drug duration can reach 180min behind the medicine.
(3) compare Danhong for injection 6mgkg with positive control drug
-1Action effect is suitable with it.Compare Danhong for injection 12mgkg with DANHONG ZHUSHEYE
-1Though to ∑-ST, NST rising, Serum LDH, CK actively increase, LVEDP raises reduction effect with to the increase effect no difference of science of statistics of dp/dtmax, CO, CAF and atrerial venous oxygen content difference value, degree has further improvement; Danhong for injection 3mgkg
-1Above index there is certain effect trend, compare there was no significant difference with DANHONG ZHUSHEYE, but the effect degree is lighter.
(4) by above pharmacodynamic study results suggest, the effective initial dose of Danhong for injection clinical application is about 1.5～3mgkg
-1, recommend clinical plan to be about 90～180mg with dosage in human body body weight 60kg.
2, Danhong for injection is to the influence of focal brain ischemia-reperfusion injury in rats model
Purpose: observe the provide protection of Danhong for injection administration to focal brain ischemia-reperfusion injury in rats.
Method: get healthy male Wistar rat, after adaptability is fed a week, be divided into 6 groups at random: sham operated rats, model group, the basic, normal, high dosage group of Danhong for injection, and positive controls by body weight.Adopt bolt line blocked method (MCAO) preparation focal brain ischemia-reperfusion injury in rats model in improved Zea Longa ' the s arteria cerebri media, and behind ischemic 1.5h, pour into again.In the process of the test, the MCAO operative failure, modeling gets nowhere and the interior dead animal of 24h is all given it up, and deficiency is scheduled to the number person according to stochastic sampling principle polishing animal and modeling again, and guarantees that the number of animals of every group of modeling success is 8.Sham operated rats gives respective volume 0.9% sodium chloride injection, and basic, normal, high group of dosage of Danhong for injection is respectively 10mgkg
-1, 20mgkg
-1And 40mgkg
-1, positive controls gives DANHONG ZHUSHEYE, and dosage is 12mlkg
-1Each group is respectively at cerebral ischemia tail vein injection relative medicine at once.After cerebral ischemia 1.5h poured into 24h again, each treated animal carried out neurologic check, and broken end is got brain and measure ischemic side cerebral tissue Infarction volume after TTC dyeing then.
Result: compare with sham operated rats, the neurologic impairment sign is obvious behind the model group focal cerebral ischemia in rats 1.5h reperfusion injury 24h, the visible significantly oyster white infarct kitchen range of ischemic side cerebral tissue naked eyes, and with oedema, ischemic vascellum laterale swelling simultaneously is more obvious, the visible significantly infarct kitchen range of TTC dyeing illustrates that model prepares successfully, satisfies this test requirements document.Compare with model group, the neurological dysfunction of positive controls and each medication group ischemical reperfusion injury rat all has improvement, and the cerebral infarction volume obviously dwindles, and ischemic side degree of cerebral edema alleviates.The positive controls brain infarction area reduces 12%, and obvious statistical significance (P＜0.05) is arranged.Low dose group cerebral infarction volume reduces 5%, but does not have tangible statistical significance; Middle dosage group cerebral infarction volume reduces by 14%, and high dose group reduces by 18%, two group all obvious statistical significance (P＜0.01).With positive controls relatively, low, middle dosage group no significant difference, high dose group has significant difference (P＜0.05), and is more effective than Danhong Injection in Treating, concrete experimental data sees Table 12 and table 13.
The influence that table 12 Danhong for injection changes the rat neuroethology
Annotate: n=8;
*P＜0.05,
*Compare with model group P＜0.01.
Table 13 Danhong for injection is to the influence of rat cerebral infarction area
Annotate: n=8;
*P＜0.05,
*Compare with model group P＜0.01;
ΔCompare with the DANHONG ZHUSHEYE group P＜0.05.
Conclusion: (1) this trial model group rat neural function is learned the standard that scoring meets the model success, and the visible obviously cerebral infarction kitchen range of TTC dyeing illustrates that model prepares successfully.Positive control drug can significantly improve cerebral ischemia.
