CN101468183B - Pharmaceutical combination and preparation method thereof - Google Patents

Pharmaceutical combination and preparation method thereof Download PDF

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CN101468183B
CN101468183B CN2007103038763A CN200710303876A CN101468183B CN 101468183 B CN101468183 B CN 101468183B CN 2007103038763 A CN2007103038763 A CN 2007103038763A CN 200710303876 A CN200710303876 A CN 200710303876A CN 101468183 B CN101468183 B CN 101468183B
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CN101468183A (en
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侯凤祥
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Heilongjiang Double Star Pharmaceutical Co Ltd
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Abstract

The present invention discloses a medicine composition which is used preventing or treating hepatic fibrosis or early hepatocirrhosis, a preparing method and a quality control method thereof. The composition is composed of ten Chinese medicine herbs of astragalus, salvia root, zedoary turmeric, bighead atractylodes rhizome, curcuma aromatica, capillary artemisia, radix bupleuri, peach kernel, rhizoma menispermi and liquorice. The methods of extracting, decocting, filtering, condensing, etc. are executed to different components in the preparing process of medicine composition for fully exerting the effective medicine. Simultaneously the invention also provides a quality control method for identifying the components of medicine composition and measuring the contents. A considerable amount of experiments shows that the medicine composition has actual curative effect for preventing or treating hepatic fibrosis or early hepatocirrhosis with safe and effective clinical use.

Description

A kind of pharmaceutical composition and preparation method thereof
Technical field
The present invention relates to a kind of pharmaceutical composition and preparation method thereof and method of quality control, pharmaceutical composition of particularly a kind of prevention or treatment hepatic fibrosis or early stage liver cirrhosis and preparation method thereof and method of quality control.
Background technology
Hepatic fibrosis is to cause on chronic hepatic injury basis due to the extracellular matrix paraplasm, and it is the only stage which must be passed by that develops into liver cirrhosis, and the blocking-up hepatic fibrosis then might be controlled the generation of liver cirrhosis.And cause chronic hepatic injury and might form hepatic fibrosis person, the most common with chronic hepatitis B.Chronic hepatitis B is common infectious disease, and the number that hepatitis B surface antigen (HBsAg) is carried in the whole world surpasses 2.8 hundred million.China is the high popular area of hepatitis B, has the crowd of 40%-60% infected by HBV, and 9.97% crowd (about 100,012,000 populations) is the HBsAg carrier.The adult be subjected to hepatitis b virus infected after, develop into chronic hepatitis person and account for 10%-20%, can develop into liver cirrhosis through hepatic fibrosis from chronic hepatitis, general only through time of 2-6.Therefore, explore the effective ways of control hepatic fibrosis and the vital task that medicine is clinical research.
Hepatic fibrosis is one of important pathological change of chronic hepatitis B, is the only stage which must be passed by that develops into liver cirrhosis.Therefore, stoping and improving hepatic fibrosis is the breach of capturing chronic hepatitis, early stage liver cirrhosis, is the difficult problem that current medical science remains to be captured.Late world-renowned expert Hans Popper once pointed out: who can stop or slow down the generation of hepatic fibrosis, and who just will cure most of chronic hepatopathys.Modern medicine has the report for the treatment of hepatic fibrosis from different perspectives, but clinical efficacy is not satisfactory, and clinical research does not have substantial progress yet.
Summary of the invention
The object of the invention is to provide a kind of pharmaceutical composition; Another object of the present invention is to provide the preparation method of said composition; The 3rd purpose of the present invention is to provide the method for quality control of said preparation.
The present invention seeks to be achieved through the following technical solutions:
The crude drug of pharmaceutical composition of the present invention consists of:
The Radix Astragali 400～600 weight portion Radix Salviae Miltiorrhizaes 150～300 weight portion Rhizoma Curcumae 150～300 weight portions
The Rhizoma Atractylodis Macrocephalae 150～300 weight portion Radix Curcumaes 100～200 weight portion Herba Artemisiae Scopariaes 100～200 weight portions
Radix Bupleuri 100～200 weight portion Semen Persicaes 100～200 weight portion Rhizoma Menispermis 50～150 weight portions
Radix Glycyrrhizae 50～150 weight portions.
The crude drug preferred group of pharmaceutical composition of the present invention becomes:
The Radix Astragali 510 weight portion Radix Salviae Miltiorrhizaes 200 weight portion Rhizoma Curcumae 200 weight portion Rhizoma Atractylodis Macrocephalaes 200 weight portions
Radix Curcumae 150 weight portions 150 weight portion Radix Bupleuri 150 weight portion Semen Persicaes 150 weight portions
Rhizoma Menispermi 100 weight portion Radix Glycyrrhizaes 100 weight portions.
The crude drug preferred group of pharmaceutical composition of the present invention becomes:
The Radix Astragali 450 weight portion Radix Salviae Miltiorrhizaes 200 weight portion Rhizoma Curcumae 200 weight portions
The Rhizoma Atractylodis Macrocephalae 200 weight portion Radix Curcumaes 120 weight portion Herba Artemisiae Scopariaes 120 weight portions
Radix Bupleuri 120 weight portion Semen Persicaes 120 weight portion Rhizoma Menispermis 80 weight portions
Radix Glycyrrhizae 80 weight portions.
The crude drug preferred group of pharmaceutical composition of the present invention becomes:
The Radix Astragali 550 weight portion Radix Salviae Miltiorrhizaes 250 weight portion Rhizoma Curcumae 250 weight portions
The Rhizoma Atractylodis Macrocephalae 250 weight portion Radix Curcumaes 180 weight portion Herba Artemisiae Scopariaes 180 weight portions
Radix Bupleuri 180 weight portion Semen Persicaes 180 weight portion Rhizoma Menispermis 120 weight portions
Radix Glycyrrhizae 120 weight portions.
Pharmaceutical composition of the present invention adds conventional adjuvant, according to common process, make tablet, capsule, powder, soft capsule, drop pill, honeyed pill, pill, granule, soft extract with bee honey agent, slow releasing preparation, quick releasing formulation, controlled release preparation, oral liquid or ejection preparation.
Pharmaceutical composition of the present invention can also be prepared by following method:
Get Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae extraction volatile oil, the water solution A after distillation device is in addition preserved, and medicinal residues A is standby; Radix Salviae Miltiorrhizae is added 4～8 times of amount 60～90% ethanol extractions 2～4 times, and each 1～3 hour, merge extractive liquid, filtered, and decompression recycling ethanol is concentrated into thick paste, in 40～80 ℃ of oven dry, and the powder A that gets dry extract, medicinal residues B is standby; With Radix Astragali extracting in water 2～4 times, the time is each 1～3 hour, and water consumption is each 8～10 times, collecting decoction filters, and filtrate is concentrated into every parts by volume and is equivalent to 0.2～0.8 weight portion medical material, with 3000 rev/mins centrifugal, remove partial impurities, supernatant concentration is standby to thick paste A; All the other Radix Bupleuri, Semen Persicae, Herba Artemisiae Scopariae, Rhizoma Menispermi, Radix Glycyrrhizae five kinds of Chinese medicine and Radix Salviae Miltiorrhizae decoction dregs B and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, the medicinal residues A of Radix Curcumae three flavor medicines merges, decoct with water 2～4 times, water consumption is each 8～10 times, time is each 1～3 hour, collecting decoction, filter, filtrate and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, the water solution A of Radix Curcumae three flavor merges, and being concentrated into and recording relative density in the time of 60 ℃ is 1.0～1.5, adds ethanol and makes and contain the alcohol amount and reach 60%, leave standstill, filter, decompression filtrate recycling ethanol also is concentrated into thick paste B, and thick paste B and Radix Astragali water extract thick paste A merge, in 40～80 ℃ of oven dry, the powder B that gets dry extract is standby; Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae volatile oil are made beta-CD inclusion, above-mentioned dried cream powder A is mixed with dried cream powder B, be ground into 60～100 order fine powders and with volatile oil beta-CD inclusion mixing, add conventional adjuvant, according to common process, make clinical or pharmaceutically acceptable tablet, capsule, powder, soft capsule, drop pill, honeyed pill, pill, granule, soft extract with bee honey agent, slow releasing preparation, quick releasing formulation, controlled release preparation, oral liquid or ejection preparation.
Preparation of drug combination method of the present invention is preferably:
Get Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae extraction volatile oil, the water solution A after distillation device is in addition preserved, and medicinal residues A is standby; Radix Salviae Miltiorrhizae is added 6 times of amount 80% ethanol extractions 3 times, each 1 hour, merge 3 times extracting solution, filter, decompression recycling ethanol is concentrated into the thick paste shape, in 60 ℃ of oven dry, the powder A that gets dry extract, medicinal residues B is standby; With Radix Astragali extracting in water 3 times, the time was respectively 2,1,1 hours, and water consumption is respectively 10,8,8 times, collecting decoction filters, and filtrate is concentrated into every parts by volume and is equivalent to 0.5 weight portion medical material, with 3000 rev/mins centrifugal, remove partial impurities, supernatant concentration is standby to thick paste A; All the other Radix Bupleuri, Semen Persicae, Herba Artemisiae Scopariae, Rhizoma Menispermi, Radix Glycyrrhizae five kinds of Chinese medicine and Radix Salviae Miltiorrhizae decoction dregs B and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, the medicinal residues A of Radix Curcumae three flavor medicines merges, decoct with water 3 times, water consumption is respectively 10,8,8 times, time is respectively 2,1,1 hour, merge 3 times decocting liquid, filter, filtrate and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, the water solution A of Radix Curcumae three flavors merges, being concentrated into and recording relative density in the time of 60 ℃ is 1.13, adds ethanol and makes and contain alcohol amount and reach 60%, standing over night, filter, decompression filtrate recycling ethanol also is concentrated into thick paste thick paste B, and thick paste B and Radix Astragali water extract thick paste A are incorporated in 60 ℃ of oven dry, and the powder B that gets dry extract is standby; Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae volatile oil are made beta-CD inclusion, above-mentioned dried cream powder A is mixed with dried cream powder B, be ground into 80 order fine powders and with volatile oil beta-CD inclusion mixing, add conventional adjuvant, according to common process, make clinical or pharmaceutically acceptable tablet, capsule, powder, soft capsule, drop pill, honeyed pill, pill, granule, soft extract with bee honey agent, slow releasing preparation, quick releasing formulation, controlled release preparation, oral liquid or ejection preparation.
