CN100569231C - A kind of red sage root salvianolic acid A tablet for the treatment of cardiovascular disease and preparation method thereof - Google Patents
A kind of red sage root salvianolic acid A tablet for the treatment of cardiovascular disease and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of red sage root salvianolic acid A tablet for the treatment of cardiovascular disease and preparation method thereof, it is characterized in that tablet adopts high effective liquid chromatography for measuring, get 3 total peaks, with the danshen root salvianolic acid A is object of reference, its retention time is that the relative retention time of 1,2 impurity is respectively 0.71,1.36; With the danshen root salvianolic acid A is contrast, and relative retention time is 0.71 impurity, and its content is 0.10-1.96%, and relative retention time is that 1.36 impurity content is 0.11-5.97%, is defined as salvia miltiorrhiza tanshinoate C, and the content of total impurities is not more than 8%; The tablet material medicated bag is drawn together danshen root salvianolic acid A, filler, disintegrating agent, lubricant, wetting agent dehydrated alcohol, dissolution was greater than 80% in 45 minutes, need not add antioxidant and can prevent that preparation raw material danshen root salvianolic acid A content from reducing and degraded, make preparation safety, stable; Pharmacological evaluation shows that the present invention respectively organizes preparation and has great pharmacological effects.
Description
Technical field
The present invention relates to technical field of traditional Chinese medicine pharmacy, be specifically related to a kind of red sage root salvianolic acid A tablet for the treatment of cardiovascular disease and preparation method thereof.
Background technology
Radix Salviae Miltiorrhizae is the root of Labiatae salvia Radix Salviae Miltiorrhizae.Bitter in the mouth, cold nature, Radix Salviae Miltiorrhizae is one of the most frequently used medical material, being used for the treatment of cardiovascular and cerebrovascular diseases such as angina pectoris, hypertension, coronary heart disease and apoplexy, having the effect of blood circulation promoting and blood stasis dispelling, nourishing blood to tranquillize the mind, removing heat from blood row's carbuncle and toxin expelling granulation promoting, is the flavour of a drug commonly used of Chinese medicine blood circulation promoting and blood stasis dispelling.Have the red sage formulation major part now and all be the crude extract that extracts in the Radix Salviae Miltiorrhizae or directly use salvia piece, it is fast to absorb metabolism in the body, and preparation is difficult to keep the stability of oral administration blood drug level.Salvianolic acid A [salvianolic acid A] is one of effective component in red sage, and its structural formula is:
Du Guanhua [preclinical medicine and clinical; 2000; 20 (5): 10～14] etc. the people has carried out big quantity research to the activity of salvianolic acid A, finds that salvianolic acid A has significant protective effect to the isolated rat myocardial ischemia reperfusion injury, therefore in the cardiovascular direction good curative effect is arranged.
Salvianolic acid A is the stronger chemical compound of nature antioxidant activity, be easy to oxidized, easier degraded during high temperature, therefore, it is that our medical research personnel need difficult problems to be solved that danshen root salvianolic acid A is developed to adaptable clinically Chinese medicine preparation.Discloses danshen root salvianolic acid A among Chinese patent CN1397276A, the CN1352985A and be prepared into tablet, but it just discloses simple preparation method, and not to preparation carry out rationally, the scientific quantitative analysis; In the preparation processing process; added the antioxidant protection; but the simple superposition of carrying out just at the fundamental property of salvianolic acid A; and the antioxidant that adds is the variation of carrying out according to the total amount of preparation; its ratio can't have quantitative relation in the preparation process; and the relation of its amount is from 0.01%-95%; differ nearly 10000 times; this range of choice to adjuvant in the preparation process is excessive; can produce very big difficulty to preparation preparation process; and whether this patent particularly needs to add this problem of antioxidant and does not carry out deep research carrying out deep research at preparation nature in the preparation tablet.In order to prepare a kind of good stability, safe and reliable red sage root salvianolic acid A tablet, must carry out more deep research at the specific (special) requirements of the preparation process of physics, chemical property and the Chinese medicinal tablet of danshen root salvianolic acid A, obtain safe and reliable preparation.
Prior art adopts the method for disintegration in the Chinese medicinal tablet quality testing, check that the evaluation criteria that absorbs in vivo as all tablets obviously is perfect inadequately disintegration but rely on, because be everlasting between the 1.6-2.0mm by disintegration tester screen cloth particle diameter behind the medicine dissolution, could be absorbed by body and medicine need be solution state, its particle size with
Calculate, so disintegrate only is the initial period of medicine stripping, and the continuation dispersion and the course of dissolution of back, disintegration, inspection was uncontrollable, and the disintegrate of tablet also will be subjected to prescription design, formulation preparation, the influence of many complicated factors in storage process and the body, so check the relation and the influence that can not objectively respond between medicine and the excipient disintegration, and dissolution test has comprised disintegrate and course of dissolution, therefore studying the Chinese medicinal tablet dissolution just has prior meaning.
Summary of the invention
For these reasons, we are prepared into qualified red sage root salvianolic acid A tablet by long term studies, and preparation is carried out the high-efficient liquid phase technique analysis: get 3 total peaks; Be 90 minutes writing time, is object of reference with the danshen root salvianolic acid A, and its retention time is that the relative retention time of 1,2 impurity is respectively 0.71,1.36; With the danshen root salvianolic acid A is contrast, and relative retention time is 0.71 impurity, and its content is 0.10-1.96%, and relative retention time is that 1.36 impurity content is 0.11-5.97%, determines that it is salvia miltiorrhiza tanshinoate C, and the content of total impurities is not more than 10.0%.According to danshen root salvianolic acid A physics and chemical property, in the preparation preparation, select filler, disintegrating agent, lubricant, wetting agent, particularly do not add any antioxidant, by of the protective effect of other pharmaceutic adjuvant to salvia miltiorrhiza tanshinoate, danshen root salvianolic acid A can not be degraded, to reach the purpose of stable effective ingredients; In the selection of wetting agent, process according to distinctive character of danshen root salvianolic acid A (low amounts of water viscosity is big especially) and preparation granulation, do not use conventional wetting agent water and 30%-70% ethanol, and be to use dehydrated alcohol, be prepared into qualified tablet, carry out the dissolution experimentation, the effective ingredient in the time of 45 minutes more than 80% can stripping.Pharmacological evaluation shows that tablet in treatment of the present invention and/or angiocardiopathy preventing disease aspect have good effect.
The present invention is achieved through the following technical solutions.
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae water or alcoholic solution extract and obtain aqueous extract or alcohol extract, alcohol extract concentrates ethanol to most, the accent pH value is to 7.5-9.0,30-80 ℃ temperature, heating 1-6 hour or transfer pH value to 3.5-6.0,110-130 ℃ temperature, gauge pressure 0.05MPa-0.17MPa pressure, heats 1-6 hour; Solution filters, and filtrate is separated through nonpolar or low pole macroporous resin column chromatography, behind the water eluting, uses the eluant eluting, collects eluent, and eluent reclaims eluant to most; Concentrated solution is used the eluant eluting with silica gel or sephadex lh-20 or the separation of polyamide chromatography post, collects eluent, and eluent reclaims eluant to most; Concentrated solution transfers pH value to 2-5, through organic solvent extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
Macroporous resin column described in its preparation method is HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101,1300-I, 1400 or AB-8.
