CN102145075A

CN102145075A - Pharmaceutical composition for regulating liver and supplementing kidney, eliminating blood stasis and removing spot and preparation method of pharmaceutical composition

Info

Publication number: CN102145075A
Application number: CN2010101095566A
Authority: CN
Inventors: 陈永泰
Original assignee: Individual
Current assignee: Shandong Danhong Pharmaceutical Co Ltd
Priority date: 2010-02-09
Filing date: 2010-02-09
Publication date: 2011-08-10
Anticipated expiration: 2030-02-09
Also published as: CN102145075B

Abstract

The invention discloses a pharmaceutical composition for regulating liver and supplementing kidney, eliminating blood stasis and removing spot, a preparation method, a quality testing method and use of the pharmaceutical composition. The pharmaceutical composition comprises the following raw material medicines of: radix bupleuri, red sage root, radix rehmanniae preparata, angelica dahurica, paenoia suffruticosa and glycyrrhiza. The preparation method comprises the following steps of: distilling the paeonia suffruticosa to get crystal, decocting the other raw material medicines with water, concentrating to obtain concrete, mixing the concrete with the crystal to prepare into various preparation formulations which are acceptable clinically. The quality testing method of the pharmaceutical composition comprises identifying one or more of methods for determining contents of propanoic acid and paeonol. A pharmacological experiment shows that the pharmaceutical composition of the invention has the function of nourishing skin and removing spot, and can effectively restrain the proliferation of B16 cells, the activity of tyosinase of B-16 melanoma cell strain, the formation of melanin, and the formation of the skin injury model mouse serum lipid peroxide caused by ultraviolet irradiation so as to cure the chloasma.

Description

A kind of pharmaceutical composition of transferring liver the kidney invigorating, stasis-resolving spot-removing and preparation method thereof

Invention field

The present invention relates to a kind of pharmaceutical composition and preparation method thereof, particularly a kind ofly can transfer the liver the kidney invigorating, pharmaceutical composition of stasis-resolving spot-removing and preparation method thereof, quality determining method and purposes.

Background technology

Chloasma, the women from the adolescence to the menopause all has generation, and particularly owing to operating pressure, life is nervous, and easier causing falls ill.This disease has had influence on numerous women's psychosomatic health, and therefore the treatment to this disease one draws attention more more.

The cause of disease of chloasma and pathogenesis are still indeterminate.Its pathogenic factors is more, and pathogenic process is comparatively complicated, clinical show as the melanin overexpression mostly and facial pigmentation that dysbolismus caused excessively calm.It is generally acknowledged that influencing melanin cell internal and external factor synthetic and metabolic process all may bring out primary disease, study at present more comprise that vivo oxidation antioxygen dysequilibrium, endocrine disturbance, the little disruption of ecological balance in skin lesion district, trace element anomaly, hemorheology properties are unusual, sunshine and heredity.

The therapeutic modality of chloasma mainly comprises: pharmacotherapy, biotherapy, chemotherapy, physiotherapy etc.Still do not have special effective medicine at present, can give to handle respectively according to the different causes of disease, the treatment approach be the hypertrophy that stops melanocyte mostly, suppresses the formation of melanosome and impels its decomposition.Based on symptomatic treatment, though certain curative effect is arranged, toxic and side effects is also obvious, as the generation exogenous ochronosis, and atrophoderma, " the stippling sample " that permanent decolouring causes, some also can cause nephrotoxicity and nerve, mental symptom.

Topical application hydroquinone (Hydroquinone) treatment chloasma is the most frequently used, and curative effect is also more sure, typical concentrations 2%-4%.Hydroquinone may pass through the restraint of tyrosinase activity, blocking-up melanin generates, and it is synthetic also may to suppress DNA and RNA, and the degraded pigment granule destroys pigment cell's structure, to reach therapeutic effect.The long-time external of hydroquinone can occur that pigment excessively goes down and dermatitis after pigmentation.The Pigmented medicine of similar interference also comprises phenolic compounds, retinoic acid, Azelaic Acid and hydroquinone compound preparation etc.In addition, chemical peeling and laser therapy all have application.