(2) Danhong for injection 10mgkg
-1Damage is had some improvement, but statistically with the model group no significant difference.20mgkg
-1Focal brain ischemia-reperfusion injury in rats is had significant protective effect, can improve ischemic 1.5h and pour into neurologic impairment symptom behind the 24h again, significantly cerebral infarct volume alleviates ischemic side degree of cerebral edema.40mgkg
-1Can further reduce the brain injury degree, and good than the positive controls result of treatment.With the increase of dosage, the cerebral infarction volume reduces gradually, presents certain dose-effect relationship.
(3) compare Danhong for injection low dosage 10mgkg with the positive control drug DANHONG ZHUSHEYE
-1DeGrain, middle dosage 20mgkg
-1Curative effect is suitable with it; High dosage 40mgkg
-1Can obviously improve the neurological dysfunction of ischemical reperfusion injury rat, the cerebral infarction volume significantly dwindles, and ischemic side degree of cerebral edema alleviates, and is effective than Danhong Injection in Treating.
(4) by above pharmacodynamic study results suggest, the effective initial dose of Danhong for injection clinical application is about 1.7～3.3mgkg
-1, recommend the clinical treatment cerebral apoplexy to intend being about 100～200mg with dosage in human body body weight 60kg.
3, the influence of Danhong for injection chmice acute pulmonary infarction that ADP is brought out
Purpose: observe Danhong for injection repeatedly the tail administration to the influence of ADP induced mice acute pulmonary embolism.
Method: get 50 of healthy adult kunming mices, be divided into blank group (Veh), the basic, normal, high dosage group of Danhong for injection and positive controls, 10 every group, male and female half and half at random by body weight.Continuously the tail administration is 3 days, and the Veh group gives 0.9% sodium chloride injection of respective volume, and the Danhong for injection dosage is respectively 15,30 and 60mgkg
-1, positive controls gives DANHONG ZHUSHEYE, and dosage is 20mlkg
-11h tail vein injection ADP 0.25gkg after the last administration
-1, make the acute pulmonary embolism model, normal to mouse breathing behind the record injection inductor, four limbs recover the required time of autonomic activities.
The result: compare with the Veh group, the positive controls mouse recovers the autonomic activities required time and reduces 131s, and remarkable significant difference (P＜0.01) is arranged.The basic, normal, high dosage group of the Danhong for injection mouse recovery required time of autonomic activities obviously shortens, and wherein low dose group shortens 98s, and middle and high dosage shortens 136s, 165s respectively, and remarkable significant difference (P＜0.01) is all arranged.Compare with positive controls, basic, normal, high dosage group does not all have obvious significant difference, and concrete experimental data sees Table 14.
Table 14 Danhong for injection to ADP cause the chmice acute pulmonary infarction influence (
, s)
Annotate: n=10;
*Compare with the Veh group P＜0.01.
Conclusion: (1) Danhong for injection 15mgkg
-1Recover autonomic activities required time (P＜0.01), middle dosage 30mgkg after can significantly shortening the chmice acute pulmonary infarction that ADP brings out
-1With high dosage 60mgkg
-1Also can obviously shorten and recover autonomic activities required time (P＜0.01).And with the increase of dosage, recover the autonomic activities required time after the chmice acute pulmonary infarction that ADP is brought out and shorten gradually, have tangible dose-effect relationship.
(2) compare with the positive control drug DANHONG ZHUSHEYE, the low dose group action effect is not remarkable, and middle and high dosage group curative effect has improvement than it, but not statistically significant.
(3) by above pharmacodynamic study results suggest, the effective initial dose of Danhong for injection clinical application is about 1.5～3mgkg
-1, recommend the clinical treatment congestion type pulmonary heart disease to intend being about 90～180mg with dosage in human body body weight 60kg.
4, Danhong for injection is to the influence of clotting time of mice
Purpose: observe Danhong for injection repeatedly the tail administration to the influence of clotting time of mice.
Method: 50 of healthy adult kunming mices are divided into blank group (Veh), the basic, normal, high dosage group of Danhong for injection and positive controls, 10 every group, male and female half and half at random by body weight.The tail administration is 3 days continuously, and the Veh group gives 0.9% sodium chloride injection of respective volume, and the Danhong for injection dosage is respectively 15mgkg
-1, 30mgkg
-1And 60mgkg
-1, positive controls gives DANHONG ZHUSHEYE, and dosage is 20mlkg
-1, after the last administration, plucked a branch hole ball rapidly with the curved tweezer of ophthalmology in 1 hour, give up the First Blood, on the slide glass of cleaning, diameter is about 5～10mm with droplet of blood, manual time-keeping, provoke gently inwards 1 time with cleaning pin autoblood edge every 30s, observation has or not the trace of blood to provoke.Be the clotting time from the blood sampling beginning to provoking a time blood.