Macroporous resin column chromatography described in its preparation method separates, and first water, 10-30% Diluted Alcohol eluting are removed impurity, the ethanol elution of reuse 30-70% concentration.
Silica gel column chromatography adopts chloroform in its preparation method: methanol: formic acid=5: 1: 0.1 is eluting solvent.
Sephadex lh-20 described in its preparation method or polyamide chromatography post separate, and first water, 20-50% alcoholic solution eluting discard eluent, reuse 50-95% alcoholic solution eluting.
Organic solvent described in its preparation method is selected from a kind of in ethyl acetate, propyl acetate, butyl acetate, n-butyl alcohol, the isopropyl alcohol.
The preparation of preparation:
Crude drug is: (weight of wetting agent is to obtain promptly according to the density of dehydrated alcohol and volume calculation: m=v* ρ) for danshen root salvianolic acid A 40-60 weight portion, filler 50-200 weight portion, disintegrating agent 1-6 weight portion, lubricant 1-4 weight portion, wetting agent dehydrated alcohol 20-30 weight portion.
Crude drug is: danshen root salvianolic acid A 40-60 weight portion, filler 150-200 weight portion, disintegrating agent 4-6 weight portion, lubricant 1-4 weight portion, wetting agent dehydrated alcohol 20-30 weight portion.
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains the tablet in the danshen root salvianolic acid A unit dose;
Filler is a kind of in lactose, starch, dextrin, Icing Sugar, microcrystalline Cellulose, pregelatinized Starch, mannitol, calcium hydrogen phosphate, the calcium sulfate;
Disintegrating agent is a kind of in carboxymethyl starch sodium, hydroxypropyl starch, hyprolose, starch, polyvinylpolypyrrolidone, cross-linking sodium carboxymethyl cellulose, the microcrystalline Cellulose;
Lubricant is a kind of in magnesium stearate, Pulvis Talci, micropowder silica gel, the Polyethylene Glycol;
Unit dose is counted 5-1000mg with danshen root salvianolic acid A; Unit dose is very little less than 5mg clinical practice effect, and unit dose is greater than 1000mg, and clinical practice has the problem of safety; Unit dose is counted 20-500mg with danshen root salvianolic acid A.
Red sage root salvianolic acid A tablet at 45 minutes dissolution greater than 80%.
Red sage root salvianolic acid A tablet treats and/or prevents application in the cardiovascular disease medicine in preparation.
The invention reside in provides a kind of red sage root salvianolic acid A tablet for the treatment of cardiovascular disease, preparation to carry out the high performance liquid chromatography detection, contains 2 impurity peaks, and wherein 1 impurity is salvia miltiorrhiza tanshinoate C, and total impurities content is not more than 10.0%.
The invention reside in provide a kind of and do not need to add antioxidant, wetting agent is the red sage root salvianolic acid A tablet of dehydrated alcohol preparation.
The present invention also is to provide a kind of preparation method for the treatment of the red sage root salvianolic acid A tablet of cardiovascular disease.
Two. detection method
Experimental technique:
Chromatographic column: C
18Reversed phase chromatographic column, NUCLEODUR, 250*4.6mm, ODS pre-column;
Chromatographic condition and system suitability experiment: with the octadecylsilane chemically bonded silica is filler; Flow velocity 1.0ml/min; 35 ℃ of column temperatures; Detect wavelength 286nm; Number of theoretical plate should be not less than 60000 by salvianolic acid A; With acetonitrile-0.2% aqueous acetic acid is mobile phase, carries out gradient elution by following condition of gradient elution, moves 90 minutes: in the time of 0-15 minute, the ratio of acetonitrile reduces to 80% by 10% ratio that rises to 20%, 0.2% aqueous acetic acid by 90%; In the time of 15-55 minute, the ratio of acetonitrile reduces to 70% by 20% ratio that rises to 30%, 0.2% aqueous acetic acid by 80%; In the time of 55-65 minute, the ratio of acetonitrile reduces to 50% by 30% ratio that rises to 50%, 0.2% aqueous acetic acid by 70%; In the time of 65-72 minute, the ratio of acetonitrile reduces to 20% by 50% ratio that rises to 80%, 0.2% aqueous acetic acid by 50%; In the time of 72-77 minute, the ratio 20% of ratio 80%, 0.2% aqueous acetic acid of acetonitrile; In the time of 77-80 minute, the ratio of acetonitrile rises to 90% by 80% ratio of reducing to 10%, 0.2% aqueous acetic acid by 20%; In the time of 80-90 minute, keep acetonitrile-0.2% aqueous acetic acid to carry out eluting with 10: 90 ratio.
The preparation of danshen root salvianolic acid A reference substance solution: precision takes by weighing salvianolic acid A reference substance 10mg in the 50ml volumetric flask, adds dissolve with methanol and shakes up, and be diluted to scale.
The preparation of need testing solution: precision takes by weighing sample, and standardize solution adds dissolve with methanol and shakes up in volumetric flask after treatment, and is diluted to scale;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, write down 90 minutes chromatogram, adopt by external standard method with calculated by peak area, promptly
Obtain red sage root salvianolic acid A tablet according to the application's process, testing result sees Table 1:
Table 1 red sage root salvianolic acid A tablet sample determination result
Experiment conclusion: show that by above-mentioned experiment danshen root salvianolic acid A and impurity are in the rule of content, retention time in the application's red sage root salvianolic acid A tablet: adopt high effective liquid chromatography for measuring, obtain 3 total peaks, wherein comprise 2 impurity; Be 90 minutes writing time, is object of reference with the danshen root salvianolic acid A, and its retention time is that the relative retention time of 1,2 impurity is respectively 0.71,1.36; With the danshen root salvianolic acid A is contrast, and relative retention time is 0.71 impurity, and its content is 0.10-1.96%, and relative retention time is that 1.36 impurity content is 0.11-5.97%, determines that it is salvia miltiorrhiza tanshinoate C, and the content of total impurities is not more than 10.0%.