Zoopery at present or clinical observation report to the effective Chinese medicine of chloasma have PAIDU YANGYAN JIAONANG etc. several, clinical report increases gradually in the recent period, but lack objective criterion of therapeutical effect and it is carried out the research of pharmacodynamics aspect in depth, it is satisfied that the curative effect of chloasma is tested title, so rarely have utilization.Endo-medicine seldom, and curative effect is extremely imprecise.The external liniment mostly is health product and skin care item, more can not obtain satisfied effect.The advantage that the performance Chinese medicine and pharmacy is regulated human body integral is developed Chinese medicine speckle dispelling new drug for clinical essential, and feasible.

Summary of the invention

The object of the invention is to disclose the kidney invigorating of a kind of accent liver, the pharmaceutical composition of stasis-resolving spot-removing, the present invention also aims to disclose this preparation of drug combination method, the present invention also aims to disclose the quality determining method of this pharmaceutical composition, the present invention also aims to disclose the purposes of this pharmaceutical composition.

The objective of the invention is to be achieved through the following technical solutions:

The crude drug of pharmaceutical composition of the present invention consists of:

Radix Bupleuri 300-400 weight portion Radix Salviae Miltiorrhizae 300-800 weight portion Radix Rehmanniae Preparata 300-800 weight portion

Radix Angelicae Dahuricae 300-400 weight portion Cortex Moutan 300-400 weight portion Radix Glycyrrhizae 50-150 weight portion.

The crude drug composition of pharmaceutical composition of the present invention is preferably:

Radix Bupleuri 333.3 weight portion Radix Salviae Miltiorrhizaes 500 weight portion Radix Rehmanniaes Preparata 500 weight portions

The Radix Angelicae Dahuricae 333.3 weight portion Cortex Moutans 333.3 weight portion Radix Glycyrrhizaes 100 weight portions.

Radix Bupleuri 310 weight portion Radix Salviae Miltiorrhizaes 750 weight portion Radix Rehmanniaes Preparata 350 weight portions

The Radix Angelicae Dahuricae 390 weight portion Cortex Moutans 310 weight portion Radix Glycyrrhizaes 130 weight portions.

Radix Bupleuri 390 weight portion Radix Salviae Miltiorrhizaes 350 weight portion Radix Rehmanniaes Preparata 750 weight portions

The Radix Angelicae Dahuricae 310 weight portion Cortex Moutans 390 weight portion Radix Glycyrrhizaes 70 weight portions.

Get the above-mentioned raw materials medicine, add conventional adjuvant, make clinical pharmaceutical dosage form: granule, pill, powder, capsule, tablet, slow releasing agent, oral liquid or lyophilized injectable powder according to common process.

Preparation of drug combination method of the present invention comprises the steps:

Get crude drug, Cortex Moutan is collected the distillate that Cortex Moutan 5-15 doubly measures with the water distillation that 10-20 doubly measures, in 0～10 ℃ of following cold preservation 6-12 hour, filter, get paeonol crystallization I, filtrate adds the sodium chloride of Cortex Moutan weight 3-8%, and redistillation is collected the distillate that Cortex Moutan 2-4 doubly measures, in 0～10 ℃ of following cold preservation 6-12 hour, filter, get the paeonol crystal II, merge paeonol crystallization I and II,, promptly get paeonol in 30-50 ℃ of cold drying, standby, filtrate A device in addition collects medicinal residues and all the other Radix Bupleuri, Radix Salviae Miltiorrhizae, Radix Rehmanniae Preparata, the Radix Angelicae Dahuricae and Radix Glycyrrhizae five tastes crude drug, decoct with water 1-3 time, add water 5-12 times of weight at every turn, decocted 1-3 hour at every turn, filter, merging filtrate gets liquor B, mix filtrate A and B, be concentrated into 50～70 ℃ of following relative densities and be 1.10～1.35 thick paste, drying, pulverize, cross 60 mesh sieves, get extract powder, standby; Get above-mentioned paeonol porphyrize, with the magnesium stearate mix homogeneously that accounts for extract powder weight 1-1.5%, add in the above-mentioned extract powder, mixing, add conventional adjuvant, make clinical pharmaceutical dosage form: granule, pill, powder, capsule, tablet, slow releasing agent, oral liquid or lyophilized injectable powder according to common process.