The method of quality control of drug combination preparation of the present invention comprises following assay and/or following one or more discriminatings:
Assay: get drug combination preparation 3～8 weight portions of the present invention, the accurate title, decide, put in the apparatus,Soxhlet's, add 40～60 parts by volume, 1～3% potassium hydroxide methanol solution, heating and refluxing extraction 4～8 hours, merceration spends the night again, and the extracting solution reclaim under reduced pressure is to doing, residue adds 10～30 parts by volume water dissolutioies, use chloroform: the mixed solution of n-butyl alcohol=2: 1, extract three times, be respectively 30～40 parts by volume, combined chloroform: following butanols=1～3: 0.5～1 extract, with the washing of 1～3% potassium dihydrogen phosphate aqueous solution, 50～80 parts by volume, aqueous phase discarded, organic facies evaporated under reduced pressure, it is fixed molten to add 1～3 parts by volume methanol solution, as need testing solution; Precision takes by weighing the astragaloside reference substance in addition, adds methanol and makes the solution that 1 parts by volume contains 0.001～0.002 weight portion, in contrast product solution; Test according to thin layer chromatography, accurate need testing solution 0.001～0.003 parts by volume of drawing, reference substance solution 0.001～0.003 parts by volume and 0.002～0.005 parts by volume, the cross point is on same silica gel g thin-layer plate respectively, under the room temperature with chloroform: methanol: water=60～80: 20～30: the solution of 5～10 ratios was placed after 12 hours, took off layer and was developing solvent, launched once, take out, dry, spray 5～15% concentrated sulphuric acid alcoholic solution, 80～120 ℃ were heated 3～8 minutes down again, take out, cover onesize glass plate again on the lamellae, fix with hinge on every side, scan according to thin layer chromatography, wavelength X s=530nm, λ R=650nm measures test sample trap integrated value and reference substance trap integrated value, calculates, that is, drug combination preparation of the present invention per diem taking dose meter contains astragaloside C44H6804, must not be lower than 2.700mg;
Differentiate: A, according to assay item preparation need testing solution down, get the reference substance astragaloside and add methanol to make the solution that 1 parts by volume contains 0.001～0.003 weight portion be reference substance solution; Other gets Milkvetch Root 1～3 weight portion, put in the Sha Shi extractor, adding 1～3% potassium hydroxide methanol solution extracted after 6～10 hours, one night of merceration, reclaim and extract solution to doing, add water 10～30 parts by volume, again with chloroform: n-butyl alcohol=1～3: the extraction of 0.5～1.5 proportioning concentration is respectively 40,30,30 parts by volume three times, combining extraction liquid adds 1～3% potassium dihydrogen phosphate 60 parts by volume washing, aqueous phase discarded, again once with 60 parts by volume water washings, organic facies is recycled to dried, adds 1～3 parts by volume dissolve with methanol, makes control medicinal material solution; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.001～0.002 parts by volume of above-mentioned three kinds of solution, put respectively in same 15cm * 10cm silica gel G aluminium sheet, under the room temperature with chloroform: methanol: water=60～80: 20～30: the solution of 5～10 ratios was placed after 12 hours, took off layer and was developing solvent, launched once, take out, dry, spray 5～15% concentrated sulphuric acid alcoholic solution are heated to clear spot; In the test sample chromatograph, with reference substance and the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
B, get drug combination preparation 8～10 weight portions of the present invention and place round-bottomed flask, add water 200～300 parts by volume and be heated to boiling and extract volatile oil, after about 1～3 hour, collect volatile oil, with petroleum ether volatile oil extractor tube wall, and join in the volatile oil, as test sample; Other gets Rhizoma Atractylodis Macrocephalae control medicinal material 1～3 weight portion, pulverizes, and crosses 40 mesh sieves, shines the medical material need testing solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.001～0.002 parts by volume of above-mentioned two kinds of solution, put respectively in same block of silica gel G aluminium sheet, with petroleum ether: ethyl acetate=5: 0.1 is developing solvent, launch at normal temperatures, take out, dry, spray 1～3% vanillin ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
C, get drug combination preparation 4～8 weight portions of the present invention, put in the triangular flask, add the vibration of ether 30～50 parts by volume, after leaving standstill 1～2 hour, filter, reclaim ether extraction solution to doing, residue adds 1～3 parts by volume ethyl acetate makes dissolving, standby as need testing solution; Other gets Radix Salviae Miltiorrhizae control medicinal material 1 weight portion, pulverizes, and crosses 40 mesh sieves, shines medical material solution in pairs with legal system, and it is standby to do the control medicinal material test liquid; Take by weighing Tanshinone I Ia reference substance 0.001～0.003 weight portion, add ethyl acetate solution and make concentration to be that every parts by volume contains the reference substance solution of 0.0008～0.0012 weight portion Tanshinone I Ia standby; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.003～0.008 parts by volume of above-mentioned three kinds of solution, put respectively in same block of silica gel G aluminium sheet, with benzene: ethyl acetate=9～10: 0.5～1 solution is developing solvent, launch at normal temperatures, take out, dry; In the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color;
D; get drug combination preparation 8～10 weight portions of the present invention; put in the conical flask; add 90～100% methanol, 40～60 parts by volume; use power 150W; the supersonic oscillations of frequency 20kHz are after 20～50 minutes; filter, get subsequent filtrate 20～40 parts by volume, decompression and solvent recovery is put dried; residue adding distil water 10～30 parts by volume slight fevers dissolving; use water saturation n-butanol extraction three times, volume is respectively 10～20 parts by volume merges butanol extraction liquid; twice of reuse 1～3% potassium hydroxide washing butanol extraction liquid; volume is respectively 10～20 parts by volume, discards alkali liquor, and butanol extraction liquid is washed till neutrality with the n-butyl alcohol saturated aqueous solution; discard water layer; the reclaim under reduced pressure butanol extraction liquid is done, residue adds the dissolving of 90～100% ethanol, 1～3 parts by volume, as need testing solution; Other gets Radix Bupleuri medical material 1 weight portion, shines medical material solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.004～0.008 parts by volume of above-mentioned two kinds of solution, put respectively in same block of silica gel G aluminium sheet, with chloroform: methanol: water=5～8: 2～5: 1～3 solution is developing solvent, launch at normal temperatures, take out, dry, spray 5～15% ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
E, get drug combination preparation 3～8 weight portions of the present invention, put in the triangular flask, add the vibration of ether 30～50 parts by volume, leave standstill 1～2 hour after, filter, discard the ether layer, add methanol 20～40 parts by volume, reflux 1～2 hour is filtered, reclaim methanol solution to doing, residue adds water 30～50 parts by volume makes dissolving, uses water saturation n-butanol extraction 3 times, each 10～30 parts by volume, merge butanol solution, wash with water 3 times, each 10～30 parts by volume are with the n-butanol layer evaporate to dryness, residue adds the dissolving of methanol 1～3 parts by volume, as need testing solution; Extracting liquorice control medicinal material 1～3 weight portion shines medical material solution in pairs with legal system in addition; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.003～0.008 parts by volume of above-mentioned two kinds of solution, put respectively in same block of silica gel G aluminium sheet, with chloroform: methanol: formic acid=2～8: 1～3: 0.1～0.3 solution is developing solvent, launch at normal temperatures, take out, dry, spray 30～80% sulphuric acid methanol solutions, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
The method of quality control of drug combination preparation of the present invention is preferably following assay and/or discriminating:
Assay: get drug combination preparation 5 weight portions of the present invention, the accurate title, decide, put in the apparatus,Soxhlet's, add 50 parts by volume, 2% potassium hydroxide methanol solution, heating and refluxing extraction 6 hours, merceration spends the night again, and the extracting solution reclaim under reduced pressure is to doing, residue adds 15 parts by volume water dissolutioies, use chloroform: the mixed solution of n-butyl alcohol=2: 1, extract three times, be respectively 40,30,30 parts by volume, combined chloroform: the extract of following butanols=2: 1, with the washing of 1% potassium dihydrogen phosphate aqueous solution, 60 parts by volume, aqueous phase discarded, organic facies evaporated under reduced pressure, it is fixed molten to add 1 parts by volume methanol solution, as need testing solution; Precision takes by weighing the astragaloside reference substance in addition, adds methanol and makes the solution that 1 parts by volume contains 0.001 weight portion, in contrast product solution; Test according to thin layer chromatography, accurate need testing solution 0.001 parts by volume of drawing, reference substance solution 0.002 parts by volume and 0.004 parts by volume, the cross point is on same silica gel g thin-layer plate respectively, under the room temperature with chloroform: methanol: the solution of water=75: 25: 5 ratio was placed after 12 hours, took off layer and was developing solvent, launched once, take out, dry, spray 10% concentrated sulphuric acid alcoholic solution, 100 ℃ were heated 5 minutes down again, take out, cover onesize glass plate again on the lamellae, fix with hinge on every side, scan according to thin layer chromatography, wavelength X s=530nm, λ R=650nm measures test sample trap integrated value and reference substance trap integrated value, calculates, that is, drug combination preparation of the present invention per diem taking dose meter contains astragaloside C44H6804, must not be lower than 2.700mg;
Differentiate: A, according to assay item preparation need testing solution down, get the reference substance astragaloside and add methanol to make the solution that 1 parts by volume contains 0.001 weight portion be reference substance solution; Other gets Milkvetch Root 1.5 weight portions, put in the Sha Shi extractor, adding 2% potassium hydroxide methanol solution extracted after 8 hours, one night of merceration, reclaim and extract solution to doing, add water 15 parts by volume, again with chloroform: n-butyl alcohol=proportioning concentration extraction in 2: 1 is respectively 40,30,30 parts by volume three times, combining extraction liquid adds 1% potassium dihydrogen phosphate 60 parts by volume washing, aqueous phase discarded, again once with 60 parts by volume water washings, organic facies is recycled to dried, adds 1 parts by volume dissolve with methanol, makes control medicinal material solution; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.002 parts by volume of above-mentioned three kinds of solution, put respectively in same 15cm * 10cm silica gel G aluminium sheet, under the room temperature with chloroform: methanol: the solution of water=75: 25: 5 ratio was placed after 12 hours, took off layer and was developing solvent, launched once, take out, dry, spray 10% concentrated sulphuric acid alcoholic solution, be heated to clear spot; In the test sample chromatograph, with reference substance and the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
B, get drug combination preparation 9 weight portions of the present invention and place round-bottomed flask, add water 250 parts by volume and be heated to boiling and extract volatile oil, after about 1～2 hour, collect volatile oil, with petroleum ether volatile oil extractor tube wall, and join in the volatile oil, as test sample; Other gets Rhizoma Atractylodis Macrocephalae control medicinal material 2 weight portions, pulverizes, and crosses 40 mesh sieves, shines the medical material need testing solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.002 parts by volume of above-mentioned two kinds of solution, put respectively in same block of silica gel G aluminium sheet, with petroleum ether: ethyl acetate=5: 0.1 is developing solvent, launch at normal temperatures, take out, dry, spray 1% vanillin ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
C, get drug combination preparation 6 weight portions of the present invention, put in the triangular flask, add the vibration of ether 40 parts by volume, leave standstill 1 hour after, filter, reclaim ether extraction solution to doing, residue adds 2 parts by volume ethyl acetate makes dissolving, standby as need testing solution; Other gets Radix Salviae Miltiorrhizae control medicinal material 1 weight portion, pulverizes, and crosses 40 mesh sieves, shines medical material solution in pairs with legal system, and it is standby to do the control medicinal material test liquid; Take by weighing Tanshinone I Ia reference substance 0.002 weight portion, add ethyl acetate solution and make concentration to be that every parts by volume contains the reference substance solution of 0.001 weight portion Tanshinone I Ia standby; According to the test of an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.005 parts by volume of above-mentioned three kinds of solution, put respectively in same block of silica gel G aluminium sheet, with benzene: the solution of ethyl acetate=9.5: 0.5 is developing solvent, launch at normal temperatures, take out, dry; In the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color;
D; get drug combination preparation 9 weight portions of the present invention; put in the conical flask; add 95% methanol, 50 parts by volume; use power 150W; the supersonic oscillations of frequency 20kHz are after 40 minutes; filter, get subsequent filtrate 30 parts by volume, decompression and solvent recovery is put dried; residue adding distil water 15 parts by volume slight fevers dissolving; use water saturation n-butanol extraction three times, volume is respectively 20; 20; 10 parts by volume merges butanol extraction liquid; twice of reuse 1% potassium hydroxide washing butanol extraction liquid; volume is respectively 20; 20 parts by volume, discards alkali liquor, and butanol extraction liquid is washed till neutrality with the n-butyl alcohol saturated aqueous solution; discard water layer; the reclaim under reduced pressure butanol extraction liquid is done, residue adds the dissolving of 95% ethanol, 1 parts by volume, as need testing solution; Other gets Radix Bupleuri medical material 1 weight portion, shines medical material solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.006 parts by volume of above-mentioned two kinds of solution, put respectively in same block of silica gel G aluminium sheet, with chloroform: methanol: the solution of water=7: 3: 1 is developing solvent, launch at normal temperatures, take out, dry, spray 10% ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
E, get drug combination preparation 5 weight portions of the present invention, put in the triangular flask, add the vibration of ether 40 parts by volume, leave standstill 1 hour after, filter, discard the ether layer, add methanol 30 parts by volume, reflux 1 hour is filtered, and reclaims methanol solution to doing, residue adds water 40 parts by volume makes dissolving, uses water saturation n-butanol extraction 3 times, each 20 parts by volume, merge butanol solution, wash each 20 parts by volume with water 3 times, with the n-butanol layer evaporate to dryness, residue adds the dissolving of methanol 1 parts by volume, as need testing solution; Extracting liquorice control medicinal material 1 weight portion shines medical material solution in pairs with legal system in addition; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.005 parts by volume of above-mentioned two kinds of solution, put respectively in same block of silica gel G aluminium sheet, with chloroform: methanol: the solution of formic acid=5: 1: 0.1 is developing solvent, launch at normal temperatures, take out, dry, spray 50% sulphuric acid methanol solution, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
The ratio of weight portion of the present invention and parts by volume is a grams per milliliter.Herba Artemisiae Scopariae is the dry aerial parts of feverfew BINHAO or Herba Artemisiae Scopariae in the crude drug, should meet pertinent regulations under the 192nd page of Herba Artemisiae Scopariae item of Pharmacopoeia of People's Republic of China of version in 2000.Rhizoma Menispermi is the dry rhizome of menispermaceous plants Caulis menispermi in the crude drug, should meet the pertinent regulations under the 74th page of Rhizoma Menispermi item of 2000 editions Chinese Pharmacopoeias.Radix Glycyrrhizae is glycyrrhizic legume Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L. in the crude drug, meets pertinent regulations under the 65th page of Radix Glycyrrhizae item of Pharmacopoeia of People's Republic of China of version in 2000.Milkvetch Root is 0.114%, 0.113%, 0.043% by three batches of medical material astragaloside content of determination mensuration under the Chinese Pharmacopoeia Radix Astragali item in the crude drug.
Use the human albumin and duplicate the rat liver fibrosis model, observe the therapeutical effect of granule of the present invention to this model, be 3 months and 6 months the course of treatment.Get the liver immunohistochemical staining when finish the course of treatment and observe the variation of hepatic tissue I, III, IV Collagen Type VI.Colchicine and granule of the present invention all can make I, III, the degraded of IV Collagen Type VI, but more obvious with the latter.Granule of the present invention can impel the degraded of I, III Collagen Type VI, and is more obvious to the degraded of type i collagen especially, 1 month course of treatment and when finished the course of treatment in 3 months, with model group significant difference arranged more all; Granule of the present invention also can promote the degraded of IV Collagen Type VI, reduces the IV type collagen content, has the important clinical meaning.
Show that through rat test when finishing respectively at 1st month, the 3rd a month course of treatment, femoral artery is got blood 3-5ml and detected D-II aggressiveness, AT-III and erythrocyte deformability.The result: compare with model group and matched group, granule of the present invention can obviously improve hypercoagulability, and different course all can improve erythrocyte deformability, and makes D-II aggressiveness, AT-III reduce, and dose-effect and timeliness dependency are arranged.Illustrate that this medicine has function of promoting blood circulation to disperse blood clots preferably, especially improve the effect of erythrocyte deformability, during to hepatic fibrosis in the liver blood stasis state can play the effect of unobstructed blood vessels.
Set up the low drag of immune function of mice and under the normal immune state of mice, observe granule of the present invention with prednisone effect of immunologic function, granule of the present invention as a result is to normal mouse and when using prednisone and causing immune function of mice low, all can improve NK cytoactive and the lymhocyte transformation rate of mice to a certain extent, but little to cytophagous phagocytosis influence.At normal mouse, granule of the present invention reduces TNF-α, IL-2 activity, and to the obviously influence of the active nothing of TNF-α, the IL-2 of immunologic hypofunction state.
Following experimental example is used to further specify but is not limited to the present invention.
The extraction conditions of experimental example 1 Radix Astragali is investigated
A. the selection of extraction solvent
Get Radix Astragali decoction pieces 20g, extract with 50% ethanol extraction, 70% ethanol extraction, water extraction and four kinds of methods of the water extraction 50% alcohol precipitation removal of impurity respectively, extracting solution adopts thin layer chromatography scanning (Chinese Pharmacopoeia nineteen ninety-five version, one one, the 272nd page) measure the content of astragaloside in four kinds of method extracting solution.Interpretation of result as a result, astragaloside content height in the sample of water and 50% ethanol extraction, and also data are very approaching.And it is all lower to adopt water to carry the sample astragaloside content of 50% ethanol precipitation and 70% ethanol extraction, so should adopt water or 50% ethanol extraction.Because made from the water extraction decapacitation outside astragaloside fully proposes, can also keep astragalus polysaccharides to greatest extent, so the method for decision employing water extraction.
B. the investigation of Radix Astragali decocting condition
Get Radix Astragali decoction pieces 10g, totally 9 parts, press factor level and experimental condition experiment arrangement, soak 1 hour respectively after, benefit adds water to aequum, heating extraction, and extracting solution merges, and filters, water-bath is concentrated into below the 50ml, is placed to room temperature, is transferred in the 50ml volumetric flask, is diluted to scale with water.Press the content of tlc scanning determination astragaloside.And the result carried out variance analysis.As can be known, A factor (extraction time) does not have significance influence, A to the result 3Be optimum condition; C factor (extraction time) has appreciable impact to the result, C 3Be optimum condition; D factor (amount of water) does not have significance influence, D to the result 3Be optimum condition.(because the sum of deviation square and the void item of D factor are close, so merge as Error processing with void item) its optimised process is A 3C 3D 3, also promptly: add 26 times in water (10,8,8) heating extraction 3 times, 2 hours for the first time, the 2nd, 3 time each 1 hour.