Three. danshen root salvianolic acid A structural identification data
Salvianolic acid A (
1HNMR, D-acetone, ppm, Hz): 6.83 (1H, d, J=8.7Hz, A-5), 7.21 (1H, d, J=8.4Hz, A-6), 8.06 (1H, d, J=15.8Hz, A-7), 6.33 (1H, d, J=15.8Hz, A-8), 6.82 (1H, d, J=2.4Hz, B-2), 6.71 (1H, d, J=8.0Hz, B-5), 6.62 (1H, dd, J=2.0,8.0Hz, B-6), 3.10 (1H, dd, J=4.2,14.3Hz, B-7 α), 2.99 (1H, dd, J=8.7,14.3Hz, B-7 β), 5.21 (1H, dd, J=4.2,8.7Hz, B-8), 7.13 (1H, d, J=2.0Hz, C-2), 6.85 (1H, d, J=8.4Hz, C-5), 6.93 (1H, dd, J=1.9,8.2Hz, C-6), 7.16 (1H, d, J=16.4Hz, C-7), 6.72 (1H, d, J=16.4Hz, C-8).(
13CNMR, D-acetone, ppm, Hz): 125.9 (A-1), 130.9 (A-2), 146.1 (A-3), 147.4 (A-4), 116.0 (A-5), 121.7 (A-6), 145.8 (A-7), 116.1 (A-8), 166.9 (A-9), 129.1 (B-1), 117.1 (B-2), 144.7 (B-3), 143.9 (B-4), 114.8 (B-5), 120.3 (B-6), 37.5 (B-7), 73.7 (B-8), 171.1 (B-9), 127.6 (C-1), 119.8 (C-2), 145.7 (C-3), 146.4 (C-4), 113.7 (C-5), 120.2 (C-6), 137.4 (C-7), 116.2 (C-8).
Four. salvia miltiorrhiza tanshinoate C-structure conclusive evidence data
Salvia miltiorrhiza tanshinoate C (
1HNMR, CD
3OD, ppm, Hz): 6.87 (d, J=8.2, A-5), 7.35 (d, J=8.3, A-6), 7.91 (d, J=16.0, A-7), 6.45 (d, J=16.0, A-8), 6.79 (d, J=1.7, B-2), 6.72 (d, J=9.4, B-5), 6.65 (d, J=8.1,1.7, B-6), 3.13 (dd, J=14.3,4.2, B-7 α), 3.06 (dd, J=14.3,8.2, B-7 β), 5.24 (dd, J=8.1,4.3, B-8), 7.38 (d, J=1.9, C-2), 6.72 (d, J=9.4, C-5), 7.36 (dd, J=8.2,2.1, C-6), 7.19 (s, C-8).(
13CNMR，CD
3OD，ppm，Hz)：123.7(A-1)，132.7(A-2)，146.1(A-3)，148.0(A-4)，111.8(A-5)，118.8(A-6)，145.2(A-7)，116.7(A-8)，168.7(A-9)，129.4(B-1)，117.7(B-2)，145.3(B-3)，144.1(B-4)，116.3(B-5)，121.9(B-6)，38.0(B-7)，74.8(B-8)，173.8(B-9)，119.6(C-1)，114.9(C-2)，146.0(C-3)，146.7(C-4)，113.4(C-5)，126.3(C-6)，159.4(C-7)，99.3(C-8)。
Five. with the dissolution rate index optimization experiment of writing out a prescription
Filler is granulated during less than 40 weight portions very difficult, and during greater than 200 weight portions, slice, thin piece is waste adjuvant during Datong District too; Disintegrating agent is during less than 1 weight portion, dissolubility is undesirable, therefore the waste adjuvant carries out optimization experiment during greater than 6 weight portions in danshen root salvianolic acid A 40-60 weight portion, filler 40-200 weight portion, disintegrating agent 1-6 weight portion, lubricant 1-4 weight portion, wetting agent dehydrated alcohol 20-30 weight portion scope.
Scheme 1: danshen root salvianolic acid A 40 weight portions, filler 40 weight portions, disintegrating agent 1 weight portion, lubricant 1 weight portion, wetting agent dehydrated alcohol 20 weight portions;
Scheme 2: danshen root salvianolic acid A 40 weight portions, filler 45 weight portions, disintegrating agent 2 weight portions, lubricant 2 weight portions, wetting agent dehydrated alcohol 25 weight portions;
Scheme 3: danshen root salvianolic acid A 40 weight portions, filler 50 weight portions, disintegrating agent 1 weight portion, lubricant 1 weight portion, wetting agent dehydrated alcohol 20 weight portions;
Scheme 4: danshen root salvianolic acid A 40 weight portions, filler 100 weight portions, disintegrating agent 1 weight portion, lubricant 2 weight portions, wetting agent dehydrated alcohol 25 weight portions;
Scheme 5: danshen root salvianolic acid A 40 weight portions, filler 200 weight portions, disintegrating agent 1 weight portion, lubricant 1 weight portion, wetting agent dehydrated alcohol 20 weight portions;
Scheme 6: danshen root salvianolic acid A 50 weight portions, filler 40 weight portions, disintegrating agent 3 weight portions, lubricant 2 weight portions, wetting agent dehydrated alcohol 25 weight portions;
Scheme 7: danshen root salvianolic acid A 50 weight portions, filler 40 weight portions, disintegrating agent 6 weight portions, lubricant 3 weight portions, wetting agent dehydrated alcohol 28 weight portions;
Scheme 8: danshen root salvianolic acid A 50 weight portions, filler 50 weight portions, disintegrating agent 1 weight portion, lubricant 1 weight portion, wetting agent dehydrated alcohol 20 weight portions;
Scheme 9: danshen root salvianolic acid A 50 weight portions, filler 100 weight portions, disintegrating agent 4 weight portions, lubricant 2 weight portions, wetting agent dehydrated alcohol 25 weight portions;
Scheme 10: danshen root salvianolic acid A 50 weight portions, filler 200 weight portions, disintegrating agent 6 weight portions, lubricant 3 weight portions, wetting agent dehydrated alcohol 30 weight portions;
Scheme 11: danshen root salvianolic acid A 60 weight portions, filler 40 weight portions, disintegrating agent 6 weight portions, lubricant 4 weight portions, wetting agent dehydrated alcohol 30 weight portions;
Scheme 12: danshen root salvianolic acid A 60 weight portions, filler 50 weight portions, disintegrating agent 1 weight portion, lubricant 1 weight portion, wetting agent dehydrated alcohol 20 weight portions;
Scheme 13: danshen root salvianolic acid A 60 weight portions, filler 120 weight portions, disintegrating agent 2 weight portions, lubricant 3 weight portions, wetting agent dehydrated alcohol 28 weight portions;
Scheme 14: danshen root salvianolic acid A 60 weight portions, filler 200 weight portions, disintegrating agent 6 weight portions, lubricant 4 weight portions, wetting agent dehydrated alcohol 30 weight portions.