Preparation of drug combination method of the present invention preferably includes following steps:

Get crude drug, Cortex Moutan is collected the distillate of 10 times of amounts of Cortex Moutan with the water distillation of 15 times of amounts, in 0～10 ℃ of following cold preservation 8 hours, filter, get paeonol crystallization I, filtrate adds the sodium chloride of Cortex Moutan weight 5%, and redistillation is collected the distillate of 3 times of amounts of Cortex Moutan, in 0～10 ℃ of following cold preservation 8 hours, filter, get the paeonol crystal II, merge paeonol crystallization I and II,, promptly get paeonol in 40 ℃ of cold drying, standby, filtrate A device in addition collects medicinal residues and all the other Radix Bupleuri, Radix Salviae Miltiorrhizae, Radix Rehmanniae Preparata, the Radix Angelicae Dahuricae and Radix Glycyrrhizae five tastes crude drug, decoct with water secondary, add for the first time water 10 times of weight, add water 8 times of weight the 2nd time, the each decoction 2 hours, filter, merging filtrate gets liquor B, mixes filtrate A and B, be concentrated into 60～65 ℃ of following relative densities and be 1.20～1.25 thick paste, drying is pulverized, and crosses 60 mesh sieves, get extract powder, standby; Get above-mentioned paeonol porphyrize, with the magnesium stearate mix homogeneously that accounts for extract powder weight 0.5%, add in the above-mentioned extract powder, mixing, add conventional adjuvant, make clinical pharmaceutical dosage form: granule, pill, powder, capsule, tablet, slow releasing agent, oral liquid or lyophilized injectable powder according to common process.

The quality determining method of pharmaceutical composition of the present invention comprises one or more in following discriminating and the content assaying method:

Differentiate:

A. get 1/12 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 20-30 parts by volume and make dissolving, filter, filtrate is put in the separatory funnel, extracts 2-4 time with water saturated n-butyl alcohol jolting, uses the 10-20 parts by volume at every turn, merge n-butanol extracting liquid, add isopyknic ammonia solution, shake up, place and make layering, divide and get n-butyl alcohol liquid, evaporate to dryness, residue add methanol 0.5-1.5 parts by volume makes dissolving, as need testing solution;

Other gets Radix Bupleuri control medicinal material 1 weight portion, adds methanol 10-30 parts by volume, reflux 20-40 minute, filter, filtrate evaporate to dryness, residue add water 10-30 parts by volume, reflux 20-40 minute, filter, filtrate evaporate to dryness, residue add water 10-30 parts by volume makes dissolving, reflux 0.5-1.5 hour, put in the separatory funnel, extract 2-4 time, use the 10-20 parts by volume at every turn with water saturated n-butyl alcohol jolting, merge n-butanol extracting liquid, add isopyknic ammonia solution, shake up, place and make layering, divide and get n-butyl alcohol liquid, evaporate to dryness, residue add methanol 0.5-1.5 parts by volume makes dissolving, preparation control medicinal material solution;

Test according to thin layer chromatography (appendix VI B), draw above-mentioned each 0.003-0.008 parts by volume of two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, is developing solvent with chloroform-methanol-water=10-15: 5-10: 1-3 at lower floor's solution of placing below 5-15 ℃, launch, take out, dry, spray is with 0.5-1.5% paradime thylaminobenzaldehyde ethanol solution of sulfuric acid, get 0.5-1.5% paradime thylaminobenzaldehyde sulfuric acid solution 1mL, to 10mL, get this solution spray silica gel g thin-layer plate with ethanol dilution, it is clear to be heated to the speckle colour developing at 60-80 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of same color; Put under the uviol lamp 365nm and inspect, show identical xanchromatic fluorescence speckle;

B. get 1/12 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 20-40 parts by volume and make dissolving, filter, filtrate transfers pH value to 1-2 with dilute hydrochloric acid, puts in the separatory funnel, extract 2-4 time with the ethyl acetate jolting, each 10-30 parts by volume merges ethyl acetate liquid, and the reclaim under reduced pressure ethyl acetate is to doing, residue adds methanol 1-3 parts by volume makes dissolving, as need testing solution;

Other gets Radix Salviae Miltiorrhizae control medicinal material 0.5 weight portion, adds methanol 10-30 parts by volume, and supersound process 20-40 minute, filter, filtrate is concentrated into the 1-3 parts by volume, in contrast medical material solution;

Test according to thin layer chromatography (appendix VIB), draw above-mentioned each 0.003-0.008 parts by volume of two kinds of solution, put respectively on same silica gel g thin-layer plate with the preparation of 0.5-1.5% sodium hydroxide solution, with ethyl acetate-formic acid-glacial acetic acid-water=10-20: 0.5-1.5: 0.5-1.5: 1-3 is developing solvent, launch, take out, dry, put under the uviol lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical fluorescence speckle;