The optimum extraction condition of experimental example 2 Radix Salviae Miltiorrhizaes
The chemical constituent of Radix Salviae Miltiorrhizae comprises liposoluble constituent and water soluble ingredient two parts.Liposoluble constituent such as tanshinone, danshensu class, iso tanshinone class etc., water soluble ingredient such as protocatechualdehyde class, protocatechuic acid etc.Pharmacological evaluation proves, Tanshinone I I AHave the obvious suppression coagulation function Deng liposoluble constituent, explanation is the active component of blood circulation promoting and blood stasis dispelling.So adopt Different concentrations of alcohol to extract Radix Salviae Miltiorrhizae respectively, with Tanshinone I I ABe index, the optimal determining alcohol of selective extraction fat soluble ingredient of red sage root.Adopt the orthogonal design scheme again, select the optimum extraction condition of fat soluble ingredient of red sage root.
A. the selection of concentration of alcohol
Get a certain amount of Radix Salviae Miltiorrhizae, respectively with 60%, 70%, 80% and 95% ethanol extraction, with Tanshinone I I ABe index, the optium concentration of selective extraction as seen, with 80% ethanol extraction person, Tanshinone I I AContent is the highest, and extraction ratio has also surpassed 60%, so adopt 80% ethanol extraction.
B. Radix Salviae Miltiorrhizae 80% ethanol extraction orthogonal test
Take by weighing 4 parts of salvia pieces, every part of 5.0g uses 80% ethanol extraction, and extracting solution filters, and puts to room temperature, is settled in the 100ml volumetric flask, measures Tanshinone I I in each sample AContent, the result as can be known, A factor (ethanol consumption) and C factor (extraction time) are to influence Tanshinone I I AThe principal element of extracted amount, in this group sample, the Tanshinone I I of No. 2 samples AThe highest (the A of extracted amount 1B 2C 2), so select for use condition to be: extract each 1 hour 3 times with 6 times of amounts of 80% ethanol.The extraction of experimental example 3 Rhizoma Curcumae, Radix Curcumae, Rhizoma Atractylodis Macrocephalae effective ingredient
Rhizoma Curcumae, Radix Curcumae, the Rhizoma Atractylodis Macrocephalae three flavor medicines contain volatile oil.Rhizoma Curcumae is a drug for invigorating blood circulation and eliminating stasis, and modern study shows that Rhizoma Curcumae injection have antitumaous effect, and its main effective ingredient is a volatile oil.Radix Curcumae is the vital energy regualting and blood circulation-promoting medicine, has regulating qi to disperse stagnation, effects such as promoting the function of the gallbladder to alleviate jaundice, and its main effective ingredient is a volatile oil.The Rhizoma Atractylodis Macrocephalae has effects such as invigorating the spleen and benefiting QI dampness diuretic.Modern study shows that Rhizoma Atractylodis Macrocephalae decoct has and protects the liver choleretic effect.So above-mentioned three flavor medicines extract volatile oil earlier, regather its decocting liquid and handle with group's medicine, to guarantee curative effect.
The investigation of Rhizoma Curcumae, Radix Curcumae, Rhizoma Atractylodis Macrocephalae volatile oil extraction conditions:
Get Rhizoma Curcumae, Radix Curcumae, each 30g of Rhizoma Atractylodis Macrocephalae decoction pieces, 4 each 30g of order granule and 8 each 30g of order granule add 6 times of water respectively, soak 0.5,1,1.5 hour respectively after, extract volatile oil, the volatile oil of record different extraction times must be measured.The result: the soak time of medical material, little to the influence of volatile oil extracted amount, also occur long (1.5 hours) volatile oil of soak time in last group experiment and extract phenomenon slowly.And the degree of grinding of medical material influences bigger to the speed of volatile oil extraction and the amount of extraction.Along with the prolongation of extraction time, pulverize 8 thinner order powder, not only extract soon, and the amount of extracting is many, extracts just carried in 4 hours 5.5ml, and the speed that decoction pieces and 4 order powder all extract than 8 order powder under the same conditions is slow and carry to such an extent that measure and lack.So the condition that three flavor medicines extract volatile oil is decided to be employing 8 order powder, times water gaging soaks to extract in 30 minutes was advisable in 6 hours.
The investigation of experimental example 4 prescription decocting for Chinese herbal medicine methods
Except that aforementioned several drugs, also have Radix Bupleuri, Herba Artemisiae Scopariae, Semen Persicae, Rhizoma Menispermi and Radix Glycyrrhizae in the prescription.Radix Bupleuri is drugs for soothing liver and regulating QI flow, and its main component is a Saponin, volatile oil, organic acid, bupleurumol etc.Modern study shows that saikoside has tangible antipyretic-antalgic and antiinflammatory action.Adopt the decocting method Saponin can be fried, fry in shallow oil extraction so close with group medicine.Herba Artemisiae Scopariae is the key medicine of clearing liver-gallbladder, has the effect of clearing away damp-heat jaundice eliminating subcutaneous ulcer.The main component of Herba Artemisiae Scopariae is a coumarin, flavonoid, and coumaric acids etc. contain a spot of volatile oil.Because it is light that the Radix Bupleuri Herba Artemisiae Scopariae contains the few body of volatile ingredient amount, thus volatile oil do not extracted separately, so the effective ingredient of Herba Artemisiae Scopariae is not very clearly to adopt traditional decocting method to extract with Radix Bupleuri simultaneously.Adjuvant drug in the Rhizoma Menispermi side of being is heat and toxic materials clearing away medicine, and its main effective ingredient is an alkaloid, also adopts the traditional extraction process of decocting in water to handle.Semen Persicae is a drug for invigorating blood circulation and eliminating stasis, its main component is fatty oil, flavone, amygdalin etc., pharmacological evaluation shows, Semen Persicae can obviously increase animal brain blood flow microcirculation improvement, especially liver surface microcirculation there is the improvement effect, and can promote bile secretion etc., and obvious because of there not being clear and definite effective ingredient decocting liquid pharmacological action, so adopt the method for frying in shallow oil to extract with group's liquid medicine.Messenger drug in the Radix Glycyrrhizae side of being, its main effective ingredient is a glycyrrhizin, modern study shows to have and protects the liver, and anticancerly waits effect, also with group's medicine with after decocting, adopt the ethanol precipitation of debita spissitudo, remove partial impurities.Adopt orthogonal design to arrange group's medicine extraction conditions to investigate experiment: it is an amount of to get group medicine by prescription, adopt the condition of above-mentioned orthogonal design to divide 9 groups to experimentize, and respectively at being concentrated into 100ml in the water-bath with after interior, put to room temperature, be transferred in the 100ml volumetric flask, and be diluted to scale, shake up standby with water.Above-mentioned sample is fully shaken up, and the accurate respectively medicinal liquid 20ml that draws is all with water saturated n-butanol extraction 3 times (20ml, 10ml, 10ml), to extract colourless substantially after, reclaim n-butyl alcohol, residue constant weight, the result as seen, A factor (extraction time) does not have significance influence, A to the result 3Be optimum condition; B factor (extraction time) has utmost point appreciable impact to the result, with B 3Be the best; C factor (amount of water) does not have obvious influence to the result, determines D 3Be the best.So optimum condition is A 3B 3C 3, also promptly: extracting in water 3 times, extraction time was respectively 2,1,1 hours, and the water multiple is respectively 12,10,8 times.
The selection of experimental example 5 decoction liquor precipitate with ethanol conditions
Get Radix Bupleuri, Herba Artemisiae Scopariae, Rhizoma Menispermi, Semen Persicae, the Radix Glycyrrhizae five kinds of Chinese medicine is totally 21 grams (one pair of dose), pressing optimum experimental condition decocts, decocting liquid is equivalent to crude drug in whole 1 gram (21ml) respectively at being concentrated into every ml in the water-bath, add 95% ethanol respectively, make that to contain alcohol amount be 50%, 60%, 70%, 80% cold preservation, standing over night, reclaim ethanol after the filtration respectively, and be concentrated into the thick paste shape, after drying under 80 ℃ of conditions,, in exsiccator, put to room temperature and weigh by 95 editions officinal 105 ℃ of dryings 3 hours that are specified in, experimental result: the paste-forming rate soprano is 50% precipitate with ethanol person, next is 60% ethanol precipitation sample, considers from the production efficiency angle, filters very slow with 50% ethanol precipitation, and with 60% ethanol precipitation, the rate of filtration is faster, and simultaneously according to preceding surface analysis, group's the effective elements of the medicine can both stripping with 50% and 60% ethanol, the removal of impurity then is that 60% ethanol better effects if is a little, so select for use 60% ethanol precipitation to be advisable.
20 routine patients with liver fibrosis of experimental example 6 pharmaceutical composition doors of the present invention have been carried out following observation:
(1) measures serum IgG, IgA, IgM, complement C3, and compare with healthy volunteer 10 people.The result: the meansigma methods that the patient organizes IgG, IgA, IgM, complement C3 content (g/L) is respectively: 16.38,1.85,2.92,0.43; The meansigma methods of healthy group IgG, IgA, IgM, complement C3 content (g/L) is respectively 8.91,0.89,1.67,1.31.Two groups are compared P＜0.01, and it is lower to illustrate that the patient organizes the immunologic function level.12.61,1.22,2.03,0.78. and the preceding comparison of treatment P＜0.05 patient group treat after 3 months through taking granule of the present invention, and the meansigma methods of check serum IgG, IgA, IgM, complement C3 content (g/L) is respectively:, significant difference is arranged.Illustrate that granule of the present invention has the function of human body immunity improving power.(2) SOD measures situation: the healthy group total SOD of serum (Nu/ml) average is 19.4 ± 4.3; The total SOD of patient (Nu/ml) average is 14.78 ± 7.13 before treating, and is 22.64 ± 3.39 (taking medicine March) after the treatment, compares P＜0.05, significant difference before and after the treatment.Illustrate that patients with liver fibrosis organism metabolism state after the treatment of this medicine is improved, the ability of removing toxic metabolite product free radical in the body improves.
Experimental example 7 adopts the stratified random double blind method to observe the clinical effectiveness of granule therapy hepatitis B of the present invention hepatic fibrosis
By 3 months by a definite date, 6 months treatment of 209 routine hepatitis B and hepatic fibrosis is observed, and with the contrast of Western medicine colchicine and Chinese patent medicine FUFANG BIEJIA RUANGAN PIAN, the result is as follows: clinical symptoms is improved: according to statistics that cardinal symptom is marked, it is 76.47% that the clinical symptoms of treatment group is as a result improved 3 months total effective rates, total effective rate was 92.65% in 6 months, was excellent (P＜0.01) than 53.03% and 63.64% of matched group.To cardinal symptom such as abdominal distention, indigestion and loss of appetite, weak, sighing frequently, stool abnormity to improve effect more excellent than western medicine group, wherein sighing frequently, abdominal distention, effect indigestion and loss of appetite, stool abnormity also are better than correlated Chinese patent medicine.Improving aspect patient's picture of the tongue, granule of the present invention has remarkable advantages, and especially the elimination to red tongue, yellowish and greasy fur is better than two kinds of contrast medicines.Illustrate that granule of the present invention has the effect of eliminating preferably and alleviating hepatitis B patients with liver fibrosis clinical symptoms.Improve liver function: by analysis to liver function index of correlation before and after the treatment, it is better that granule of the present invention improves the effect of hepatitis B patients with liver fibrosis liver function, especially obvious to the influence of ALT, the Western medicine group that is better than contrasting (P＜0.01), but little to the influence of AST, ALB, GLB, A/G.Influence to hepatitis B virus: through treatment half a year, groups of grains HBV-DNA negative conversion rate of the present invention is 47.22%, and the negative conversion rate of HBeAg is 35.13%, and the negative conversion rate of HBsAg is 25.00%.Western medicine group HBV-DNA negative conversion rate is 18.18%, and the negative conversion rate of HBeAg is 12.50%, and the negative conversion rate of HBsAg is 12.50%.The anti-HBV effect of correlated Chinese patent medicine is also not obvious.Illustrate that granule of the present invention has certain anti-HBV effect.Reverse hepatic fibrosis: paired observation before and after the treatment, granule of the present invention can obviously alleviate the degree of hepatic fibrosis, to early stage hepatic fibrosis such as S 1, S 2Phase, reaching fully of having reverses.For the S that enters early stage liver cirrhosis 4Phase, the fibrous septum is dwindled.Wear histopathology relatively from liver, it is preceding 7.53 ± 2.21 that inflammation mobility integration treatment group is treated, and is 4.56 ± 3.47 after the treatment, and inflammation alleviates obviously, with matched group comparison P＜0.01; The degree of hepatic fibrosis integration is 6.54 ± 2.78 before treating, and is 4.12 ± 2.86 after the treatment, and hepatic fibrosis alleviates, and compares P＜0.01 with matched group.Its effect of anti hepatic fibrosis is better than the control drug group.
Experimental example 8 granules of the present invention are to the preventive effect of rat carbon tetrachloride Liver Fibrosis Model
Rat is bought the back adaptability back and fed for 1 week, is divided into 6 groups at random by sex then, and grouping and prophylactic are as follows.A: model group (20), each Mus gavages the isometric(al) distilled water; B: colchicine prevention matched group (10), gavage colchicine every day 1 time, consumption is 250ug/kg; C: the granule heavy dose of group of prevention of the present invention (10), gavage granule of the present invention 1 time every day, consumption is 6g/kg (quite 20 times of the clinical application amount); D: dosage group (10) in the granule prevention of the present invention, gavage granule of the present invention 1 time every day, consumption is 3g/kg (quite 10 times of the clinical application amount); E: granule prevention small dose group (10) of the present invention, gavage granule of the present invention 1 time every day, consumption is 1.5g/kg (quite 5 times of the clinical application amount); F: normal control group (9), equal conditions is fed down, and preventive drug is not given in not modeling.
(1) to the observation of animal performance in the experimentation and death condition
Method: modeling before measurement body weight, after this, per injection CCl 4Before all survey body weight, to understand the variation of rat body weight during the modeling.Experimental session examines situations such as the mobility, hair color, diet of rat, to understand the health status of rat.
The result: the model group rat is at CCl 4Attack withered nothings of the hair that becomes after 1 week pool, diet reduces, and most companions see diarrhoea, weight increase minimizing, the appearance negative growth that has.Colchicine prevention group and granule prevention group of the present invention are all light than model group, and be the lightest with the heavy dose of group of granule of the present invention especially.Experimental session, each is organized the rats death situation and sees Table 1.