Experimental technique: prepare tablet according to above-mentioned experimental program, the tablet that obtains is according to the dissolution (45 minutes sampling and measuring) of " two ones of Pharmacopoeias of People's Republic of China " (version in 2005) appendix dissolution experimental technique (first method) mensuration different schemes tablet, and experimental result sees Table 2:
The dissolution experimentation of table 2 different schemes tablet
Experiment conclusion: by above-mentioned experimentation, we determine when danshen root salvianolic acid A during at the 40-60 weight portion, filler should be greater than 50 weight portions, disintegrating agent should be greater than 1 weight portion the time, dissolution can reach more than 80%, 45 minutes dissolutions of pharmacopeia regulation should be greater than 70%, therefore, we determine that the preparation raw material medicine is: danshen root salvianolic acid A 40-60 weight portion, filler 50-200 weight portion, disintegrating agent 1-6 weight portion, lubricant 1-4 weight portion, wetting agent dehydrated alcohol 20-30 weight portion; The preferred formulation prescription is: danshen root salvianolic acid A 40-60 weight portion, filler 150-200 weight portion, disintegrating agent 4-6 weight portion, lubricant 1-4 weight portion, wetting agent dehydrated alcohol 20-30 weight portion
Five. pharmaceutic adjuvant is selected the experiment of antioxidant
Those skilled in the art consults document can learn that danshen root salvianolic acid A is the stronger chemical compound of nature antioxidant activity, be easy to oxidized, therefore think and in preparation tablets, should add a certain amount of antioxidant, we find through experiment, in tablet, do not add antioxidant, the content of the composition danshen root salvianolic acid A of yet well remaining valid.
Experimental program:
Scheme 1: danshen root salvianolic acid A 40 grams, filler 150 grams, disintegrating agent 4 grams, lubricant 1 gram, wetting agent dehydrated alcohol 30 grams, antioxidant 40 grams;
Scheme 2: danshen root salvianolic acid A 60 grams, filler 200 grams, disintegrating agent 6 grams, lubricant 4 grams, wetting agent dehydrated alcohol 30 grams, antioxidant 30 grams;
Scheme 3: danshen root salvianolic acid A 50 grams, filler 180 grams, disintegrating agent 5 grams, lubricant 2 grams, wetting agent dehydrated alcohol 25 grams, antioxidant 12.5 grams;
Scheme 4: danshen root salvianolic acid A 45 grams, filler 160 grams, disintegrating agent 4.5 grams, lubricant 3 grams, wetting agent dehydrated alcohol 22 grams, antioxidant 9 grams;
Scheme 5: danshen root salvianolic acid A 55 grams, filler 190 grams, disintegrating agent 5.5 grams, lubricant 2.5 grams, wetting agent dehydrated alcohol 28 grams, antioxidant 5 grams;
Scheme 6: danshen root salvianolic acid A 50 grams, filler 180 grams, disintegrating agent 5 grams, lubricant 3.5 grams, wetting agent dehydrated alcohol 25 grams, antioxidant 0 gram;
Experimental technique: be prepared tablet according to above-mentioned experimental program crude drug prescription, prepare qualified after according to the above analysis experimental program carry out check and analysis, obtain danshen root salvianolic acid A content; Tablet was placed 3 months at normal temperatures, detected, obtain danshen root salvianolic acid A content, experimental result sees Table 3:
Table 3 different schemes danshen root salvianolic acid A changes of contents
Experiment conclusion: show by above-mentioned experiment, in the preparation process, add antioxidant and do not add the content not influence of antioxidant, consider that from formulation preparation process and economic angle we determine not add any antioxidant in preparation prescription to danshen root salvianolic acid A.
Six. wetting agent is selected experiment
Experimental program:
Scheme 1: danshen root salvianolic acid A 40 grams, filler 150 grams, disintegrating agent 4 grams, lubricant 1 gram, wetting agent water 30 grams;
Scheme 2: danshen root salvianolic acid A 60 grams, filler 200 grams, disintegrating agent 6 grams, lubricant 4 grams, wetting agent 30% ethanol 30 grams;
Scheme 3: danshen root salvianolic acid A 50 grams, filler 180 grams, disintegrating agent 5 grams, lubricant 2 grams, wetting agent 40% ethanol 25 grams;
Scheme 4: danshen root salvianolic acid A 45 grams, filler 160 grams, disintegrating agent 4.5 grams, lubricant 3 grams, wetting agent 50% ethanol 22 grams;
Scheme 5: danshen root salvianolic acid A 55 grams, filler 190 grams, disintegrating agent 5.5 grams, lubricant 3 grams, wetting agent 60% ethanol 28 grams;
Scheme 6: danshen root salvianolic acid A 50 grams, filler 180 grams, disintegrating agent 5 grams, lubricant 3 grams, wetting agent 70% ethanol 25 grams;
Scheme 7: danshen root salvianolic acid A 50 grams, filler 180 grams, disintegrating agent 5 grams, lubricant 2 grams, wetting agent 80% ethanol 25 grams;
Scheme 8: danshen root salvianolic acid A 50 grams, filler 180 grams, disintegrating agent 5 grams, lubricant 3 grams, wetting agent 90% ethanol 25 grams;
Scheme 9: danshen root salvianolic acid A 50 grams, filler 180 grams, disintegrating agent 5 grams, lubricant 2 grams, wetting agent 95% ethanol 25 grams;
Scheme 10: danshen root salvianolic acid A 50 grams, filler 180 grams, disintegrating agent 5 grams, lubricant 3 grams, wetting agent 100% ethanol 25 grams;
Experimental result: carry out formulation preparation according to above-mentioned experimental program, in pelletization, scheme 1-scheme 8 basic systems do not go out granule, and all raw materials bond together; Though scheme 9 can be prepared granule, indexs such as its granular size are defective; Scheme 10 can be prepared qualified granule.
Experiment conclusion: show that by above-mentioned experiment wetting agent should be defined as dehydrated alcohol in the crude drug.
Seven. the dissolution experiment
Experimental technique: obtain tablet according to the application's prepared, carry out check and analysis (analytical method is carried out according to the application's experiment detection method) according to " two ones of Pharmacopoeias of People's Republic of China " (version in 2005) dissolution experimental technique (first method), experimental result sees Table 4:
Table 4 the application tablet dissolution
Experiment conclusion: the application's preparation prescription determines under optimization experiment, carries out above-mentioned experiment and shows by the write out a prescription preparation of respectively organizing of preparation of the application: greater than 80%, prove absolutely that the application's preparation prescription has scientific meaning at 45 minutes dissolutions.