C. get 1/6 of the daily amount of formulation of pharmaceutical composition of the present invention, add ethanol 10-30 parts by volume, close plug, jolting 5-15 minute, filter, filtrate is concentrated into the 0.5-1.5 parts by volume, as need testing solution;

Other gets the paeonol reference substance, adds acetone and makes the solution that per 1 parts by volume contains the 0.003-0.008 weight portion, product solution in contrast;

According to thin layer chromatography (appendix VI B test), draw above-mentioned each 0.008-0.012 parts by volume of two kinds of solution, put respectively on same silica gel g thin-layer plate, with cyclohexane extraction-ethyl acetate=2-4: 0.5-1.5 is developing solvent, launch, take out, dry, spray is with the acid 3-8% ferric chloride of hydrochloric acid alcoholic solution, and it is clear to be heated to the speckle colour developing; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical blue brown speckle;

D. get 1/12 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 20-30 parts by volume and make dissolving, filter, filtrate is put in the separatory funnel, extracts 2-4 time with water saturated n-butyl alcohol jolting, uses the 10-20 parts by volume at every turn, merge n-butanol extracting liquid, add isopyknic ammonia solution, shake up, place and make layering, divide and get n-butyl alcohol liquid, evaporate to dryness, residue add methanol 0.5-1.5 parts by volume makes dissolving, as need testing solution;

Other gets Radix Angelicae Dahuricae control medicinal material 0.5 weight portion, adds methanol 5-15 parts by volume, and supersound process 20-40 minute, filter, filtrate is concentrated into the 1-3 parts by volume, in contrast medical material solution;

Test according to thin layer chromatography (appendix VI B), draw above-mentioned each 0.003-0.008 parts by volume of two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, with 30-60 ℃ of petroleum ether-ethyl acetate=0.5-1.5: 0.5-1.5 is developing solvent, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

E. get 1/6 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 20-40 parts by volume and make dissolving, filter, filtrate is put in the separatory funnel, extract 2-4 time with water saturated n-butyl alcohol jolting, each 10-30 parts by volume merges n-butyl alcohol liquid, water 10-30 parts by volume washing 1-3 time, discard water layer, divide and get n-butyl alcohol liquid, evaporate to dryness, residue adds methanol 0.5-1.5 parts by volume makes dissolving, as need testing solution;

Extracting liquorice control medicinal material 0.5 weight portion adds methanol 3-5 parts by volume in addition, supersound process 20-40 minute, gets supernatant medical material solution in contrast;

Test according to thin layer chromatography (appendix VI B), draw above-mentioned each 0.003-0.008 parts by volume of two kinds of solution, put respectively on same silica gel g thin-layer plate with the preparation of 0.5-1.5% sodium hydroxide solution, with ethyl acetate-formic acid-glacial acetic acid-water=10-20: 0.5-1.5: 0.5-1.5: 1-3 is developing solvent, launches, and takes out, dry, spray is with the 5-15% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, inspects under ultra-violet lamp 365nm; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

Assay: measure according to high performance liquid chromatography (appendix VI D)

A. danshensu:

Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; Methanol-0.5% glacial acetic acid=10-30: 60-100 is a mobile phase; The detection wavelength is 280nm; Number of theoretical plate calculates by the danshensu peak should be not less than 2000;

The reference substance solution preparation: get the danshensu sodium reference substance, the accurate title, decide, and adds methanol and make the solution that per 1 parts by volume contains the 0.00004-0.00008 weight portion, promptly;

The preparation of need testing solution: get 1/12 of the daily amount of formulation of pharmaceutical composition of the present invention, put in the 100 parts by volume triangular flasks, precision adds methanol 40-60 parts by volume, supersound process 20-40 minute, put coldly, claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 5-15 parts by volume, puts in the 50 parts by volume round-bottomed flasks, reclaims solvent to doing, residue adds the mobile phase dissolving and changes in the 5-15 parts by volume measuring bottle, add mobile phase to scale, shake up, promptly;

Algoscopy: accurate respectively absorption reference substance solution 0.003-0.008 parts by volume and 0.005-0.015 parts by volume, need testing solution 0.005-0.015 parts by volume, inject chromatograph of liquid, measure, promptly;