Table 1 is respectively organized the rats death situation
Grouping Beginning laboratory animal number Death toll Mortality rate (%)
Dosage group (D) small dose group (E) normal group (F) in the heavy dose of group of model group (A) colchicine group (B) (C) 20 10 10 10 10 9 8 3 1 2 2 0 40.00 30.00 10.00 20.00 20.00 0
(2) the liver general form is observed
Method: cut open get liver after, observe the variations such as profile, color of fresh liver, and accurately claim its weight, calculate the ratio of liver and body weight then.
The result: the normal rat liver surface is smooth, and matter is soft, and color is dark red.Model group has 5 liver color grizzles, and wherein 2 colors are khaki, and white little cyst is seen on 1 liver surface, the part liver with every myosynizesis.Colchicine prevention group has 2 rats'liver to be lark, 2 visible tuberositys in Hepar Mus surface.The heavy dose of group of granule prevention of the present invention has 1 Hepar Mus gray, and white cyst is seen on 1 liver surface; Middle dosage group has 3 rats'liver grays, and sees white cyst; Small dose group has 3 rats'liver grays, and wherein tuberosity regeneration is seen on 2 liver surfaces.Each is organized liver weight and sees Table 3-2 with the ratio of body weight.The liver that rat is respectively organized in prevention heavily reaches the heavy ratio with body weight of liver all greater than normal rat (all P＜0.05), but changes irregularities (P＞0.05) between each group of prevention
Table 2 respectively organize liver heavily reach liver heavy with body weight frequently ( ) *
Group Number of animals Liver weight (g) The ratio (%) of liver percentage of liveweight
Little dose of group of agent group normal group in big dose of group of model group colchicine group 11 7 9 8 8 9 13.94±1.74 15.24±3.35 12.95±1.89 15.29±2.70 14.23±1.51 9.68±1.05 5.99±0.58 6.54±1.07 5.74±0.57 6.25±0.79 5.87±0.56 3.78±0.32
Expression mean ± standard deviation, its significance test are all with the t check, down together.
(3) hepatic tissue HE colored light sem observation
Method: each Mus is all drawn materials at right lobe of liver, 10% formalin fixed, and paraffin embedding, serial section, slice thick 4 μ m, HE dyeing, light microscopic is observed hepatic injury and fibrosis down.
The result:
1. normal rat: hepatocyte is that the center is radial arrangement towards periphery with the central vein, and hepatocyte is polyhedron shape, and various organelles are abundant and flourishing in the kytoplasm, and liver cell nuclear is big and the garden is positioned at cell central authorities, and the portal area does not have expansion, no pathologic collagenation.
2. model group rat: each Mus all sees the obvious cloudy swelling of hepatocyte, necrosis, serious steatosis, and the portal area enlarges, and accompanies tangible fibroplasia, and extends towards periphery, and what have holds whole lobules of liver, reaches 3 of hepatic fibrosis III level persons.Illustrate that replication of Model is successful.
3. colchicine prevention group: each Mus all sees hepatocyte cloudy swelling, steatosis, does not relatively have significant difference with model group, and wherein 6 are also shown in liver point-like, lamellar necrosis.The portal area enlarges, and fibroplasia is obvious, and extends towards periphery, wherein reaches 2 of hepatic fibrosis III level persons.
4. each group of granule prevention of the present invention: each Mus all sees hepatocyte cloudy swelling, steatosis in various degree, only 1 of heavy dose of group, and 3 of middle dosage groups, 5 visible hepatic necrosis of rat of small dose group, but degree is all light than model group.Each Mus all has hepatic fibrosis to some extent, but mostly is the 1-2 level, reaches 2 of III level person small dose group.
Each organizes the hepatic injury degree and the hepatic fibrosis situation sees Table 3.As can be known, the heavy dose of group of granule of the present invention hepatic injury index is starkly lower than model group (P＜0.01) from table.
Table 3 granule of the present invention is to the influence of rats'liver damage and hepatic fibrosis
Group Number of animals The hepatic injury index (X ± SD) Liver fiber formation rate (%) Degree of hepatic fibrosis (only)
0 grade 1 grade 2 grades 3 grades
Little dose of group of agent group normal group in big dose of group of model group colchicine group 11 7 9 8 8 9 6.91±0.94 6.14±1.07 5.00±2.45* 6.88±1.25 6.93±1.13 / 100 100 100 100 100 0 0 0 0 0 0 9 2 2 7 4 3 0 6 3 2 4 3 0 3 2 0 0 2 0
Compare * P＜0.01 with model group (A)
(4) hepatic tissue Masson dyeing is observed and graphical analysis
Method: fresh liver specimen is through 10% formalin fixed, paraffin embedding, and serial section, slice thick 4um presses the dyeing of Masson method, measures the liver collagen content with MPEAS-500 type image analyzer, and calculates the percentage ratio that collagen accounts for liver by computer.Select the many relatively places of fibrosis to measure under 200 times of light microscopics, every sheet is surveyed 5 visuals field, and its meansigma methods is the percentage ratio of this sample.
The result: under Masson dyeing, background takes on a red color.Liver collagen is dyed blueness, and only in the portal area or visible a little blue stock-dye around the central vein, color is light and carefully be its characteristics in the normal rat liver.Model group visible portal area expansion, fiber is extended towards periphery by the portal area, even holds whole lobules of liver formation pseudolobuli, and fiber is thick and dyeing is dark.Colchicine prevention group hepatic fibrosis and model group relatively do not have significant difference.Granule prevention group of the present invention hepatic fibrosis is light slightly than model group, and is the lightest with heavy dose group degree of hepatic fibrosis especially.Each is organized the percentage ratio that the liver fiber accounts for liver and sees Table 4.Colchicine group and model group compare, and collagen content reduces (P＜0.05), and each group of granule prevention of the present invention all is starkly lower than model group (P＜0.01-P＜0.001).
Table 4 is respectively organized collagen relative amount graphical analysis result ()
Group Number of animals Collagen relative amount (%)
Little dose of group of agent group normal group in big dose of group of model group colchicine group 11 7 9 8 8 9 8.78±5.58 6.52±2.18# 2.19±1.63** 2.95±0.97** 4.33±2.72* 1.19±0.64
Compare * P＜0.01 * * P＜0.001 #P＜0.05 with model group
(5) to the influence of liver function
Method: get blood 3-4ml through femoral artery before each sacrifice of animal, get the serum enzyme process after centrifugal and detect alanine aminotransferase (ALT), r-glutamyl transpeptidase (r-GT), biuret method detects total protein (TP), the bromocresol green method detects albumin (ALB), globulin (GLB) and calculates albumins/globulins ratio (A/G ratio).
The result: the heavy dose of group of granule of the present invention can obviously reduce ALT and r-GT, with model group P＜0.01 relatively, but to each group of proteic influence and other relatively difference do not have significance (equal P＞0.05).
Table 5 granule of the present invention is to the influence () of CCL4 Liver Fibrosis Model rats'liver function
Group Number of animals ALT(IU/L) r-GT(IU/L)
Little dose of group of agent group normal group in big dose of group of model group colchicine group 11 7 9 8 8 9 153.91±87.48 142.00±103.01 63.00±52.43** 106.75±67.37 143.25±79.92 44.44±13.99 58.62±17.33 52.78±20.26 40.73±16.83* 49.84±22.17 53.61±18.47 36.08±16.56
Compare * P＜0.05 * * P＜0.01 with model group
Table 6 granule of the present invention is to the influence () of CCL4 Liver Fibrosis Model rat protein
Group Number of animals ?T-P(g/dl) ?ALB(g/dl) ?GLB(g/dl) ?A/G
Little dose of group of agent group normal group in big dose of group of model group colchicine group 11 7 9 8 8 9 ?7.93±0.42?7.66±0.48?8.20±0.27?8.10±0.45?7.90±0.82?8.20±0.56 ?3.37±0.23?3.23±0.25?3.40±0.52?3.30±0.52?3.31±0.24?3.79±0.12 ?4.55±0.38?4.43±0.37?4.60±0.49?4.80±0.32?4.79±0.48?4.63±0.52 ?0.75±0.10?0.73±0.10?0.70±0.13?0.70±0.09?0.70±0.11?0.83±0.11
(6) detection of serum liver fibrosis index of correlation
Method: gets blood 3-4ml through femoral artery before each sacrifice of animal, get the content that serum detects III Collagen Type VI propetide (PIIIP), IV Collagen Type VI (IV-C) and laminin (LN) after centrifugal.Three kinds of test kits are produced by Dutch Monosan company, and the packing of the gloomy male Industrial Co., Ltd. in Shanghai is all operated by medicine box description method.
Result: see Table 7.
Table 7 granule of the present invention to the influence of experimental rat blood-serum P IIIP, IV-C and LN content (
Figure S2007103038763D00151
)
Group Number of animals PIIIP(ng/ml) IV-C(ng/ml) LN(ng/ml)
Little dose of group of agent group normal group in big dose of group of model group colchicine group 11 7 9 8 8 9 43.62±9.96 35.2±8.26** 30.5±5.40** 43.5±15.22 41.95±5.20 24.47±10.10*** 211.6±44.97 229.53±32.70 201.98±97.15 200.84±93.37 197.69±44.68 120.78±53.30 95.75±40.56 83.85±47.23 72.85±57.38* 82.00±20.94 86.25±21.95 42.8±12.69***
Compare * P＜0.05 * * P＜0.01 * * * P＜0.001 with model group;
Carbon tetrachloride is a kind of selectivity hepatotoxic agent, causes the hepatocyte acute injury, even downright bad.After the prolonged and repeated damage of hepatocyte, in the liver network subside, poly-ly disturb, adhesion, fibroblast growth factor generates too much simultaneously, impels pathologic collagen hypertrophy, a large amount of hypertrophy of collagen fiber also are deposited on liver and promptly form Liver Fibrosis Model.The hepatopathy reason of this experimental model rat is checked the obvious hypertrophy of prompting fibrous tissue, and stretches in lobules of liver, and part also forms pseudolobuli, hepatocyte companion point-like, the necrosis of kitchen range shape in various degree simultaneously, and steatosis illustrates that it is successful that Liver Fibrosis Model is duplicated.
Give granule prevention of the present invention in the time of the rat modeling; three dosage groups all can reduce the mortality rate of experimental rat as a result; liver function protecting; alleviate the degree of injury and the degree of hepatic fibrosis of liver; wherein more obvious with the effect of heavy dose group; relatively have notable statistics difference (P＜0.05) with model group, granule prevention ccl of the present invention is described 4Liver Fibrosis Model is effectively, and this just provides experimental basis for this medicine treatment hepatic fibrosis of clinical practice.As can be seen, colchicine also has certain effect of anti hepatic fibrosis from this experiment, but a little less than its effect.
Experimental example 8 granules of the present invention are to the preventive effect of rat albumin Liver Fibrosis Model
The positive rat of albumin antibody is divided into 5 groups at random: 1. model group (18), each Mus gavages the isometric(al) distilled water; 2. colchicine prevention matched group (10) gavages colchicine every day 1 time, and consumption is 250ug/kg; 3. granule of the present invention prevents large, medium and small dosage group (every group equal 10), gavage granule of the present invention 1 time every day, consumption is respectively 6g/kg, 3g/kg and 1.5g/kg (respectively be equivalent to clinical application amount 20 times, 10 times and 5 times), feeds in contrast down with 5 normal rats (without the sensitization stage) equal conditions in addition.
(1) to the observation of animal performance in the experimentation and death condition
Result: behind each tail vein injection, rat all is the reaction of anaphylactic shock sample, showing as rapid breathing can not lie prostrate, side or lie on the back mostly recovered normal in 30 minutes, each organizes rat recovery time like indifference, therefore and dead but also there is the minority animal, animal dead is many, and especially the mortality rate in the 1st week is the highest in the 1st, 2 weeks, and each is organized mortality rate and sees Table 3-8.
Whole experimental session, model group and granule small dose group rat of the present invention show slightly the withered nothing of hair pool, and mobility reduces, and each group and normal group body weight gain do not have significant difference.
Table 8 is respectively organized the rats death situation
Group Number of animals Death toll (only) Mortality rate (%)
Little dose of group of agent group normal group in big dose of group of model group colchicine group 18 10 10 10 10 5 9 3 3 3 2 0 50.00 30.00 30.00 30.00 20.00 0
(2) the liver general form is observed
The result: the normal rat liver surface is smooth, matter is soft, color is dark red.Model group and the equal smooth surface of each prevention group rats'liver, no tuberosity, most colors are dark red, and wherein model group is 4,2 of colchicine groups, 2 of the heavy dose of groups of granule of the present invention, 2 of middle dosage groups, 4 rats'liver colors of small dose group show slightly Lycoperdon polymorphum Vitt.Each is organized liver weight and sees Table 9 with the ratio of body weight.Credit is analysed by statistics, there was no significant difference (P＞0.05) between each group.
Table 9 respectively organize liver heavily reach liver heavy with body weight frequently (
Figure S2007103038763D00161
) *
Group Number of animals Liver weight (g) The ratio (%) of liver percentage of liveweight
Little dose of group of agent group normal group in big dose of group of model group colchicine group 9 7 7 7 8 5 11.61±0.94 12.43±1.18 12.27±0.83 10.46±0.86 11.96±1.287 11.04±0.54 2.82±0.32 2.96±0.38 2.95±0.27 2.67±0.14 2.90±0.26 2.73±0.27
* represent mean ± standard deviation, its significance test is all with the t check, down together.
(3) hepatic tissue pathology is observed
The result:
1. normal rat: hepatocyte is that the center is radial arrangement towards periphery with the central vein, does not have obvious cloudy swelling, steatosis, and the visible local cell infiltration of idol is not seen necrosis.The portal area does not have expansion, visible small amount of fibers tissue, no pathologic collagenation.
2. model group: each Mus all sees cloudy swelling, steatosis and cell infiltration in various degree, but degree is heavy.Do not see obvious hepatic necrosis.Proliferation of fibrous tissue is obvious, extends towards periphery from the portal area, holds to have suffered lobules of liver, and pseudolobuli appears in the lobules of liver structural deterioration that has.
3. prevention is respectively organized: compare with model group, also see hepatocyte cloudy swelling, steatosis and cell infiltration, but do not have significant difference on the degree, there is no hepatic necrosis.Colchicine prevention group, the hepatic fibrosis of the big or middle dosage group of granule of the present invention is all light than model group, but does not have significant difference between three groups, and granule small dose group of the present invention hepatic fibrosis and model group are close and heavier.
Each is organized the hepatic fibrosis situation and sees Table 10, table 11.