Eight. pharmacological evaluation
Experiment 1
1. to the protective effect of anesthetized rat myocardial ischemia reperfusion injury
Experimental technique:
Get 108 of healthy SD rats, body weight 240-260g, random packet: blank group, Composite Salvia Dropping Pill group, the application's salvia miltiorrhiza tanshinoate tablet group.Place the pre-raising of equivalent environment 2 days, free diet.After pre-raising finishes, test, animal is weighed, and 20% urethane is pressed the 0.6ml/100g lumbar injection, after treating that anesthesia is satisfied, lie on the back and be fixed on the Mus plate, tracheal intubation connects respirator, by 10～12ml tidal volume, 70 times/minute frequency is exhaled, and continuous positive pressure breathing is inhaled: exhale than being 1: 1.Adjust respiration parameter according to the respiratory frequency and the degree of depth.Connect electrocardiograph subsequently, survey normal ECG.Cut off front field of operation hair, iodine disinfection, cut off skin, subcutaneous tissue, front muscle and fascia 3～4cm, it is long to separate Intercostal muscle 3cm with the 18# vascular forceps along the 3rd intercostal passivity, open thoracic cavity and pericardium, recording ecg, strut 3,4 ribs, refer to hold thoracic cavity, rat right side with left hand four, the assistant upwards pushes away thymus with the ophthalmology tweezer, finds ligation sign blood vessel great cardiac vein between left auricle and pulmonary conus, 2mm place noinvasive roundlet pin band 6-0 silk thread threading below left auricle, depth of needle is 1～1.5mm, wide 2～3mm, recording ecg behind the threading, give corresponding medicinal liquid through the tail vein, recording ecg behind the administration 10min, and with one the band groove little plastics pipe pad at the ligation position, the ligation thereon of two rear line heads.At once recording ecg after the ligation is cyanosis or the II S-T section back of a bow that leads with left chamber antetheca and upwards raises greater than 0.1mv and be that ligation successfully indicates (it is superseded that the S-T section does not have the changer) more than the lasting 0.5h.10min recording ecg is once more cut off ligature behind the ligation 30min after the ligation, realizes perfusion again, and record pours into electrocardiogram at once again, removes in the thoracic cavity layer-by-layer suture thoracic wall behind the hematocele, removes respirator, animal recovery autonomous respiration, and incision of trachea does not process.Irritate again at once, 10min, 20min, 40min, 1h, 2h, 3h recording ecg respectively.With heart after refrigerator and cooled is frozen 10min, from the apex of the heart entad the parallel coronary sulcus direction in the end 5 of equal thickness are cut in left chamber, put into the 1%TTC dye liquor, 37 ℃ of dyeing 10min, the necrotic area is not a kermesinus, the necrotic area is canescence.Digital camera is taken pictures.Weighed respectively in necrotic area and non-necrotic area, calculate the percentage ratio that the necrotic area accounts for left ventricular mass, i.e. infarction size.
Table 5 salvianolic acid A tablet is to the influence of myocardial ischemia myocardial infarct size (%) due to the logical rat ramus descendens anterior arteriae coronariae sinistrae of ligation/again (x ± s)
Annotate: compare * * P＜0.01 with the blank group; Compare #P＜0.05 with positive Composite Salvia Dropping Pill group
Experiment 2
Influence to SOD activity, MDA content in the rat heart muscle ischemia/reperfusion model serum due to the logical rat ramus descendens anterior arteriae coronariae sinistrae of ligation/again
Experimental result sees Table 6:
The influence of SOD activity, MDA content in the rat heart muscle ischemia/reperfusion model serum due to table 6 pair ligation/logical again rat ramus descendens anterior arteriae coronariae sinistrae
Annotate: compare * * P＜0.01 with the blank group; Compare #P＜0.05 with positive Composite Salvia Dropping Pill group
Experiment conclusion: the application's preparation taking dose is defined as 5-1000mg, and preferred taking dose is 20-500mg, shows that by above-mentioned experiment the application's preparation has great pharmacological effects.
Description of drawings
1. Fig. 1 abscissa unit is time (min), and vertical coordinate unit is voltage (mv); Fig. 1 is the high-efficient liquid phase chromatogram of danshen root salvianolic acid A standard substance.
2. Fig. 2 abscissa unit is time (min), and vertical coordinate unit is voltage (mv); Fig. 2 is the high-efficient liquid phase chromatogram of red sage root salvianolic acid A tablet.
3. Fig. 3 abscissa unit is time (min), and vertical coordinate unit is voltage (mv); Fig. 3 is the high-efficient liquid phase chromatogram of salvia miltiorrhiza tanshinoate C standard substance.
The present invention compared with the prior art distinguishing characteristics is:
1. prior art limits the danshen root salvianolic acid A active constituent content in preparation research, to system Impurity in the agent does not carry out any research, and the present invention is ensuring on the basis of effective component content, in the preparation Impurity is studied fully, has found impurity and active ingredient in the rule of content, retention time, and has carried out Restriction: simultaneously determined that tentatively an impurity is salvia miltiorrhiza tanshinoate F or its isomer, an impurity is true Be decided to be salvia miltiorrhiza tanshinoate C.
In the prior art according to the character of danshen root salvianolic acid A, in preparation prescription, added a certain amount of antioxygen Agent, and the present invention finds in preparation research, does not add antioxidant in the preparation prescription and also can well stop pellet The degraded of ginseng salviandic acid A; The wetting agent that prior art is used is water or 30%-70% ethanol, and we Research finds that being chosen in the preparation process of wetting agent is extremely important, and experiment determines that absolute ethyl alcohol can be used as preparation The wetting agent of preparation.
3. general employing is analyzed disintegration time limited in the existing Chinese medicinal tablet quality testing, and we scientific research personnel adopts dissolution rate Experiment has determined that the application's preparation prescription has scientific meaning, and the dissolution rate of the application's tablet can in the time of 45 minutes Reach more than 80%, good bioavilability is arranged.
Six. preparation embodiment
Embodiment 1
Danshen root salvianolic acid A extracts purification:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, transfers pH value to 5.0,120 ℃ of temperature, and gauge pressure 0.10MPa pressure heated 4 hours; Solution filters, and filtrate is separated through the HPD-300 macroporous resin column chromatography, and first water, 20% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 50% concentration is collected and contained the salvianolic acid A part, and eluent is concentrated into does not have the alcohol flavor; Concentrated solution separates with polyamide chromatography post, first water, 35% alcoholic solution eluting, and eluent discards, and reuse 75% alcoholic solution eluting concentrates ethanol to most; Concentrated solution is transferred pH value to 3, through the organic solvent n-butyl acetate extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
Crude drug is: danshen root salvianolic acid A 20 grams, filler starch 25 grams, disintegrating agent carboxymethyl base Starch Sodium 0.5 gram, magnesium stearate lubricant 0.5 gram, (weight of wetting agent is to obtain promptly according to the density of dehydrated alcohol and volume calculation to wetting agent dehydrated alcohol 10 grams: m=v* ρ).
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains 4000 in the tablet of unit dose; Unit dose is 5mg; Preparation adopts high effective liquid chromatography for measuring, is object of reference with the danshen root salvianolic acid A, and relative retention time is that 0.71 impurity content is 0.10%, and relative retention time is that 1.36 impurity content is 0.11%, the content 1.2% of total impurities; Dissolution is 83.2% after measured.
Embodiment 2
Danshen root salvianolic acid A extracts purification:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, transfers pH value to 9.0,80 ℃ of temperature, heating 6 hours; Solution filters, and filtrate is separated through the HPD-400A macroporous resin column chromatography, and first water, 30% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 70% concentration is collected and contained the salvianolic acid A part, and eluent is concentrated into does not have the alcohol flavor; Concentrated solution separates with sephadex lh-20, and first water, 50% alcoholic solution eluting discard eluent, and reuse 95% alcoholic solution eluting is collected eluent and concentrated ethanol to most; Concentrated solution is transferred pH value to 5, through the extraction of organic solvent isopropyl alcohol, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
Crude drug is: danshen root salvianolic acid A 40 grams, filler dextrin 150 grams, disintegrating agent hydroxypropyl starch 4 grams, lubricant Pulvis Talci 1 restrain, (weight of wetting agent is to obtain promptly according to the density of dehydrated alcohol and volume calculation to wetting agent dehydrated alcohol 20 grams: m=v* ρ).