Pharmaceutical composition of the present invention every day is with containing Radix Salviae Miltiorrhizae in the amount of formulation with danshensu (C ₉H ₁₀O ₅) meter, must not be less than 19.2mg;

B. paeonol:

Chromatograph vitta and system suitability test: with octadecylsilane chemically bonded silica is filler; Methanol-water-glacial acetic acid=40-80: 30-50: 0.5-1.5 is a mobile phase; The detection wavelength is 27nm; Number of theoretical plate calculates by the paeonol peak should be not less than 2000;

The preparation of reference substance solution: get the paeonol reference substance, the accurate title, decide, and adds methanol and make the solution that per 1 parts by volume contains the 0.000003-0.000008 weight portion, promptly;

The preparation of need testing solution: get 1/24 of the daily amount of formulation of pharmaceutical composition of the present invention, put in the 100 parts by volume triangular flasks, precision adds methanol 40-60 parts by volume, excusing from death was handled 20-40 minute, put coldly, claimed to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 0.5-1.5 parts by volume, put in the 10 parts by volume measuring bottles, add methanol to scale, shake up, promptly;

Algoscopy: accurate respectively reference substance solution and each 0.005-0.015 parts by volume of need testing solution drawn, injecting chromatograph is measured, promptly;

Pharmaceutical composition of the present invention every day is with containing Cortex Moutan in the amount of formulation with paeonol (C ₉H ₁₀O ₃) meter, must not be less than 31.2mg.

Drug regimen of the present invention quality determining method preferably include in following discriminating and the content assaying method one or more:

Differentiate:

A. get 1/12 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 25 parts by volume and make dissolving, filter, filtrate is put in the separatory funnel, extracts 3 times with water saturated n-butyl alcohol jolting, uses 15 parts by volume at every turn, merge n-butanol extracting liquid, add isopyknic ammonia solution, shake up, place and make layering, divide and get n-butyl alcohol liquid, evaporate to dryness, residue add methanol 1 parts by volume makes dissolving, as need testing solution;

Other gets Radix Bupleuri control medicinal material 1 weight portion, adds methanol 20 parts by volume, reflux 30 minutes, filter, filtrate evaporate to dryness, residue add water 20 parts by volume, reflux 30 minutes, filter, filtrate evaporate to dryness, residue add water 20 parts by volume makes dissolving, reflux 1 hour, put in the separatory funnel, extract 3 times, use 15 parts by volume at every turn with water saturated n-butyl alcohol jolting, merge n-butanol extracting liquid, add isopyknic ammonia solution, shake up, place and make layering, divide and get n-butyl alcohol liquid, evaporate to dryness, residue add methanol 1 parts by volume makes dissolving, preparation control medicinal material solution;

Test according to thin layer chromatography (appendix VI B), draw each 0.005 parts by volume of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, so that chloroform-methanol-water=the lower floor's solution in placement below 10 ℃ was developing solvent in 13: 7: 2, launch, take out, dry, spray is with 1% paradime thylaminobenzaldehyde ethanol solution of sulfuric acid, get 0.5-1.5% paradime thylaminobenzaldehyde sulfuric acid solution 1mL, to 10mL, get this solution spray silica gel g thin-layer plate with ethanol dilution, it is clear to be heated to the speckle colour developing at 70 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of same color; Put under the uviol lamp 365nm and inspect, show identical xanchromatic fluorescence speckle;

B. get 1/12 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 30 parts by volume and make dissolving, filter, filtrate transfers pH value to 1-2 with dilute hydrochloric acid, puts in the separatory funnel, extract 3 times with the ethyl acetate jolting, each 20 parts by volume merge ethyl acetate liquid, and the reclaim under reduced pressure ethyl acetate is to doing, residue adds methanol 2 parts by volume makes dissolving, as need testing solution;

Other gets Radix Salviae Miltiorrhizae control medicinal material 0.5 weight portion, adds methanol 20 parts by volume, and supersound process 30 minutes filters, and filtrate is concentrated into 2 parts by volume, in contrast medical material solution;

Test according to thin layer chromatography (appendix VI B), draw each 0.005 parts by volume of above-mentioned two kinds of solution, put respectively on the silica gel g thin-layer plate of same usefulness 1% sodium hydroxide solution preparation, with ethyl acetate-formic acid-glacial acetic acid-water=15: 1: 1: 2 was developing solvent, launch, take out, dry, put under the uviol lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical fluorescence speckle;