Table 10 granule of the present invention is to the influence of rat liver fibrosis grading
Group Number of animals Degree of hepatic fibrosis (only)
0 grade 1 grade 2 grades 3 grades 4 grades
Little dose of group of agent group normal group in big dose of group of model group colchicine group 9 7 7 7 8 5 0 4 4 4 1 5 2 0 1 0 2 0 1 0 0 1 1 0 3 1 2 1 1 0 3 2 0 1 3 0
Table 11 granule of the present invention is to the influence of rats'liver fiber formation rate and collagen content ratio
Figure S2007103038763D00171
Compare * P＜0.05 with model group
Table 12 granule of the present invention is to the influence () of albumin hepatic fibrosis rats blood-serum P IIIP, IV-C and LN content
Group Number of animals PIIIP(ng/ml) IV-C(ng/ml) LN(ng/ml)
Little dose of group of agent group normal group in big dose of group of model group colchicine group 9 7 7 7 8 5 48.96±10.37 37.26±8.64** 30.42±6.78*** 38.14±7.78** 46.36±9.14 25.67±5.57 246.33±53.26 206.57±28.90* 196.98±60.72** 216.33±50.42 257.87±90.56 132.47±49.51 98.54±40.78 86.73±52.64 88.84±50.26 89.42±38.64 90.63±70.56 77.57±30.62
Compare * P＜0.05 * * p＜0.01 * * * P＜0.001 with model group
In hepatic fibrosis and liver cirrhosis formation and development process, immune factor plays an important role, and albumin causes that the blood flow circulating immune complex increases repeatedly after continuing repeatedly to inject, can cause circulating immune complex to be deposited on kiernan's space blood capillary tip, the progressive widely chronic inflammatory disease pathological changes of appearance causes the hepatocellular degeneration necrosis around making lobules of liver, fibroblast proliferation and collagen fiber pathological proliferation, and stretch around lobules of liver from the portal area, form hepatic fibrosis and liver cirrhosis.This model stability is reliable, and similar with human chronic viral hepatitis B heptic fibrosis pathogeny, so be widely used in the screening of anti-hepatic fibrosis medicines.
PIIIP is the polypeptide that the III Collagen Type VI gets off through aminoterminal restriction endonuclease effect hydrolysis, it is the good index of early stage degree of hepatic fibrosis of reflection and mobility, the increase of serum I V-C content also is proportionate with hepatic fibrosis, and detects the degree that these indexs can reflect hepatic fibrosis from serum.From this experimental result, the content of model group P of Rats IIIP, IV-C all is higher than normal rat, and is consistent with the document conclusion.Confirm that in conjunction with the hepatic tissue pathology analysis after granule prevention of the present invention, big or middle dosage group can obviously reduce the hepatic fibrosis formation rate, the liver protecting and alleviate degree of hepatic fibrosis has shown the good preventive effect of this medicine to human albumin's Liver Fibrosis Model.
Experimental example 9 granules of the present invention are to the inhibitory action of hepatitis B virus
(1) cytotoxicity of granule of the present invention in the 2.2.15 cell culture
Be to observe the toxicity of granule of the present invention, behind inoculation 2.2.15 cell 24 hours, add 2 times of dilute liquid medicines people's hepatocarcinoma 2.2.15 cell of hepatitis B virogene transfection.Experiment begins from 16mg/ml: 8; 4; 2; 1; 0.5; 0.25mg/ml, establish the normal cell contrast simultaneously.Change medicinal liquid in 4 days one time, kept 8 days, use the microscope observing cell pathological changes, by formula calculate the poisonous concentration (TC of half 50) and maximal non-toxic concentration (TC 0), see Table 1.TC 50Two batches of experiments are respectively 2mg/ml, 2mg/ml, the average poisonous concentration (TC of half 50) be 2mg/ml, maximal non-toxic concentration (TC 0) be 1mg/ml.See Table 4-13.
(2) effect in the 2.2.15 cell culture, HBeAg and HBsAg expressed of granule of the present invention
Granule 1mg/ml of the present invention; 0.5mg/ml; 0.25mg/ml, adding in the 2.2.15 cell and cultivate, the inhibition effect to HBsAg and HBeAg saw Table 4-14, table 4-15,4-16 in the 8th day.
1. to the suppression ratio of HBsAg: three batches of experiments of granule of the present invention are respectively the average suppression ratio of HBsAg: 1mg/ml suppresses 23.63 ± 9.25% (P＜0.05-.001); 0.5mg/ml suppress 5.27 ± 3.14% (P＞0.05).Three crowdes of experiment IC 50All＞2.0mg/ml.
2. to the suppression ratio of HBeAg: three batches of each the concentration groups of experiment granule of the present invention are respectively the average suppression ratio of HBeAg; 1mg/ml inhibition 37.60 ± 8.31% (P＜0.01), 0.5mg/ml inhibition 23.33 ± 5.75%, 0.25mg/ml suppress 17.90 ± 1.66% (P＞0.05).Three crowdes of empirical average IC 50Be 3.23 ± 2.64mg/ml.
3. selection index [SI]: granule of the present invention is 0.62 to the SI of HBeAg suppression ratio, can't calculate the SI of HBsAg suppression ratio.
The toxicity of table 13 granule of the present invention in the 2.2.15 cell culture
The experiment batch Experimental technique Different pharmaceutical concentration mg/ml/ cytopathy TC 50(mg/ml) TC 0
16 8 4 ?2 1 0.5 0.25 0
1 CPE destroys % 444100 4 4 4 100 4 4 4 100 ?2?2?2?50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1
2 CPE destroys % 444100 4 4 4 100 4 4 4 100 ?2?2?2?50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1
Two batches average 2 1
Table 14 granule of the present invention is the 8th day inhibitory action to HBsAg (three batches of experiments) in the 2.2.15 cell
Figure S2007103038763D00191
Annotate: experimental group and matched group be * * P＜0.01 relatively, * P＜0.05.
Table 15 granule of the present invention is the 8th day inhibitory action to HBeAg (three batches of experiments) in the 2.2.15 cell
Annotate: experimental group and matched group be * * P＜0.01 relatively, * P＜0.05.
Table 16 granule of the present invention in the 2.2.15 cell culture to the inhibition effect analysis of HBsAg and HBeAg
Figure S2007103038763D00201
Table 17 granule of the present invention in the 2.2.15 cell culture to the inhibitory action conclusive table of HBeAg and HBsAg
Cytotoxicity (mg/ml) HBeAg HBsAg IC50(mg/ml)
TC50 TC0 IC50(mg/ml) SI
2 1 3.23±2.64 0.62 ＞2
The result: granule of the present invention is to the toxicity of 2.2.15 cell culture: granule of the present invention adds in the 2.2.15 cell to be cultivated 8 days, was index with the cytopathy, two batches of empirical averages: median toxic concentration TC 50Be 2mg/ml, maximal non-toxic concentration TC 0Be 1mg/ml.Granule of the present invention in the 2.2.15 cell culture to HBeAg and the excretory inhibitory action of HBsAg: granule of the present invention was cultivated 8 days in the 2.2.15 cell, to the IC of HBeAg 50Three batches of empirical averages are 3.23 ± 2.64mg/ml, and SI is 0.62.To the suppression ratio average out to 23.63 ± 9.25% of HBsAg maximal non-toxic concentration 1mg/ml (P＜0.05-0.01).The at present clinical arabinofuranosyl adenine monophsphate (Ara-AMP) that is used for the treatment of hepatitis B virus, acycloguanosine (ACV), phosphorus formic acid (PFA) though etc. in the 2.2.15 cell, can suppress HBV-DNA, the HBeAg expression inhibiting is lower than 50%, can not calculate IC 50This experiment positive control drug.
Following embodiment all can realize the effect of above-mentioned experimental example
The specific embodiment
Embodiment 1: the preparation of granule
Radix Astragali 510kg Radix Salviae Miltiorrhizae 200kg Rhizoma Curcumae 200kg art 200kg
Radix Curcumae 150kg Herba Artemisiae Scopariae 150kg Radix Bupleuri 150kg Semen Persicae 150kg
Rhizoma Menispermi 100kg Radix Glycyrrhizae 100kg
Get Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae extraction volatile oil, the water solution A after distillation device is in addition preserved, and medicinal residues A is standby; Radix Salviae Miltiorrhizae is added 6 times of amount 80% ethanol extractions 3 times, each 1 hour, merge 3 times extracting solution, filter, decompression recycling ethanol is concentrated into the thick paste shape, in 60 ℃ of oven dry, the powder A that gets dry extract, medicinal residues B is standby; With Radix Astragali extracting in water 3 times, the time was respectively 2,1,1 hours, and water consumption is respectively 10,8,8 times, collecting decoction filters, and filtrate is concentrated into every milliliter and is equivalent to 0.5 gram medical material, with 3000 rev/mins centrifugal, remove partial impurities, supernatant concentration is standby to thick paste A; All the other Radix Bupleuri, Semen Persicae, Herba Artemisiae Scopariae, Rhizoma Menispermi, Radix Glycyrrhizae five kinds of Chinese medicine and Radix Salviae Miltiorrhizae decoction dregs B and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, the medicinal residues A of Radix Curcumae three flavor medicines merges, decoct with water 3 times, water consumption is respectively 10,8,8 times, time is respectively 2,1,1 hour, merge 3 times decocting liquid, filter filtrate and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, the water extract A of Radix Curcumae three flavors merges, and being concentrated into and recording relative density in the time of 60 ℃ is 1.13, adding ethanol makes and contains alcohol amount and reach 60%, standing over night is filtered, and decompression filtrate recycling ethanol also is concentrated into thick paste thick paste B, thick paste B and Radix Astragali water extract thick paste A are incorporated in 60 ℃ of oven dry, and the powder B that gets dry extract is standby; Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae volatile oil are made beta-CD inclusion, and above-mentioned dried cream powder A is mixed with dried cream powder B, be ground into 80 order fine powders and with volatile oil beta-CD inclusion mixing, add conventional adjuvant, according to common process, make granule, pack, every bag of 9g, one time 1 bag, 3 times on the one.
Embodiment 2: the preparation of capsule
Radix Astragali 450kg Radix Salviae Miltiorrhizae 200kg Rhizoma Curcumae 200kg
Rhizoma Atractylodis Macrocephalae 200kg Radix Curcumae 120kg Herba Artemisiae Scopariae 120kg
Radix Bupleuri 120kg Semen Persicae 120kg Rhizoma Menispermi 80kg
Radix Glycyrrhizae 80kg
Get Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae extraction volatile oil, the water solution A after distillation device is in addition preserved, and medicinal residues A is standby; Radix Salviae Miltiorrhizae is added 5 times of amount 85% ethanol extractions 2 times, and each 3 hours, merge extractive liquid, filtered, and decompression recycling ethanol is concentrated into the thick paste shape, in 40 ℃ of oven dry, and the powder A that gets dry extract, medicinal residues B is standby; With Radix Astragali extracting in water 4 times, the time is each 1 hour, and water consumption is each 10 times, and collecting decoction filters, and filtrate is concentrated into every milliliter and is equivalent to 0.2 gram medical material, with 3000 rev/mins centrifugal, remove partial impurities, supernatant concentration is standby to thick paste A; The medicinal residues A of all the other Radix Bupleuri, Semen Persicae, Herba Artemisiae Scopariae, Rhizoma Menispermi, Radix Glycyrrhizae five kinds of Chinese medicine and Radix Salviae Miltiorrhizae decoction dregs B and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae three flavor medicines merges, decoct with water 4 times, water consumption is each 8 times, time is each 3 hours, collecting decoction, filter, the water extract A of filtrate and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae three flavors merges, being concentrated into and recording relative density in the time of 60 ℃ is 1.0, adds ethanol and makes and contain alcohol amount and reach 60%, standing over night, filter, decompression filtrate recycling ethanol also is concentrated into thick paste thick paste B, and thick paste B and Radix Astragali water extract thick paste A are incorporated in 80 ℃ of oven dry, and the powder B that gets dry extract is standby; Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae volatile oil are made beta-CD inclusion, and above-mentioned dried cream powder A is mixed with dried cream powder B, be broken into 60 order fine powders and with volatile oil beta-CD inclusion mixing, add conventional adjuvant, according to common process, add conventional adjuvant, incapsulate, promptly.
Embodiment 3: the preparation of tablet
Radix Astragali 550kg Radix Salviae Miltiorrhizae 250kg Rhizoma Curcumae 250kg
Rhizoma Atractylodis Macrocephalae 250kg Radix Curcumae 180kg Herba Artemisiae Scopariae 180kg
Radix Bupleuri 180kg Semen Persicae 180kg Rhizoma Menispermi 120kg
Radix Glycyrrhizae 120kg
Get Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae extraction volatile oil, the water solution A after distillation device is in addition preserved, and medicinal residues A is standby; Radix Salviae Miltiorrhizae is added 7 times of amount 65% ethanol extractions 4 times, and each 1 hour, merge extractive liquid, filtered, and decompression recycling ethanol is concentrated into the thick paste shape, in 80 ℃ of oven dry, and the powder A that gets dry extract, medicinal residues B is standby; With Radix Astragali extracting in water 2 times, the time is each 3 hours, and water consumption is each 8 times, and collecting decoction filters, and filtrate is concentrated into every milliliter and is equivalent to 0.8 gram medical material, with 3000 rev/mins centrifugal, remove partial impurities, supernatant concentration is standby to thick paste A; All the other Radix Bupleuri, Semen Persicae, Herba Artemisiae Scopariae, Rhizoma Menispermi, Radix Glycyrrhizae five kinds of Chinese medicine and Radix Salviae Miltiorrhizae decoction dregs B and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, the medicinal residues A of Radix Curcumae three flavor medicines merges, decoct with water 2 times, water consumption is each 10 times, time is each 1 hour, collecting decoction, filter, filtrate and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, the water solution A of Radix Curcumae three flavors merges, being concentrated into and recording relative density in the time of 60 ℃ is 1.5, adds ethanol and makes and contain alcohol amount and reach 60%, standing over night, filter, decompression filtrate recycling ethanol also is concentrated into thick paste thick paste B, and thick paste B and Radix Astragali water extract thick paste A are incorporated in 40 ℃ of oven dry, and the powder B that gets dry extract is standby; Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae volatile oil are made beta-CD inclusion, and above-mentioned dried cream powder A is mixed with dried cream powder B, be ground into 100 order fine powders and with volatile oil beta-CD inclusion mixing, add conventional adjuvant, according to common process, make tablet.