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains 2000 in the tablet of unit dose; Unit dose 20mg; Preparation adopts high effective liquid chromatography for measuring, is object of reference with the danshen root salvianolic acid A, and relative retention time is that 0.71 impurity content is 1.96%, and relative retention time is that 1.36 impurity content is 5.97%, the content 10.0% of total impurities; Dissolution is 85.0% after measured.
Embodiment 3
Danshen root salvianolic acid A extracts purification:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, transfer pH value to 3.5,110 ℃ of temperature, gauge pressure 0.05MPa pressure, heated 1 hour: solution filters, filtrate is separated through the HPD-100 macroporous resin column chromatography, elder generation's water, 10% Diluted Alcohol eluting are removed impurity, the ethanol elution of reuse 30% concentration, collection contains the salvianolic acid A part, and eluent is concentrated into does not have the alcohol flavor; Concentrated solution separates with polyamide chromatography post, first water, 20% alcoholic solution eluting, and eluent discards, and reuse 95% alcoholic solution eluting is collected eluent, concentrates ethanol extremely to the greatest extent; Concentrated solution is transferred pH value to 2, through ethyl acetate extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
Crude drug is: danshen root salvianolic acid A 50 grams, filler Icing Sugar 180 grams, disintegrating agent hyprolose 5 grams, lubricant micropowder silica gel 3 restrain, (weight of wetting agent is to obtain promptly according to the density of dehydrated alcohol and volume calculation to wetting agent dehydrated alcohol 25 grams: m=v* ρ).
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains 1000 in the tablet of unit dose; Unit dose 50mg; Preparation adopts high effective liquid chromatography for measuring, is object of reference with the danshen root salvianolic acid A, and relative retention time is that 0.71 impurity content is 0.35%, and relative retention time is that 1.36 impurity content is 1.22%, the content 2.21% of total impurities; Dissolution is 84.7% after measured.
Danshen root salvianolic acid A extracts purification:
Radix Salviae Miltiorrhizae extracts with alcoholic solution and obtains alcohol extract, concentrates ethanol to the greatest extent, transfers pH value to 7.5, and 30 ℃ of temperature heated 1 hour; Solution filters, and filtrate is separated through the HPD-400 macroporous resin column chromatography, and first water, 10% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 30% concentration is collected and contained the salvianolic acid A part, and eluent is concentrated into does not have the alcohol flavor; Concentrated solution silica gel, eluant adopt chloroform: methanol: formic acid=5: 1: 0.1 be the eluting solvent eluting, collects eluent, concentrates, and drying or lyophilization must danshen root salvianolic acid As;
Crude drug is: danshen root salvianolic acid A 60 grams, filler microcrystalline Cellulose 200 grams, disintegrating agent starch 6 grams, lubricant, Polyethylene Glycol 4 restrain, (weight of wetting agent is to obtain promptly according to the density of dehydrated alcohol and volume calculation to wetting agent dehydrated alcohol 30 grams: m=v* ρ).
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains 1000 in the tablet of unit dose; Unit dose 60mg; Preparation adopts high effective liquid chromatography for measuring, is object of reference with the danshen root salvianolic acid A, and relative retention time is that 0.71 impurity content is 0.87%, and relative retention time is that 1.36 impurity content is 4.30%, the content 7.38% of total impurities; Dissolution is 84.2% after measured.
Embodiment 5
Danshen root salvianolic acid A extracts purification:
Radix Salviae Miltiorrhizae extracts with alcoholic solution and obtains alcohol extract, concentrates ethanol to the greatest extent, transfers pH value to 6.0,130 ℃ of temperature, and gauge pressure 0.17MPa pressure heated 6 hours; Solution filters, and filtrate HPD-100A macroporous resin column chromatography separates, and first water, 30% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 70% concentration is collected and contained the salvianolic acid A part, and eluent is concentrated into does not have the alcohol flavor; The concentrated solution sephadex lh-20, first water, 20% alcoholic solution eluting, eluent discards, reuse 95% alcoholic solution eluting; Collect eluent, concentrate ethanol to most; Concentrated solution is transferred pH value to 5, through the extraction of organic solvent propyl acetate, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
Crude drug is: danshen root salvianolic acid A 450 grams, filler pregelatinized Starch 1700 grams, disintegrating agent polyvinylpolypyrrolidone 55 grams, magnesium stearate lubricant 30 restrain, (weight of wetting agent is to obtain promptly according to the density of dehydrated alcohol and volume calculation to wetting agent dehydrated alcohol 230 grams: m=v* ρ).
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains 10000 in the tablet of unit dose; Unit dose 450mg; Preparation adopts high effective liquid chromatography for measuring, is object of reference with the danshen root salvianolic acid A, and relative retention time is that 0.71 impurity content is 1.82%, and relative retention time is that 1.36 impurity content is 0.42%, the content 3.04% of total impurities; Dissolution is 87.0% after measured.
Embodiment 6
Danshen root salvianolic acid A extracts purification:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, transfers pH value to 5.0,120 ℃ of temperature, gauge pressure 0.10MPa pressure, heats 3.5 hours; Solution filters, and filtrate is separated through the AB-8 macroporous resin column chromatography, and first water 20% Diluted Alcohol eluting is removed impurity, and the ethanol elution of reuse 45% concentration is collected and contained the salvianolic acid A part, and eluent is concentrated into does not have the alcohol flavor; Concentrated solution separates with polyamide chromatography post, first water, 35% alcoholic solution eluting, and eluent discards, and reuse 85% alcoholic solution eluting concentrates ethanol to most; Eluent is transferred pH value to 3, through the organic solvent n-butyl acetate extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
Crude drug is: danshen root salvianolic acid A 1000 grams, filler mannitol 3600 grams, disintegrating agent cross-linking sodium carboxymethyl cellulose 100 grams, lubricant Pulvis Talci 60 restrain, (weight of wetting agent is to obtain promptly according to the density of dehydrated alcohol and volume calculation to wetting agent dehydrated alcohol 500 grams: m=v* ρ).
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains 20000 in the tablet of unit dose; Unit dose 1000mg; Preparation adopts high effective liquid chromatography for measuring, is object of reference with the danshen root salvianolic acid A, and relative retention time is that 0.71 impurity content is 1.45%, and relative retention time is that 1.36 impurity content is 5.24%, the content 7.8% of total impurities; Dissolution is 84.1% after measured.