C. get 1/6 of the daily amount of formulation of pharmaceutical composition of the present invention, add ethanol 20 parts by volume, close plug, jolting 10 minutes filters, and filtrate is concentrated into 1 parts by volume, as need testing solution;

Other gets the paeonol reference substance, adds acetone and makes the solution that per 1 parts by volume contains 0.005 weight portion, product solution in contrast;

According to thin layer chromatography (appendix VI B test), draw each 0.010 parts by volume of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with cyclohexane extraction-ethyl acetate=3: 1 was developing solvent, launch, take out, dry, spray is with the acid 5% ferric chloride alcoholic solution of hydrochloric acid, and it is clear to be heated to the speckle colour developing; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical blue brown speckle;

D. get 1/12 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 25 parts by volume and make dissolving, filter, filtrate is put in the separatory funnel, extracts 3 times with water saturated n-butyl alcohol jolting, uses 15 parts by volume at every turn, merge n-butanol extracting liquid, add isopyknic ammonia solution, shake up, place and make layering, divide and get n-butyl alcohol liquid, evaporate to dryness, residue add methanol 1 parts by volume makes dissolving, as need testing solution;

Other gets Radix Angelicae Dahuricae control medicinal material 0.5 weight portion, adds methanol 10 parts by volume, and supersound process 30 minutes filters, and filtrate is concentrated into 2 parts by volume, in contrast medical material solution;

Test according to thin layer chromatography (appendix VI B), draw each 0.005 parts by volume of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, with 30-60 ℃ of petroleum ether-ethyl acetate=1: 1.1 was developing solvent, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

E. get 1/6 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 30 parts by volume and make dissolving, filter, filtrate is put in the separatory funnel, extract 3 times with water saturated n-butyl alcohol jolting, each 20 parts by volume merge n-butyl alcohol liquid, water 20 parts by volume washing 2 times, discard water layer, divide and get n-butyl alcohol liquid, evaporate to dryness, residue adds methanol 1 parts by volume makes dissolving, as need testing solution;

Extracting liquorice control medicinal material 0.5 weight portion adds methanol 4 parts by volume in addition, and supersound process 30 minutes is got supernatant medical material solution in contrast;

Test according to thin layer chromatography (appendix VI B), draw each 0.005 parts by volume of above-mentioned two kinds of solution, put respectively on the silica gel g thin-layer plate of same usefulness 1% sodium hydroxide solution preparation, with ethyl acetate-formic acid-glacial acetic acid-water=15: 1: 1: 2 was developing solvent, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, inspects under ultra-violet lamp 365nm; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

A. danshensu:

Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; Methanol-0.5% glacial acetic acid=20: 80 is a mobile phase; The detection wavelength is 280nm; Number of theoretical plate calculates by the danshensu peak should be not less than 2000;

The reference substance solution preparation: get the danshensu sodium reference substance, the accurate title, decide, and adds methanol and make the solution that per 1 parts by volume contains 0.00006 weight portion, promptly;

The preparation of need testing solution: get 1/12 of the daily amount of formulation of pharmaceutical composition of the present invention, put in the 100 parts by volume triangular flasks, precision adds methanol 50 parts by volume, supersound process 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 10 parts by volume, puts in the 50 parts by volume round-bottomed flasks, reclaims solvent to doing, residue adds the mobile phase dissolving and changes in the 10 parts by volume measuring bottles, add mobile phase to scale, shake up, promptly;

Algoscopy: accurate respectively absorption reference substance solution 0.005 parts by volume and 0.010 parts by volume, need testing solution 0.010 parts by volume, inject chromatograph of liquid, measure, promptly;

B. paeonol:

Chromatograph vitta and system suitability test: with octadecylsilane chemically bonded silica is filler; Methanol-water-glacial acetic acid=60: 40: 1 is a mobile phase; The detection wavelength is 27nm; Number of theoretical plate calculates by the paeonol peak should be not less than 2000;

The preparation of reference substance solution: get the paeonol reference substance, the accurate title, decide, and adds methanol and make the solution that per 1 parts by volume contains 0.000005 weight portion, promptly;

The preparation of need testing solution: get 1/24 of the daily amount of formulation of pharmaceutical composition of the present invention, put in the 100 parts by volume triangular flasks, precision adds methanol 50 parts by volume, excusing from death was handled 30 minutes, put coldly, claimed to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 1 parts by volume, put in the 10 parts by volume measuring bottles, add methanol to scale, shake up, promptly;

Algoscopy: accurate respectively reference substance solution and each 0.010 parts by volume of need testing solution drawn, injecting chromatograph is measured, promptly;

The ratio of weight portion of the present invention and parts by volume is a grams per milliliter.