Embodiment 4: the preparation of drop pill
Radix Astragali 510kg Radix Salviae Miltiorrhizae 200kg Rhizoma Curcumae 200kg Rhizoma Atractylodis Macrocephalae 200kg
Radix Curcumae 150kg Herba Artemisiae Scopariae 150kg Radix Bupleuri 150kg Semen Persicae 150kg
Rhizoma Menispermi 100kg Radix Glycyrrhizae 100kg
Get Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae extraction volatile oil, the water solution A after distillation device is in addition preserved, and medicinal residues A is standby; Radix Salviae Miltiorrhizae is added 4 times of amount 90% ethanol extractions 2 times, and each 3 hours, merge extractive liquid, filtered, and decompression recycling ethanol is concentrated into the thick paste shape, in 50 ℃ of oven dry, and the powder A that gets dry extract, medicinal residues B is standby; With Radix Astragali extracting in water 4 times, the time is each 2 hours, and water consumption is each 10 times, and collecting decoction filters, and filtrate is concentrated into every milliliter and is equivalent to 0.3 gram medical material, with 3000 rev/mins centrifugal, remove partial impurities, supernatant concentration is standby to thick paste A; The medicinal residues A of all the other Radix Bupleuri, Semen Persicae, Herba Artemisiae Scopariae, Rhizoma Menispermi, Radix Glycyrrhizae five kinds of Chinese medicine and Radix Salviae Miltiorrhizae decoction dregs B and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae three flavor medicines merges, decoct with water 4 times, water consumption is each 8 times, time is each 3 hours, collecting decoction, filter, the water solution A of filtrate and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae three flavors merges, being concentrated into and recording relative density in the time of 60 ℃ is 1.0, adds ethanol and makes and contain alcohol amount and reach 60%, standing over night, filter, decompression filtrate recycling ethanol also is concentrated into thick paste thick paste B, and thick paste B and Radix Astragali water extract thick paste A are incorporated in 70 ℃ of oven dry, and the powder B that gets dry extract is standby; Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae volatile oil are made beta-CD inclusion, and above-mentioned dried cream powder A is mixed with dried cream powder B, be ground into 70 order fine powders and with volatile oil beta-CD inclusion mixing, add conventional adjuvant, according to common process, make drop pill.
Embodiment 5: the preparation of unguentum
Radix Astragali 450kg Radix Salviae Miltiorrhizae 200kg Rhizoma Curcumae 200kg
Rhizoma Atractylodis Macrocephalae 200kg Radix Curcumae 120kg Herba Artemisiae Scopariae 120kg
Radix Bupleuri 120kg Semen Persicae 120kg Rhizoma Menispermi 80kg
Radix Glycyrrhizae 80kg
Get the above-mentioned raw materials medicine, add conventional adjuvant,, make unguentum according to common process.
Embodiment 6: the preparation of oral liquid
Radix Astragali 550kg Radix Salviae Miltiorrhizae 250kg Rhizoma Curcumae 250kg
Rhizoma Atractylodis Macrocephalae 250kg Radix Curcumae 180kg Herba Artemisiae Scopariae 180kg
Radix Bupleuri 180kg Semen Persicae 180kg Rhizoma Menispermi 120kg
Radix Glycyrrhizae 120kg
Get the above-mentioned raw materials medicine, add conventional adjuvant,, make oral liquid according to common process.
Embodiment 7: the preparation of injection
Radix Astragali 510kg Radix Salviae Miltiorrhizae 200kg Rhizoma Curcumae 200kg Rhizoma Atractylodis Macrocephalae 200kg
Radix Curcumae 150kg Herba Artemisiae Scopariae 150kg Radix Bupleuri 150kg Semen Persicae 150kg
Rhizoma Menispermi 100kg Radix Glycyrrhizae 100kg
Get the above-mentioned raw materials medicine, add conventional adjuvant,, make injection according to common process.
Embodiment 8: the preparation of slow releasing agent
Radix Astragali 450kg Radix Salviae Miltiorrhizae 200kg Rhizoma Curcumae 200kg
Rhizoma Atractylodis Macrocephalae 200kg Radix Curcumae 120kg Herba Artemisiae Scopariae 120kg
Radix Bupleuri 120kg Semen Persicae 120kg Rhizoma Menispermi 80kg
Radix Glycyrrhizae 80kg
Get the above-mentioned raw materials medicine, add conventional adjuvant,, make slow releasing agent according to common process.
Embodiment 9: the preparation of honeyed pill
Radix Astragali 550kg Radix Salviae Miltiorrhizae 250kg Rhizoma Curcumae 250kg
Rhizoma Atractylodis Macrocephalae 250kg Radix Curcumae 180kg Herba Artemisiae Scopariae 180kg
Radix Bupleuri 180kg Semen Persicae 180kg Rhizoma Menispermi 120kg
Radix Glycyrrhizae 120kg
Get the above-mentioned raw materials medicine, add conventional adjuvant,, make honeyed pill according to common process.
Embodiment 10: the preparation of powder
Radix Astragali 450kg Radix Salviae Miltiorrhizae 250kg Rhizoma Curcumae 250kg
Rhizoma Atractylodis Macrocephalae 250kg Radix Curcumae 150kg Herba Artemisiae Scopariae 150kg
Radix Bupleuri 150kg Semen Persicae 150kg Rhizoma Menispermi 80kg
Radix Glycyrrhizae 80kg.
Get the above-mentioned raw materials medicine, add conventional adjuvant,, make powder according to common process.
Embodiment 11: the preparation of soft capsule
Radix Astragali 500kg Radix Salviae Miltiorrhizae 200kg Rhizoma Curcumae 200kg
Rhizoma Atractylodis Macrocephalae 200kg Radix Curcumae 120kg Herba Artemisiae Scopariae 120kg
Radix Bupleuri 120kg Semen Persicae 120kg Rhizoma Menispermi 100kg
Radix Glycyrrhizae 100kg.
Get the above-mentioned raw materials medicine, add conventional adjuvant,, make powder according to common process.
Embodiment 12: the content assaying method of drug combination preparation of the present invention and discrimination method
Content assaying method: get embodiment 1 granular preparation 5g, the accurate title, decide, put in the apparatus,Soxhlet's, add the 50ml2% potassium hydroxide methanol solution, heating and refluxing extraction 6 hours, merceration spends the night again, and the extracting solution reclaim under reduced pressure is to doing, residue adds the 15ml water dissolution, use chloroform: the mixed solution of n-butyl alcohol=2: 1, extract three times, be respectively 40,30,30ml, combined chloroform: the extract of following butanols=2: 1, with 1% potassium dihydrogen phosphate aqueous solution 60ml washing, aqueous phase discarded, organic facies evaporated under reduced pressure, it is fixed molten to add the 1ml methanol solution, as need testing solution; Precision takes by weighing the astragaloside reference substance in addition, adds methanol and makes the solution that 1ml contains 0.001g, in contrast product solution; Test according to thin layer chromatography, the accurate need testing solution 0.001ml that draws, reference substance solution 0.002ml and 0.004ml, the cross point is on same silica gel g thin-layer plate respectively, under the room temperature with chloroform: methanol: the solution of water=75: 25: 5 ratio was placed after 12 hours, took off layer and was developing solvent, launched once, take out, dry, spray 10% concentrated sulphuric acid alcoholic solution, 100 ℃ were heated 5 minutes down again, take out, cover onesize glass plate again on the lamellae, fix with hinge on every side, scan according to thin layer chromatography, wavelength X s=530nm, λ R=650nm measures test sample trap integrated value and reference substance trap integrated value, calculates, that is, this drug combination preparation per diem taking dose meter contains astragaloside C44H6804, must not be lower than 2.700mg.
Discrimination method: A, according to the following preparation need testing solution of assay item, get the reference substance astragaloside and add an amount of methanol to make the solution that 1ml contains 0.001g be reference substance solution; Other gets Milkvetch Root 1.5g, puts in the Sha Shi extractor, adds 2% potassium hydroxide methanol solution and extracts after 8 hours, one night of merceration, reclaim and extract solution to doing, add water 15ml, again with chloroform: the extraction of n-butyl alcohol=2: 1 proportioning concentration is respectively 40,30 for three times, 30ml, combining extraction liquid adds 1% potassium dihydrogen phosphate 60ml washing, aqueous phase discarded, again once with the 60ml water washing, organic facies is recycled to dried, adds the 1ml dissolve with methanol, makes control medicinal material solution; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw above-mentioned three kinds of each 0.002ml of solution, put respectively in same 15cm * 10cm silica gel G aluminium sheet, under the room temperature with chloroform: methanol: the solution of water=75: 25: 5 ratio was placed after 12 hours, took off layer and was developing solvent, launched once, take out, dry, spray 10% concentrated sulphuric acid alcoholic solution, be heated to clear spot; In the test sample chromatograph, with reference substance and the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
B, get embodiment 1 granule 9g and place the 50ml round-bottomed flask, add water 250ml and be heated to boiling and extract volatile oil, after about 1～2 hour, collect volatile oil, with petroleum ether volatile oil extractor tube wall, and join in the volatile oil, as test sample; Other gets Rhizoma Atractylodis Macrocephalae control medicinal material 2g, pulverizes, and crosses 40 mesh sieves, shines the medical material need testing solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw above-mentioned two kinds of each 0.002ml of solution, put respectively in same block of silica gel G aluminium sheet, with petroleum ether: ethyl acetate=5: 0.1 is developing solvent, launch at normal temperatures, take out, dry, spray 1% vanillin ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
C, get embodiment 1 granule 6g, put in the 50ml triangular flask, add ether 40ml vibration, leave standstill 1 hour after, filter, reclaim ether extraction solution to doing, residue adds the 2ml ethyl acetate makes dissolving, standby as need testing solution; Other gets Radix Salviae Miltiorrhizae control medicinal material 1g, pulverizes, and crosses 40 mesh sieves, shines medical material solution in pairs with legal system, and it is standby to do the control medicinal material test liquid; Take by weighing Tanshinone I Ia reference substance 0.002g, add ethyl acetate solution and make concentration to be that every ml contains the reference substance solution of 1mg Tanshinone I Ia standby; According to the test of an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw above-mentioned three kinds of each 0.005ml of solution, put respectively in same block of silica gel G aluminium sheet, with benzene: the solution of ethyl acetate=9.5: 0.5 is developing solvent, launch at normal temperatures, take out, dry; In the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color;
D, get embodiment 1 granule 9g, put in the 50ml conical flask, add 95% methanol 50ml, use power 150W, the supersonic oscillations of frequency 20kHz are after 40 minutes, filter, get subsequent filtrate 30ml, decompression and solvent recovery is put dried, the dissolving of residue adding distil water 15ml slight fever, with water saturation n-butanol extraction three times, volume is respectively 20,20,10ml merges butanol extraction liquid, twice of reuse 1% potassium hydroxide washing butanol extraction liquid, volume is respectively 20,20ml discards alkali liquor, and butanol extraction liquid is washed till neutrality with the n-butyl alcohol saturated aqueous solution, discard water layer, the reclaim under reduced pressure butanol extraction liquid is done, and residue adds 95% ethanol 1ml dissolving, as need testing solution; Other gets Radix Bupleuri medical material 1g, shines medical material solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw above-mentioned two kinds of each 0.006ml of solution, put respectively in same block of silica gel G aluminium sheet, with chloroform: methanol: the solution of water=7: 3: 1 is developing solvent, launch at normal temperatures, take out, dry, spray 10% ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
E, get embodiment 1 granule 5g, put in the 50ml triangular flask, add ether 40ml vibration, leave standstill 1 hour after, filter, discard the ether layer, add methanol 30ml, reflux 1 hour is filtered, and reclaims methanol solution to doing, residue adds water 40ml makes dissolving, uses water saturation n-butanol extraction 3 times, each 20ml, merge butanol solution, wash with water 3 times, each 20ml, with the n-butanol layer evaporate to dryness, residue adds methanol 1ml dissolving, as need testing solution; Extracting liquorice control medicinal material 1g shines medical material solution in pairs with legal system in addition; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw above-mentioned two kinds of each 0.005ml of solution, put respectively in same block of silica gel G aluminium sheet, with chloroform: methanol: the solution of formic acid=5: 1: 0.1 is developing solvent, launch at normal temperatures, take out, dry, spray 50% sulphuric acid methanol solution, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
Embodiment 13: this compositions is made the discrimination method of powder
A, get powder according to method under the embodiment 12 assay items and prepare need testing solution, get the reference substance astragaloside and add an amount of methanol to make the solution that 1ml contains 0.002g be reference substance solution; Other gets Milkvetch Root 3g, puts in the Sha Shi extractor, adds 1% potassium hydroxide methanol solution and extracts after 10 hours, one night of merceration, reclaim and extract solution to doing, add water 10ml, again with chloroform: the extraction of n-butyl alcohol=3: 1.5 proportioning concentration is respectively 40,30 for three times, 30ml, combining extraction liquid adds 1% potassium dihydrogen phosphate 60ml washing, aqueous phase discarded, again once with the 60ml water washing, organic facies is recycled to dried, adds the 3ml dissolve with methanol, makes control medicinal material solution; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw above-mentioned three kinds of each 0.001ml of solution, put respectively in same 15cm * 10cm silica gel G aluminium sheet, under the room temperature with chloroform: methanol: the solution of water=80: 30: 5 ratio was placed after 12 hours, took off layer and was developing solvent, launched once, take out, dry, spray 5% concentrated sulphuric acid alcoholic solution, be heated to clear spot; In the test sample chromatograph, with reference substance and the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
B, get medicinal composition powders 10g of the present invention and place the 50ml round-bottomed flask, add water 200ml and be heated to boiling and extract volatile oil, after about 3 hours, collect volatile oil, with petroleum ether volatile oil extractor tube wall, and join in the volatile oil, as test sample; Other gets Rhizoma Atractylodis Macrocephalae control medicinal material 1g, pulverizes, and crosses 40 mesh sieves, shines the medical material need testing solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw above-mentioned two kinds of each 0.002ml of solution, put respectively in same block of silica gel G aluminium sheet, with petroleum ether: ethyl acetate=5: 0.1 is developing solvent, launch at normal temperatures, take out, dry, spray 1% vanillin ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
C, get medicinal composition powders 8g of the present invention, put in the 50ml triangular flask, add ether 30ml vibration, leave standstill 2 hours after, filter, reclaim ether extraction solution to doing, residue adds the 1ml ethyl acetate makes dissolving, standby as need testing solution; Other gets Radix Salviae Miltiorrhizae control medicinal material 1g, pulverizes, and crosses 40 mesh sieves, shines medical material solution in pairs with legal system, and it is standby to do the control medicinal material test liquid; Take by weighing Tanshinone I Ia reference substance 0.003g, add ethyl acetate solution and make concentration to be that every ml contains the reference substance solution of 0.8mg Tanshinone I Ia standby; According to the test of an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw above-mentioned three kinds of each 0.008ml of solution, put respectively in same block of silica gel G aluminium sheet, with benzene: the solution of ethyl acetate=10: 1 is developing solvent, launch at normal temperatures, take out, dry; In the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color;
D; get medicinal composition powders 10g of the present invention; put in the 50ml conical flask adds 90% methanol 60ml, uses power 150W; the supersonic oscillations of frequency 20kHz are after 30 minutes; filter, get subsequent filtrate 40ml, decompression and solvent recovery is put dried; residue adding distil water 10ml slight fever dissolving; use water saturation n-butanol extraction three times, volume is respectively 20ml merges butanol extraction liquid; twice of reuse 1% potassium hydroxide washing butanol extraction liquid; volume is respectively 20ml, discards alkali liquor, and butanol extraction liquid is washed till neutrality with the n-butyl alcohol saturated aqueous solution; discard water layer; the reclaim under reduced pressure butanol extraction liquid is done, and residue adds 90% ethanol 3ml dissolving, as need testing solution; Other gets Radix Bupleuri medical material 1g, shines medical material solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw above-mentioned two kinds of each 0.004ml of solution, put respectively in same block of silica gel G aluminium sheet, with chloroform: methanol: the solution of water=5: 2: 1 is developing solvent, launch at normal temperatures, take out, dry, spray 15% ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
E, get drug combination preparation 4g of the present invention, put in the 50ml triangular flask, add ether 50ml vibration, leave standstill 1 hour after, filter, discard the ether layer, add methanol 40ml, reflux 1 hour is filtered, and reclaims methanol solution to doing, residue adds water 50ml makes dissolving, uses water saturation n-butanol extraction 3 times, each 10ml, merge butanol solution, wash with water 3 times, each 30ml, with the n-butanol layer evaporate to dryness, residue adds methanol 1ml dissolving, as need testing solution; Extracting liquorice control medicinal material 3g shines medical material solution in pairs with legal system in addition; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw above-mentioned two kinds of each 0.003ml of solution, put respectively in same block of silica gel G aluminium sheet, with chloroform: methanol: the solution of formic acid=8: 3: 0.3 is developing solvent, launch at normal temperatures, take out, dry, spray 30% sulphuric acid methanol solution, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
Embodiment 14: this compositions is made the content assaying method of capsule
Get medicament composition capsule preparation 4g of the present invention, the accurate title, decide, put in the apparatus,Soxhlet's, add 60ml 1% potassium hydroxide methanol solution, heating and refluxing extraction 8 hours, merceration spends the night again, the extracting solution reclaim under reduced pressure as for, residue adds the 10ml water dissolution, use chloroform: the mixed solution of n-butyl alcohol=2: 1, extract three times, be respectively 40ml, combined chloroform: the extract of following butanols=1: 0.5, with 1% potassium dihydrogen phosphate aqueous solution 70ml washing, aqueous phase discarded, organic facies evaporated under reduced pressure, it is fixed molten to add the 1ml methanol solution, as need testing solution; Precision takes by weighing the astragaloside reference substance in addition, adds methanol and makes the solution that 1ml contains 0.002g, in contrast product solution; Test according to thin layer chromatography, the accurate need testing solution 0.001ml that draws, reference substance solution 0.003ml and 0.002ml, the cross point is on same silica gel g thin-layer plate respectively, under the room temperature with chloroform: methanol: the solution of water=80: 20: 10 ratio was placed after 12 hours, took off layer and was developing solvent, launched once, take out, dry, spray 6% concentrated sulphuric acid alcoholic solution, 120 ℃ were heated 4 minutes down again, take out, cover onesize glass plate again on the lamellae, fix with hinge on every side, scan according to thin layer chromatography, wavelength X s=530nm, λ R=650nm measures test sample trap integrated value and reference substance trap integrated value, calculates, that is, this medicament composition capsule preparation per diem taking dose meter contains astragaloside C44H6804, must not be lower than 2.700mg.