Embodiment 7
Danshen root salvianolic acid A extracts purification:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, transfers pH value to 4.0,115 ℃ of temperature, gauge pressure 0.09MPa pressure, heats 2 hours; Solution filters, and filtrate is separated through the D101 macroporous resin column chromatography, and first water, 15% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 35% concentration is collected and contained the salvianolic acid A part, and eluent is concentrated into does not have the alcohol flavor; Concentrated solution separates with sephadex lh-20, first water, 25% alcoholic solution eluting, and eluent discards, and reuse 65% alcoholic solution eluting is collected eluent, concentrates ethanol to most; Concentrated solution is transferred pH value to 2.5, through the organic solvent ethyl acetate extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
Crude drug is: danshen root salvianolic acid A 90 grams, filler lactose 320 grams, disintegrating agent microcrystalline Cellulose 9 grams, lubricant micropowder silica gel 4 restrain, (weight of wetting agent is to obtain promptly according to the density of dehydrated alcohol and volume calculation to wetting agent dehydrated alcohol 42 grams: m=v* ρ).
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains 2000 in the tablet of unit dose; Unit dose 90mg; Preparation adopts high effective liquid chromatography for measuring, is object of reference with the danshen root salvianolic acid A, and relative retention time is that 0.71 impurity content is 0.28%, and relative retention time is that 1.36 impurity content is 2.58%, and the content of total impurities is not more than 3.57%; Dissolution is 85.0% after measured.
Embodiment 8
Danshen root salvianolic acid A extracts purification:
Radix Salviae Miltiorrhizae extracts with alcoholic solution and obtains alcohol extract, concentrates ethanol to the greatest extent, transfers pH value to 8.5,75 ℃ of temperature, heating 5.5 hours; Solution filters, and filtrate is separated through the AB-8 macroporous resin column chromatography, and first water, 25% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 55% concentration is collected and contained the salvianolic acid A part, and eluent is concentrated into does not have the alcohol flavor; Concentrated solution sephadex lh-20 chromatographic column separates, and first water, 45% alcoholic solution eluting discard eluent, and reuse 90% alcoholic solution eluting is collected eluent, concentrates ethanol to most; Concentrated solution is transferred pH value to 4.5, through the organic solvent n-butanol extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
Crude drug is: danshen root salvianolic acid A 42 grams, filler mannitol 89 grams, disintegrating agent hydroxypropyl starch 2.0 grams, lubricant Polyethylene Glycol 1.5 restrain, (weight of wetting agent is to obtain promptly according to the density of dehydrated alcohol and volume calculation to wetting agent dehydrated alcohol 20 grams: m=v* ρ).
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains 1000 in the tablet of unit dose; Unit dose 42mg; Preparation adopts high effective liquid chromatography for measuring, is object of reference with the danshen root salvianolic acid A, and relative retention time is that 0.71 impurity content is 0.14%, and relative retention time is that 1.36 impurity content is 5.81%, and the content of total impurities is not more than 7.2%; Dissolution is 85.0% after measured.
Embodiment 9
Danshen root salvianolic acid A extracts purification:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, transfers pH value to 8.0,35 ℃ of temperature, heating 2 hours; Solution filters, and filtrate is separated through 1400 macroporous resin column chromatographies, and first water, 15% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 65% concentration is collected and contained the salvianolic acid A part, and eluent is concentrated into does not have the alcohol flavor; Concentrated solution separates with polyamide chromatography post, first water, 45% alcoholic solution eluting, and eluent discards, and reuse 55% alcoholic solution eluting concentrates ethanol to most; Concentrated solution is transferred pH value to 2.5, through the organic solvent n-butyl acetate extraction, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
Crude drug is: danshen root salvianolic acid A 49 grams, filler calcium hydrogen phosphate 140 grams, disintegrating agent starch 3.5 grams, magnesium stearate lubricant 2 restrain, (weight of wetting agent is to obtain promptly according to the density of dehydrated alcohol and volume calculation to wetting agent dehydrated alcohol 24 grams: m=v* ρ).
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains 1000 in the tablet of unit dose; Unit dose 49mg; Preparation adopts high effective liquid chromatography for measuring, is object of reference with the danshen root salvianolic acid A, and relative retention time is that 0.71 impurity content is 0.19%, and relative retention time is that 1.36 impurity content is 3.42%, and the content of total impurities is not more than 4.52%; Dissolution is 84.8% after measured.
Embodiment 10
Danshen root salvianolic acid A extracts purification:
Radix Salviae Miltiorrhizae extracts with alcoholic solution and obtains alcohol extract, concentrates ethanol to the greatest extent, transfers pH value to 5.5,125 ℃ of temperature, gauge pressure 0.15MPa pressure, heats 5.5 hours; Solution filters, and filtrate is separated through the 1300-I macroporous resin column chromatography, and first water, 25% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 65% concentration is collected and contained the salvianolic acid A part, and eluent is concentrated into does not have the alcohol flavor; Concentrated solution separates with the sephadex lh-20 chromatographic column, and first water, 45% alcoholic solution eluting discard eluent, and reuse 90% alcoholic solution eluting is collected eluent, concentrates ethanol extremely to the greatest extent; Concentrated solution is transferred pH value to 4.5, through the extraction of organic solvent propyl acetate, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
Crude drug is: danshen root salvianolic acid A 500 grams, filler calcium sulfate 1850 grams, disintegrating agent carboxymethyl base Starch Sodium 55 grams, lubricant Polyethylene Glycol 35 restrain, (weight of wetting agent is to obtain promptly according to the density of dehydrated alcohol and volume calculation to wetting agent dehydrated alcohol 280 grams: m=v* ρ).
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains 10000 in the tablet of unit dose; Unit dose 500mg; Preparation adopts high effective liquid chromatography for measuring, is object of reference with the danshen root salvianolic acid A, and relative retention time is that 0.71 impurity content is 1.87%, and relative retention time is that 1.36 impurity content is 0.21%, and the content of total impurities is not more than 3.24%; Dissolution is 84.8% after measured.
Embodiment 11
Danshen root salvianolic acid A extracts purification:
Radix Salviae Miltiorrhizae obtains aqueous extract with water extraction, transfers pH value to 4.0,125 ℃ of temperature, gauge pressure 0.06MPa pressure, heats 5.5 hours; Solution filters, and filtrate is separated through the HPD-100A macroporous resin column chromatography, and first water, 15% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 60% concentration is collected and contained the salvianolic acid A part, and eluent is concentrated into does not have the alcohol flavor; Concentrated solution separates with sephadex lh-20, and first water, 45% alcoholic solution eluting discard eluent, and reuse 55% alcoholic solution eluting concentrates ethanol to most; Concentrated solution is transferred pH value to 2.5, through the extraction of organic solvent isopropyl alcohol, separates the organic solvent phase, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
Crude drug is: danshen root salvianolic acid A 58 grams, filler lactose 197 grams, disintegrating agent hyprolose 5.9 grams, magnesium stearate lubricant 3.8 restrain, (weight of wetting agent is to obtain promptly according to the density of dehydrated alcohol and volume calculation to wetting agent dehydrated alcohol 29 grams: m=v* ρ).
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains 1000 in the tablet of unit dose; Unit dose 58mg.Preparation adopts high effective liquid chromatography for measuring, is object of reference with the danshen root salvianolic acid A, and relative retention time is that 0.71 impurity content is 0.58%, and relative retention time is that 1.36 impurity content is 3.47%, and the content of total impurities is not more than 5.68%; Dissolution is 87.2% after measured.