The quality determining method of pharmaceutical composition of the present invention can be applied to the various dosage forms of compositions, as tablet, granule, pill, capsule, drop pill, soft capsule, slow releasing agent, clinical acceptable forms such as oral liquid or lyophilized injectable powder, because be identical with suitable crude drug amount contained in the amount of formulation preparation every day of different dosage form, therefore each dosage form is when carrying out assay, selected sample size can be unified conversion for using amount of formulation every day, and this content assaying method is meant total sheet number that use every day with amount of formulation described every day, total grain number or total ball number etc.

Description of drawings

Fig. 1: the influence of pharmaceutical composition vitro inhibition B-16 propagation of the present invention;

Fig. 2: pharmaceutical composition of the present invention influences tyrosinase activity; A, pharmaceutical composition of the present invention; B, PAIDU YANGYAN JIAONANG

Fig. 3: the influence that pharmaceutical composition of the present invention generates melanin; A, pharmaceutical composition of the present invention; B, PAIDU YANGYAN JIAONANG

Fig. 4: hormone disturbance causes mice chloasma model skin melanin immunohistochemical staining, the SP method, and A: the normal control group presents brown reaction in accidental skin surface and the adnexa epithelial cell endochylema

B: model control group, easily see in the skin surface that is dispersed in and the adnexa epithelial cell endochylema to present brown reaction

C: pharmaceutical composition group of the present invention presents brown reaction in rare skin surface and the adnexa epithelial cell endochylema;

Fig. 5: the HPLC chromatogram of content of Danshensu mensuration-standard curve;

Fig. 6: the HPLC chromatogram of content of Danshensu mensuration-reference substance, test sample and negative control product;

Fig. 7: the HPLC chromatogram that content of Danshensu mensuration-sample stability is investigated;

Fig. 8: the HPLC chromatogram that content of Danshensu mensuration-method precision is investigated;

Fig. 9: the HPLC chromatogram that content of Danshensu mensuration-method repeatability is investigated;

Figure 10: the HPLC chromatogram of content of Danshensu mensuration-sample determination;

Figure 11: the HPLC chromatogram of content detection of paeonol-standard curve;

Figure 12: the HPLC chromatogram of content detection of paeonol-reference substance, test sample and negative control product;

Figure 13: the HPLC chromatogram that content detection of paeonol-sample stability is investigated;

Figure 14: the HPLC chromatogram that content detection of paeonol-method precision is investigated;

Figure 15: the HPLC chromatogram that content detection of paeonol-method repeatability is investigated;

Figure 16: the HPLC chromatogram of content detection of paeonol-sample determination.

Pharmaceutical composition of the present invention belongs to transfers the liver the kidney invigorating, and stasis-resolving spot-removing side's medicine cures mainly stagnation of liver-QI and suffers from a deficiency of the kidney, and women chloasma due to QI and blood is retarded by silt has the effect of supporting flourish speckle dispelling.Disease meeting portion skin dark stain often can be with dizziness and fatigue, and is uncomfortable in chest how tired, soreness of the waist and knees, and menoxenia etc., arteries and veins is heavy string how, and tongue is dim or ecchymosis arranged.Effect experiment shows, pharmaceutical composition of the present invention can significantly reduce the average area, target of the positive target of estrogen and progestogen induced mice skin immunization histochemical stain melanin and the ratio (surface density) of statistics area, and is inhibited to the melanic formation of mouse skin; Erythema that ultraviolet causes mouse skin, edema be can significantly alleviate, obviously degeneration, the necrosis on ultraviolet radiation mouse skin surface alleviated, and cell infiltration, hyperemia, edema; Propionibacterium acne, micrococcus luteus all there are obvious inhibition and killing action; Mutations in Skin of Different Ultraviolet damage model mice and alloxan are caused the serum of the high lipid peroxide animal model of mice and liver lipid peroxide to be formed certain inhibitory action is all arranged; Pharmaceutical composition of the present invention can also suppress and kill propionibacterium acne, micrococcus luteus, and epinephrine and frozen water are caused rat blood stasis model hemorheological property certain improvement effect.The toxicity performance judges that pharmaceutical composition of the present invention belongs to no overt toxicity medicine behind acute toxicity institute's amount of reagent of pharmaceutical composition of the present invention and the absolute value medicine.