The result: compare with the Veh group, each dosage group clotting time of mice of Danhong for injection obviously prolongs, and wherein low dose group prolongs 27s, but not statistically significant.Middle dosage group prolongs 54s, has significant difference (P＜0.05).The high dose group clotting time of mice prolongs 129s, has remarkable significant difference (P＜0.01).Compare with positive controls, low dose group has significant difference (P＜0.05), and middle and high dosage group does not have remarkable significant difference, and concrete experimental data sees Table 15.
Table 15 Danhong for injection is to the influence of clotting time of mice
Annotate: n=10;
*P＜0.05
*Compare with the Veh group P＜0.01;
ΔCompare with the DANHONG ZHUSHEYE group P＜0.05.
Conclusion: (1) Danhong for injection 15mgkg
-1Can certain prolongation effect be arranged to clotting time of mice, but compare no difference of science of statistics with the Veh group.30mgkg
-1Can obviously prolong clotting time of mice, high dose group 60mgkg
-1Effect is more obvious.And with the increase of dosage, Danhong for injection increases progressively clotting time of mice, shows tangible dose-effect relationship.
(2) compare with the positive control drug DANHONG ZHUSHEYE, the low dose group curative effect does not have it obvious, and middle and high dosage group action effect has improvement than it, but not statistically significant.
(3) by above pharmacodynamic study results suggest, the effective initial dose of Danhong for injection clinical application is about 1.5～3mgkg
-1, recommend clinical treatment to intend being about 90～180mg with dosage in human body body weight 60kg.
5, Danhong for injection is to the influence of rat platelet aggregation rate
Purpose: observe Danhong for injection repeatedly the tail administration to the influence of rat platelet aggregation rate.
Method: get 50 of healthy adult Wistar rats, be divided into blank group (Veh), the basic, normal, high dosage group of Danhong for injection and positive controls at random by body weight.Every group 10, male and female half and half.The tail administration is 3 days continuously, and the Veh group gives 0.9% sodium chloride injection of respective volume, and the Danhong for injection dosage is respectively 10mgkg
-1, 20mgkg
-1And 40mgkg
-1, positive controls gives DANHONG ZHUSHEYE, and dosage is 12mlkg
-1, the 1h jugular sinus is got blood after the last administration, 3.8% Sodium Citrate anti-freezing.With anticoagulation centrifugal (800rpm, 10min), drawing upper plasma (is platelet rich plasma, PRP) with remainder recentrifuge (3000rpm, 15min), drawing upper plasma (is platelet poor plasma, PPP), survey the variation of transmittance then with platelet aggregation instrument, ask the calculation platelet aggregation rate, concrete experimental data is shown in table 16.
Table 16 Danhong for injection is to the influence of the influence of rat platelet aggregation rate
Annotate:
*P＜0.05,
*Compare with the Veh group P＜0.01;
ΔP＜0.05,
The Δ ΔCompare with the DANHONG ZHUSHEYE group P＜0.01.
The result: compare with the Veh group, the basic, normal, high dosage group of Danhong for injection rat platelet aggregation rate all obviously descends, and wherein low dosage makes platelet aggregation rate descend 10%, but no difference of science of statistics.Middle and high dosage group descends 14%, 22%, and significant difference (P＜0.05) is all arranged, and high dose group difference extremely significantly (P＜0.01) illustrates that Danhong for injection can effectively suppress ADP and Collagen inductive platelet aggregation rate.Compare the basic, normal, high dosage group of Danhong for injection not statistically significant with positive controls.
Conclusion: (1) Danhong for injection 10mgkg
-1Can reduce the rat platelet aggregation rate to a certain extent.20mgkg
-1Remarkable anticoagulant, 40mgkg
-1Restraining effect is stronger.Increase with dosage simultaneously, the anticoagulant effect strengthens, and certain dose-effect relationship is arranged.