Annotate: the present invention's concrete technical scheme required for protection is not limited to the concrete combination of the expressed technical scheme of the foregoing description.
Claims (6)
1. red sage root salvianolic acid A tablet for the treatment of cardiovascular disease consists of the crude drug and the pharmaceutic adjuvant of danshen root salvianolic acid A, it is characterized in that preparation adopts high effective liquid chromatography for measuring, 3 total peaks; Be 90 minutes writing time, is object of reference with the danshen root salvianolic acid A, and its retention time is that the relative retention time of 1,2 impurity is respectively 0.71,1.36; With the danshen root salvianolic acid A is contrast, and relative retention time is 0.71 impurity, and its content is 0.10-1.96%, and relative retention time is that 1.36 impurity content is 0.11-5.97%, and the content of total impurities is not more than 10.0%; Wherein relative retention time is that 1.36 impurity is salvia miltiorrhiza tanshinoate C in the high-efficient liquid phase chromatogram; Wherein high-efficient liquid phase chromatogram process measuring method is:
Chromatographic condition and system suitability experiment: with the octadecylsilane chemically bonded silica is filler; Flow velocity 1.0ml/min; 35 ℃ of column temperatures; Detect wavelength 286nm; Number of theoretical plate should be not less than 60000 by salvianolic acid A; With acetonitrile-0.2% aqueous acetic acid is mobile phase, carries out gradient elution by following condition of gradient elution, moves 90 minutes;
In the time of 0-15 minute, the ratio of acetonitrile reduces to 80% by 10% ratio that rises to 20%, 0.2% aqueous acetic acid by 90%; In the time of 15-55 minute, the ratio of acetonitrile reduces to 70% by 20% ratio that rises to 30%, 0.2% aqueous acetic acid by 80%; In the time of 55-65 minute, the ratio of acetonitrile reduces to 50% by 30% ratio that rises to 50%, 0.2% aqueous acetic acid by 70%; In the time of 65-72 minute, the ratio of acetonitrile reduces to 20% by 50% ratio that rises to 80%, 0.2% aqueous acetic acid by 50%; In the time of 72-77 minute, the ratio 20% of ratio 80%, 0.2% aqueous acetic acid of acetonitrile; In the time of 77-80 minute, the ratio of acetonitrile rises to 90% by 80% ratio of reducing to 10%, 0.2% aqueous acetic acid by 20%; In the time of 80-90 minute, keep acetonitrile-0.2% aqueous acetic acid to carry out eluting with 10: 90 ratio;
The preparation of reference substance solution: precision takes by weighing the salvianolic acid A reference substance in volumetric flask, adds dissolve with methanol and shakes up, and be diluted to scale;
The preparation of need testing solution: precision takes by weighing sample, and standardize solution adds dissolve with methanol and shakes up in volumetric flask after treatment, and is diluted to scale;
Algoscopy: accurate respectively reference substance solution and the need testing solution drawn, inject chromatograph of liquid, the record chromatogram, promptly;
Crude drug is: danshen root salvianolic acid A 40-60 weight portion, and pharmaceutic adjuvant is: filler 50-200 weight portion, disintegrating agent 1-6 weight portion, lubricant 1-4 weight portion, wetting agent dehydrated alcohol 20-30 weight portion; Wherein filler is a kind of in lactose, starch, dextrin, Icing Sugar, microcrystalline Cellulose, pregelatinized Starch, mannitol, calcium hydrogen phosphate, the calcium sulfate; Wherein disintegrating agent is a kind of in carboxymethyl starch sodium, hydroxypropyl starch, hyprolose, starch, polyvinylpolypyrrolidone, cross-linking sodium carboxymethyl cellulose, the microcrystalline Cellulose; Wherein lubricant is a kind of in magnesium stearate, Pulvis Talci, micropowder silica gel, the Polyethylene Glycol; Its preparation method is:
The danshen root salvianolic acid A preparation method:
Radix Salviae Miltiorrhizae water or alcoholic solution extract and obtain aqueous extract or alcohol extract, alcohol extract concentrates ethanol to most, adjust pH to 7.5-9.0,30-80 ℃ temperature, heating 1-6 hour or adjust pH to 3.5-6.0,110-130 ℃ temperature, gauge pressure 0.05MPa-0.17MPa pressure, heated 1-6 hour; Solution filters, and filtrate is separated through nonpolar or low pole macroporous resin column chromatography, and first water, 10-30% Diluted Alcohol eluting are removed impurity, and the ethanol elution of reuse 30-70% concentration is collected eluent, and eluent reclaims eluant to most; Concentrated solution is used the eluant eluting with silica gel or sephadex lh-20 or the separation of polyamide chromatography post; Wherein silica gel column chromatography adopts chloroform: methanol: formic acid=5: 1: 0.1 is eluting solvent; Wherein sephadex lh-20 or polyamide chromatography post separate, and first water, 20-50% alcoholic solution eluting discard eluent, reuse 50-95% alcoholic solution eluting; Collect eluent, eluent reclaims eluant to most; The concentrated solution adjust pH through organic solvent extraction, separates the organic solvent phase to 2-5, must contain drug solns, concentrates, and drying or lyophilization get danshen root salvianolic acid A;
Formulation preparation:
Crude drug danshen root salvianolic acid A, filler, disintegrating agent, lubricant, wetting agent dehydrated alcohol;
Get danshen root salvianolic acid A and filler, disintegrating agent mix homogeneously, add ethanol solution and make soft material, cross 18 mesh sieves and select granule, 60 ℃ of dryings 40 minutes, granulate adds lubricant, mixing, tabletting obtains the tablet in the unit dose of danshen root salvianolic acid A.
2. a kind of red sage root salvianolic acid A tablet for the treatment of cardiovascular disease according to claim 1, wherein crude drug is: danshen root salvianolic acid A 40-60 weight portion, pharmaceutic adjuvant is: filler 150-200 weight portion, disintegrating agent 4-6 weight portion, lubricant 1-4 weight portion, wetting agent dehydrated alcohol 20-30 weight portion.
3. a kind of red sage root salvianolic acid A tablet for the treatment of cardiovascular disease according to claim 1, wherein said macroporous resin column are HPD-100, HPD-100A, HPD-300, HPD-400, HPD-400A, HPD-450, D101,1300-I, 1400 or AB-8.
4. a kind of red sage root salvianolic acid A tablet for the treatment of cardiovascular disease according to claim 1, wherein said organic solvent are selected from a kind of in ethyl acetate, propyl acetate, butyl acetate, n-butyl alcohol, the isopropyl alcohol.
5. a kind of red sage root salvianolic acid A tablet for the treatment of cardiovascular disease according to claim 1, wherein unit dose is 5-1000mg.
6. a kind of red sage root salvianolic acid A tablet for the treatment of cardiovascular disease according to claim 1, wherein unit dose is 20-500mg.
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