Embodiment 15: this compositions is made the content assaying method and the discrimination method of tablet
Content assaying method: get pharmaceutical composition tablet 5g of the present invention, the accurate title, decide, put in the apparatus,Soxhlet's, add the 45ml2% potassium hydroxide methanol solution, heating and refluxing extraction 6 hours, merceration spends the night again, and the extracting solution reclaim under reduced pressure is to doing, residue adds the 15ml water dissolution, use chloroform: the mixed solution of n-butyl alcohol=2: 1, extract three times, be respectively 35ml, combined chloroform: the extract of following butanols=2: 1, with 1.5% potassium dihydrogen phosphate aqueous solution 60ml washing, aqueous phase discarded, organic facies evaporated under reduced pressure, it is fixed molten to add the 1.5ml methanol solution, as need testing solution; Precision takes by weighing the astragaloside reference substance in addition, adds methanol and makes the solution that 1ml contains 0.002g, in contrast product solution; Test according to thin layer chromatography, the accurate need testing solution 0.002ml that draws, reference substance solution 0.002ml and 0.002ml, the cross point is on same silica gel g thin-layer plate respectively, under the room temperature with chloroform: methanol: the solution of water=70: 15: 8 ratio was placed after 12 hours, took off layer and was developing solvent, launched once, take out, dry, spray 8% concentrated sulphuric acid alcoholic solution, 90 ℃ were heated 6 minutes down again, take out, cover onesize glass plate again on the lamellae, fix with hinge on every side, scan according to thin layer chromatography, wavelength X s=530nm, λ R=650nm measures test sample trap integrated value and reference substance trap integrated value, calculates, that is, this pharmaceutical composition tablet per diem taking dose meter contains astragaloside C44H6804, must not be lower than 2.700mg;
Discrimination method: get pharmaceutical composition tablet 6g of the present invention, put in the 50ml triangular flask, add ether 35ml vibration, leave standstill 1.5 hours after, filter, reclaim ether extraction solution to doing, residue adds the 1.5ml ethyl acetate makes dissolving, standby as need testing solution; Other gets Radix Salviae Miltiorrhizae control medicinal material 1g, pulverizes, and crosses 40 mesh sieves, shines medical material solution in pairs with legal system, and it is standby to do the control medicinal material test liquid; Take by weighing Tanshinone I Ia reference substance 0.002g, add ethyl acetate solution and make concentration to be that every ml contains the reference substance solution of 1mg Tanshinone I Ia standby; According to the test of an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw above-mentioned three kinds of each 0.004ml of solution, put respectively in same block of silica gel G aluminium sheet, with benzene: the solution of ethyl acetate=10: 1 is developing solvent, launch at normal temperatures, take out, dry; In the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color.
Embodiment 16: this compositions is made the discrimination method of drop pill
Get medicament composition dropping pills preparation 9g of the present invention and place the 50ml round-bottomed flask, add water 200ml and be heated to boiling extraction volatile oil, after about 3 hours, collect volatile oil, with petroleum ether volatile oil extractor tube wall, and join in the volatile oil, as test sample; Other gets Rhizoma Atractylodis Macrocephalae control medicinal material 1g, pulverizes, and crosses 40 mesh sieves, shines the medical material need testing solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw above-mentioned two kinds of each 0.002ml of solution, put respectively in same block of silica gel G aluminium sheet, with petroleum ether: ethyl acetate=5: 0.1 is developing solvent, launch at normal temperatures, take out, dry, spray 2% vanillin ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.

Claims (11)

1. pharmaceutical composition is characterized in that the crude drug of this pharmaceutical composition consists of:
The Radix Astragali 400～600 weight portion Radix Salviae Miltiorrhizaes 150～300 weight portions
Rhizoma Curcumae 150～300 weight portion Rhizoma Atractylodis Macrocephalaes 150～300 weight portions
Radix Curcumae 100～200 weight portion Herba Artemisiae Scopariaes 100～200 weight portions
Radix Bupleuri 100～200 weight portion Semen Persicaes 100～200 weight portions
Rhizoma Menispermi 50～150 weight portion Radix Glycyrrhizaes 50～150 weight portions.
2. pharmaceutical composition as claimed in claim 1 is characterized in that the crude drug of this pharmaceutical composition consists of:
The Radix Astragali 510 weight portion Radix Salviae Miltiorrhizaes 200 weight portions
The Rhizoma Curcumae 200 weight portion Rhizoma Atractylodis Macrocephalaes 200 weight portions
Radix Curcumae 150 weight portion Herba Artemisiae Scopariaes 150 weight portions
Radix Bupleuri 150 weight portion Semen Persicaes 150 weight portions
Rhizoma Menispermi 100 weight portion Radix Glycyrrhizaes 100 weight portions.
3. pharmaceutical composition as claimed in claim 1 is characterized in that the crude drug of this pharmaceutical composition consists of:
The Radix Astragali 450 weight portion Radix Salviae Miltiorrhizaes 200 weight portions
The Rhizoma Curcumae 200 weight portion Rhizoma Atractylodis Macrocephalaes 200 weight portions
Radix Curcumae 120 weight portion Herba Artemisiae Scopariaes 120 weight portions
Radix Bupleuri 120 weight portion Semen Persicaes 120 weight portions
Rhizoma Menispermi 80 weight portion Radix Glycyrrhizaes 80 weight portions.
4. pharmaceutical composition as claimed in claim 1 is characterized in that the crude drug of this pharmaceutical composition consists of:
The Radix Astragali 550 weight portion Radix Salviae Miltiorrhizaes 250 weight portions
The Rhizoma Curcumae 250 weight portion Rhizoma Atractylodis Macrocephalaes 250 weight portions
Radix Curcumae 180 weight portion Herba Artemisiae Scopariaes 180 weight portions
Radix Bupleuri 180 weight portion Semen Persicaes 180 weight portions
Rhizoma Menispermi 120 weight portion Radix Glycyrrhizaes 120 weight portions.
5. as the arbitrary described pharmaceutical composition of claim 1-4, it is characterized in that getting this pharmaceutical composition crude drug, add conventional adjuvant,, make tablet, capsule, powder, pill, granule, soft extract with bee honey agent, controlled release preparation, oral liquid or ejection preparation according to common process.
6. as the arbitrary described pharmaceutical composition of claim 1-4, it is characterized in that getting this pharmaceutical composition crude drug, add conventional adjuvant,, make soft capsule, drop pill, honeyed pill, slow releasing preparation, quick releasing formulation according to common process.
7. as the arbitrary described preparation of drug combination method of claim 1-4, it is characterized in that this method is:
Get Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae extraction volatile oil, the water solution A after distillation device is in addition preserved, and medicinal residues A is standby; Radix Salviae Miltiorrhizae is added 4～8 times of amount 60～90% ethanol extractions 2～4 times, and each 1～3 hour, merge extractive liquid, filtered, and decompression recycling ethanol is concentrated into thick paste, in 40～80 ℃ of oven dry, and the powder A that gets dry extract, medicinal residues B is standby; With Radix Astragali extracting in water 2～4 times, the time is each 1～3 hour, and water consumption is each 8～10 times, collecting decoction filters, and filtrate is concentrated into every parts by volume and is equivalent to 0.2～0.8 weight portion medical material, with 3000 rev/mins centrifugal, remove partial impurities, supernatant concentration is standby to thick paste A; All the other Radix Bupleuri, Semen Persicae, Herba Artemisiae Scopariae, Rhizoma Menispermi, Radix Glycyrrhizae five kinds of Chinese medicine and Radix Salviae Miltiorrhizae decoction dregs B and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, the medicinal residues A of Radix Curcumae three flavor medicines merges, decoct with water 2～4 times, water consumption is each 8～10 times, time is each 1～3 hour, collecting decoction, filter, filtrate and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, the water solution A of Radix Curcumae three flavor merges, and being concentrated into and recording relative density in the time of 60 ℃ is 1.0～1.5, adds ethanol and makes and contain the alcohol amount and reach 60%, leave standstill, filter, decompression filtrate recycling ethanol also is concentrated into thick paste B, and thick paste B and Radix Astragali water extract thick paste A merge, in 40～80 ℃ of oven dry, the powder B that gets dry extract is standby; Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae volatile oil are made beta-CD inclusion, above-mentioned dried cream powder A is mixed with dried cream powder B, be ground into 60～100 order fine powders and with volatile oil beta-CD inclusion mixing, add conventional adjuvant, according to common process, make tablet, capsule, powder, pill, granule, soft extract with bee honey agent, controlled release preparation, oral liquid or ejection preparation.
8. preparation of drug combination method as claimed in claim 7 is characterized in that this method is:
Get Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae extraction volatile oil, the water solution A after distillation device is in addition preserved, and medicinal residues A is standby; Radix Salviae Miltiorrhizae is added 6 times of amount 80% ethanol extractions 3 times, each 1 hour, merge 3 times extracting solution, filter, decompression recycling ethanol is concentrated into the thick paste shape, in 60 ℃ of oven dry, the powder A that gets dry extract, medicinal residues B is standby; With Radix Astragali extracting in water 3 times, the time was respectively 2,1,1 hours, and water consumption is respectively 10,8,8 times, collecting decoction filters, and filtrate is concentrated into every parts by volume and is equivalent to 0.5 weight portion medical material, with 3000 rev/mins centrifugal, remove partial impurities, supernatant concentration is standby to thick paste A; All the other Radix Bupleuri, Semen Persicae, Herba Artemisiae Scopariae, Rhizoma Menispermi, Radix Glycyrrhizae five kinds of Chinese medicine and Radix Salviae Miltiorrhizae decoction dregs B and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, the medicinal residues A of Radix Curcumae three flavor medicines merges, decoct with water 3 times, water consumption is respectively 10,8,8 times, time is respectively 2,1,1 hour, merge 3 times decocting liquid, filter, filtrate and Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, the water solution A of Radix Curcumae three flavors merges, being concentrated into and recording relative density in the time of 60 ℃ is 1.13, adds ethanol and makes and contain alcohol amount and reach 60%, standing over night, filter, decompression filtrate recycling ethanol also is concentrated into thick paste thick paste B, and thick paste B and Radix Astragali water extract thick paste A are incorporated in 60 ℃ of oven dry, and the powder B that gets dry extract is standby; Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Curcumae volatile oil are made beta-CD inclusion, and above-mentioned dried cream powder A is mixed with dried cream powder B, are ground into
80 order fine powders and with volatile oil beta-CD inclusion mixing, add conventional adjuvant, according to common process, make tablet, capsule, powder, pill, granule, soft extract with bee honey agent, controlled release preparation, oral liquid or ejection preparation.