(2) compare with the positive control drug DANHONG ZHUSHEYE, low, middle dosage group effect is not remarkable, and the high dose group curative effect has further improvement than it, but does not have significant difference.
(3) by above pharmacodynamic study results suggest, the effective initial dose of Danhong for injection clinical application is about 1.7～3.3mgkg
-1, recommend clinical treatment to intend being about 100～200mg with dosage in human body body weight 60kg.
Claims (3)
1. the extracting method of salvianolic acid B, the extracting method that it is characterized in that salvianolic acid B carries out as follows: one, add 8～18 times of salvia piece quality to the red rooted salvia medicine materical crude slice earlier, mass concentration is that 35%～60% ethanol and volume are ethanol volume 1 ‰～4 ‰, concentration is the salt acid soak 1～2 hour of 1mol/L, refluxing extraction is 1.5～3 hours then, leach danshen solution, add 6～16 times of dregs of a decoction quality to the dregs of a decoction again, mass concentration is that 35%～60% ethanol and volume are ethanol volume 1 ‰～4 ‰, concentration is the hydrochloric acid of 1mol/L, refluxing extraction 1～2 hour, leach danshen solution, the danshen solution that merges extracted twice, to become relative density be 1.16～1.24 the clear cream of the red sage root to concentrating under reduced pressure again; Two, the water that adds 3～5 times of the clear cream weight of the red sage root in the clear cream of the red sage root, limit add the waterside and stir, and leave standstill under the room temperature 12～24 hours, refilter, and red sage root filtrate is 2.8～3.2 with the salt acid for adjusting pH value, obtains red sage root acidifying filtrate; Three, with macroporous adsorptive resins be 2.8～3.2 hcl acidifying with the pH value, the red sage root acidifying filtrate that step 2 is obtained is splined on the macroporous absorption post, with 5～7 times of resin volume pH values is 2.8～3.2 hydrochloric acid flushing impurity, be 35%～55% ethanol elution again with mass concentration, collect the red sage root elutriant of 1.5～2.5 times of resin volumes, the pH value of regulating red sage root elutriant with sodium hydroxide solution is 4.0～4.2, is evaporated to relative density and is 1.08～1.25 the clear cream of salvianolic acid; Promptly obtain salvianolic acid B.
2. the extracting method of salvianolic acid B according to claim 1 is characterized in that in the step 3 with macroporous adsorptive resins being 3.0 hcl acidifying with the pH value.
3. the extracting method of salvianolic acid B according to claim 1 is characterized in that the hydrochloric acid with 6 times of column volume pH values 3.0 washes impurity in the step 3.
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| CN101638402B (en) * | 2008-07-30 | 2013-06-19 | 天津天士力现代中药资源有限公司 | Online quality monitoring method for salvianolic acid B production |
| CN101947254B (en) * | 2010-08-20 | 2012-06-27 | 浙江大学 | Method for extraction of total salvianolic acid from salvia miltrorrhiza ethanol-precipitation precipitates |
| CN102675379B (en) * | 2011-03-15 | 2015-08-19 | 河北以岭医药研究院有限公司 | A kind of method of Hydrolysis kinetics hydroxyl radical carthamin yellow carthamus A from safflower |
| CN102357118A (en) * | 2011-11-03 | 2012-02-22 | 哈药集团中药二厂 | Extraction method of salvia extract and safflower extract and preparation method of salvia-safflower oral preparation |
| CN102662026B (en) * | 2012-05-30 | 2014-04-30 | 山东阿如拉药物研究开发有限公司 | Quality detecting method for traditional Chinese medicine Qianliening preparation |
| CN102775811A (en) * | 2012-08-01 | 2012-11-14 | 乌鲁木齐三高和药业有限公司 | Process of extracting flavonoids from Coreopsis bicolor |
| CN103923043B (en) * | 2013-01-15 | 2018-11-09 | 天津天士力现代中药资源有限公司 | A method of effectively preparing tanshin polyphenolic acid B extract |
| CN103923042B (en) * | 2013-01-15 | 2018-11-09 | 天津天士力现代中药资源有限公司 | The preparation method of tanshin polyphenolic acid B extract |
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| CN1425659A (en) * | 2002-12-31 | 2003-06-25 | 南京虹桥医药技术研究所 | Process for preparing danshen salviandic acid |
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