9. as the quality determining method of the arbitrary described pharmaceutical composition of claim 1-4, it is characterized in that this method comprises following assay and/or following one or more discriminatings:
Assay: get this pharmaceutical composition 3～8 weight portions, the accurate title, decide, put in the apparatus,Soxhlet's, add 40～60 parts by volume, 1～3% potassium hydroxide methanol solution, heating and refluxing extraction 4～8 hours, merceration spends the night again, and the extracting solution reclaim under reduced pressure is to doing, residue adds 10～30 parts by volume water dissolutioies, use chloroform: the mixed solution of n-butyl alcohol=2: 1, extract three times, be respectively 30～40 parts by volume, combined chloroform: following butanols=1～3: 0.5～1 extract, with the washing of 1～3% potassium dihydrogen phosphate aqueous solution, 50～80 parts by volume, aqueous phase discarded, organic facies evaporated under reduced pressure, it is fixed molten to add 1～3 parts by volume methanol solution, as need testing solution; Precision takes by weighing the astragaloside reference substance in addition, adds methanol and makes the solution that 1 parts by volume contains 0.001～0.002 weight portion, in contrast product solution; Test according to thin layer chromatography, accurate need testing solution 0.001～0.003 parts by volume of drawing, reference substance solution 0.001～0.003 parts by volume and 0.002～0.005 parts by volume, the cross point is on same silica gel g thin-layer plate respectively, under the room temperature with chloroform: methanol: water=60～80: 20～30: the solution of 5～10 ratios was placed after 12 hours, take off layer and be developing solvent, launch once, take out, dry, spray 5～15% concentrated sulphuric acid alcoholic solution, 80～120 ℃ were heated 3～8 minutes down again, took out, cover onesize glass plate again on the lamellae, fix with hinge on every side, scan wavelength X s=530nm according to thin layer chromatography, λ R=650nm, measure test sample trap integrated value and reference substance trap integrated value, calculate, promptly; This drug combination preparation per diem taking dose meter contains astragaloside C44H68O4, must not be lower than 2.700mg;
A, be equipped with need testing solution, get the reference substance astragaloside and add methanol to make the solution that 1 parts by volume contains 0.001～0.003 weight portion be reference substance solution according to assay item below legal system; Other gets Milkvetch Root 1～3 weight portion, put in the Sha Shi extractor, adding 1～3% potassium hydroxide methanol solution extracted after 6～10 hours, one night of merceration, reclaim and extract solution to doing, add water 10～30 parts by volume, again with chloroform: n-butyl alcohol=1～3: the extraction of 0.5～1.5 proportioning concentration is respectively 40,30,30 parts by volume three times, combining extraction liquid adds 1～3% potassium dihydrogen phosphate 60 parts by volume washing, aqueous phase discarded, again once with 60 parts by volume water washings, organic facies is recycled to dried, adds 1～3 parts by volume dissolve with methanol, makes control medicinal material solution; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.001～0.002 parts by volume of above-mentioned three kinds of solution, put respectively in same 15cm * 10cm silica gel G aluminium sheet, under the room temperature with chloroform: methanol: water=60～80: 20～30: the solution of 5～10 ratios was placed after 12 hours, took off layer and was developing solvent, launched once, take out, dry, spray 5～15% concentrated sulphuric acid alcoholic solution are heated to clear spot; In the test sample chromatograph, with reference substance and the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
B, get this pharmaceutical composition 8～10 weight portions and place round-bottomed flask, add water 200～300 parts by volume and be heated to boiling and extract volatile oil, after about 1～3 hour, collect volatile oil, with petroleum ether volatile oil extractor tube wall, and join in the volatile oil, as test sample; Other gets Rhizoma Atractylodis Macrocephalae control medicinal material 1～3 weight portion, pulverizes, and crosses 40 mesh sieves, shines the medical material need testing solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.001～0.002 parts by volume of above-mentioned two kinds of solution, put respectively in same block of silica gel G aluminium sheet, with petroleum ether: ethyl acetate=5: 0.1 is developing solvent, launch at normal temperatures, take out, dry, spray 1～3% vanillin ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
C, get this pharmaceutical composition 4～8 weight portions, put in the triangular flask, add the vibration of ether 30～50 parts by volume, leave standstill 1～2 hour after, filter, reclaim ether extraction solution to doing, residue adds 1～3 parts by volume ethyl acetate makes dissolving, standby as need testing solution; Other gets Radix Salviae Miltiorrhizae control medicinal material 1 weight portion, pulverizes, and crosses 40 mesh sieves, shines medical material solution in pairs with legal system, and it is standby to do the control medicinal material test liquid; Take by weighing Tanshinone I Ia reference substance 0.001～0.003 weight portion, add ethyl acetate solution and make concentration to be that every parts by volume contains the reference substance solution of 0.0008～0.0012 weight portion Tanshinone I Ia standby; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.003～0.008 parts by volume of above-mentioned three kinds of solution, put respectively in same block of silica gel G aluminium sheet, with benzene: ethyl acetate=9～10: 0.5～1 solution is developing solvent, launch at normal temperatures, take out, dry; In the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color;
D; get this pharmaceutical composition 8～10 weight portions; put in the conical flask; add 90～100% methanol, 40～60 parts by volume; use power 150W; the supersonic oscillations of frequency 20kHz are after 20～50 minutes; filter, get subsequent filtrate 20～40 parts by volume, decompression and solvent recovery is put dried; residue adding distil water 10～30 parts by volume slight fevers dissolving; use water saturation n-butanol extraction three times, volume is respectively 10～20 parts by volume merges butanol extraction liquid; twice of reuse 1～3% potassium hydroxide washing butanol extraction liquid; volume is respectively 10～20 parts by volume, discards alkali liquor, and butanol extraction liquid is washed till neutrality with the n-butyl alcohol saturated aqueous solution; discard water layer; the reclaim under reduced pressure butanol extraction liquid is done, residue adds the dissolving of 90～100% ethanol, 1～3 parts by volume, as need testing solution; Other gets Radix Bupleuri medical material 1 weight portion, shines medical material solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.004～0.008 parts by volume of above-mentioned two kinds of solution, put respectively in same block of silica gel G aluminium sheet, with chloroform: methanol: water=5～8: 2～5: 1～3 solution is developing solvent, launch at normal temperatures, take out, dry, spray 5～15% ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
E, get this pharmaceutical composition 3～8 weight portions, put in the triangular flask, add the vibration of ether 30～50 parts by volume, leave standstill 1～2 hour after, filter, discard the ether layer, add methanol 20～40 parts by volume, reflux 1～2 hour is filtered, reclaim methanol solution to doing, residue adds water 30～50 parts by volume makes dissolving, uses water saturation n-butanol extraction 3 times, each 10～30 parts by volume, merge butanol solution, wash with water 3 times, each 10～30 parts by volume are with the n-butanol layer evaporate to dryness, residue adds the dissolving of methanol 1～3 parts by volume, as need testing solution; Extracting liquorice control medicinal material 1～3 weight portion shines medical material solution in pairs with legal system in addition; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.003～0.008 parts by volume of above-mentioned two kinds of solution, put respectively in same block of silica gel G aluminium sheet, with chloroform: methanol: formic acid=2～8: 1～3: 0.1～0.3 solution is developing solvent, launch at normal temperatures, take out, dry, spray 30～80% sulphuric acid methanol solutions, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
10. the quality determining method of pharmaceutical composition as claimed in claim 9, it is characterized in that this method comprise following assay and differentiate in one or more:
Assay: get this pharmaceutical composition 5 weight portions, the accurate title, decide, put in the apparatus,Soxhlet's, add 50 parts by volume, 2% potassium hydroxide methanol solution, heating and refluxing extraction 6 hours, merceration spends the night again, the extracting solution reclaim under reduced pressure as for, residue adds 15 parts by volume water dissolutioies, use chloroform: the mixed solution of n-butyl alcohol=2: 1, extract three times, be respectively 40,30,30 parts by volume, combined chloroform: the extract of following butanols=2: 1, with the washing of 1% potassium dihydrogen phosphate aqueous solution, 60 parts by volume, aqueous phase discarded, organic facies evaporated under reduced pressure, it is fixed molten to add 1 parts by volume methanol solution, as need testing solution; Precision takes by weighing the astragaloside reference substance in addition, adds methanol and makes the solution that 1 parts by volume contains 0.001 weight portion, in contrast product solution; Test according to thin layer chromatography, accurate need testing solution 0.001 parts by volume of drawing, reference substance solution 0.002 parts by volume and 0.004 parts by volume, the cross point is on same silica gel g thin-layer plate respectively, under the room temperature with chloroform: methanol: the solution of water=75: 25: 5 ratio was placed after 12 hours, take off layer and be developing solvent, launch once, take out, dry, spray 10% concentrated sulphuric acid alcoholic solution, 100 ℃ were heated 5 minutes down again, took out, cover onesize glass plate again on the lamellae, fix with hinge on every side, scan wavelength X s=530nm according to thin layer chromatography, λ R=650nm, measure test sample trap integrated value and reference substance trap integrated value, calculate, promptly; This drug combination preparation per diem taking dose meter contains astragaloside C44H68O4, must not be lower than 2.700mg.
A, be equipped with need testing solution, get the reference substance astragaloside and add methanol to make the solution that 1 parts by volume contains 0.001 weight portion be reference substance solution according to assay item below legal system; Other gets Milkvetch Root 1.5 weight portions, put in the Sha Shi extractor, adding 2% potassium hydroxide methanol solution extracted after 8 hours, one night of merceration, reclaim and extract solution to doing, add water 15 parts by volume, again with chloroform: n-butyl alcohol=proportioning concentration extraction in 2: 1 is respectively 40,30,30 parts by volume three times, combining extraction liquid adds 1% potassium dihydrogen phosphate 60 parts by volume washing, aqueous phase discarded, again once with 60 parts by volume water washings, organic facies is recycled to dried, adds 1 parts by volume dissolve with methanol, makes control medicinal material solution; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.002 parts by volume of above-mentioned three kinds of solution, put respectively in same 15cm * 10cm silica gel G aluminium sheet, under the room temperature with chloroform: methanol: the solution of water=75: 25: 5 ratio was placed after 12 hours, took off layer and was developing solvent, launched once, take out, dry, spray 10% concentrated sulphuric acid alcoholic solution, be heated to clear spot; In the test sample chromatograph, with reference substance and the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
B, get this pharmaceutical composition 9 weight portions and place round-bottomed flask, add water 250 parts by volume and be heated to boiling and extract volatile oil, after about 1～2 hour, collect volatile oil, with petroleum ether volatile oil extractor tube wall, and join in the volatile oil, as test sample; Other gets Rhizoma Atractylodis Macrocephalae control medicinal material 2 weight portions, pulverizes, and crosses 40 mesh sieves, shines the medical material need testing solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.002 parts by volume of above-mentioned two kinds of solution, put respectively in same block of silica gel G aluminium sheet, with petroleum ether: ethyl acetate=5: 0.1 is developing solvent, launch at normal temperatures, take out, dry, spray 1% vanillin ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
C, get this pharmaceutical composition 6 weight portions, put in the triangular flask, add the vibration of ether 40 parts by volume, leave standstill 1 hour after, filter, reclaim ether extraction solution to doing, residue adds 2 parts by volume ethyl acetate makes dissolving, standby as need testing solution; Other gets Radix Salviae Miltiorrhizae control medicinal material 1 weight portion, pulverizes, and crosses 40 mesh sieves, shines medical material solution in pairs with legal system, and it is standby to do the control medicinal material test liquid; Take by weighing Tanshinone I Ia reference substance 0.002 weight portion, add ethyl acetate solution and make concentration to be that every parts by volume contains the reference substance solution of 0.001 weight portion Tanshinone I Ia standby; According to the test of an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.005 parts by volume of above-mentioned three kinds of solution, put respectively in same block of silica gel G aluminium sheet, with benzene: the solution of ethyl acetate=9.5: 0.5 is developing solvent, launch at normal temperatures, take out, dry; In the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color;
D; get this pharmaceutical composition 9 weight portions; put in the conical flask; add 95% methanol, 50 parts by volume; use power 150W; the supersonic oscillations of frequency 20kHz are after 40 minutes; filter, get subsequent filtrate 30 parts by volume, decompression and solvent recovery is put dried; residue adding distil water 15 parts by volume slight fevers dissolving; use water saturation n-butanol extraction three times, volume is respectively 20; 20; 10 parts by volume merges butanol extraction liquid; twice of reuse 1% potassium hydroxide washing butanol extraction liquid; volume is respectively 20; 20 parts by volume, discards alkali liquor, and butanol extraction liquid is washed till neutrality with the n-butyl alcohol saturated aqueous solution; discard water layer; the reclaim under reduced pressure butanol extraction liquid is done, residue adds the dissolving of 95% ethanol, 1 parts by volume, as need testing solution; Other gets Radix Bupleuri medical material 1 weight portion, shines medical material solution in pairs with legal system; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.006 parts by volume of above-mentioned two kinds of solution, put respectively in same block of silica gel G aluminium sheet, with chloroform: methanol: the solution of water=7: 3: 1 is developing solvent, launch at normal temperatures, take out, dry, spray 10% ethanol solution of sulfuric acid, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
E, get this pharmaceutical composition 5 weight portions, put in the triangular flask, add the vibration of ether 40 parts by volume, leave standstill 1 hour after, filter, discard the ether layer, add methanol 30 parts by volume, reflux 1 hour is filtered, and reclaims methanol solution to doing, residue adds water 40 parts by volume makes dissolving, uses water saturation n-butanol extraction 3 times, each 20 parts by volume, merge butanol solution, wash each 20 parts by volume with water 3 times, with the n-butanol layer evaporate to dryness, residue adds the dissolving of methanol 1 parts by volume, as need testing solution; Extracting liquorice control medicinal material 1 weight portion shines medical material solution in pairs with legal system in addition; Test according to an appendix VIB of Pharmacopoeia of People's Republic of China version in 2000 thin layer chromatography, draw each 0.005 parts by volume of above-mentioned two kinds of solution, put respectively in same block of silica gel G aluminium sheet, with chloroform: methanol: the solution of formic acid=5: 1: 0.1 is developing solvent, launch at normal temperatures, take out, dry, spray 50% sulphuric acid methanol solution, clear spot is put in heating; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
11. as the application of the arbitrary described pharmaceutical composition of claim 1-4 in the medicine of preparation prevention or treatment hepatic fibrosis or early stage liver cirrhosis.
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