Following experimental example and embodiment are used to further specify but are not limited to the present invention.

Experimental example 1 extraction process technology Study on Conditions

1, the research of paeonol extraction conditions

(1) the distillate volume is to the influence of paeonol extraction ratio

The volume of collecting distillate is very big to paeonol extraction ratio (rate of transform) influence, should screen.Get Cortex Moutan (paeonol content is 2.34%) 50g, add water 1000ml, add thermal distillation, collect distillate, every part of 50ml puts in the 100ml measuring bottle, adds 95% ethanol dilution to scale, shakes up, precision is measured in right amount, puts in the 25ml measuring bottle, and thin up shakes up to scale.According to spectrophotography (appendix VA), side is decided trap at the wavelength place of 274nm, presses the absorptance (E of paeonol _1cm ^1%) be 862 calculating, see the content assaying method under 2000 editions 137 pages of Cortex Moutan items of Chinese Pharmacopoeia for details, promptly get the concentration and the total amount of paeonol in every part of distillate, the results are shown in Table 1.

Table 1 distillate volume is to the influence of paeonol extraction efficiency

* extraction ratio=(total amount of paeonol in the accumulative total total amount ÷ medical material) * 100%

Total amount=the 50g of paeonol * 2.34%=1.17g in the medical material

Conclusion: the volume of collecting distillate is advisable with 10 times of amounts of Cortex Moutan, and the extraction ratio of this step operation is about 88.8%.

(2) the sodium chloride addition is to the influence of paeonol extraction ratio in the re-distilled liquid

The paeonol crystallization is separated out in the cold preservation of Cortex Moutan distillate, filters, and gets paeonol.Still contain a certain amount of paeonol in the filtrate (mother solution), should need redistillation once more.Add proper amount of sodium chloride (be equivalent to medical material amount 5%) and can improve the extraction ratio of paeonol,, study as follows so the sodium chloride addition is screened:

Get Cortex Moutan 150g, add water 2250ml, add thermal distillation, collect distillate 1500ml, put refrigerator cold-storage (about 5 ℃), spend the night, filter, cold drying gets paeonol 2.35g.Filtrate is divided into three equal parts, and every part of 500ml (paeonol content is 0.445mg/ml) adds sodium chloride (being equivalent to medical material) 0 (0%), 2.5g (5%), 5.0g (10%) respectively, and redistillation is collected distillate, every part of 50ml.Get 20ml respectively and put refrigerator cold-storage, spend the night, observation has or not the paeonol crystallization to separate out; Other gets the 25ml distillate, puts in the 50ml measuring bottle, adds 95% ethanol dilution to scale, shakes up, and precision is measured in right amount, puts in the 25ml measuring bottle, and thin up shakes up to scale.According to spectrophotography (appendix VA), measure trap at the wavelength place of 274nm, press the absorptance (E of paeonol _1cm ^1%) be 862 calculating, promptly get the concentration and the total amount of paeonol in every part of re-distilled liquid, the results are shown in Table 2.

Table 2 sodium chloride is to the influence of paeonol extraction efficiency in the re-distilled liquid

# cold preservation crystallize: +++show that crystallization is many, ++ more ,+few ,-do not have

* extraction ratio=(total amount of paeonol in the accumulative total total amount ÷ filtrate) * 100%

Total amount=the 500ml of paeonol * 0.445mg/ml=222.5mg in the filtrate

(3) conclusion

By table 2 as seen: (a) need add the sodium chloride that is equivalent to Cortex Moutan weight 5% during redistillation, it is fastest that paeonol steams, and promptly the concentration of paeonol is the highest in the re-distilled liquid, so the sodium chloride consumption is the best with 5% of Cortex Moutan weight.(b) get every part of re-distilled liquid 20ml, put refrigerator cold-storage, spend the night, the result shows that 1-3 part (accumulative total 150ml) has the paeonol crystallization to separate out, and 4-7 part does not have the paeonol crystallization and separates out, so collection re-distilled liquid 150ml, promptly 3 times of amounts of Cortex Moutan are advisable.The ashamed extraction ratio of this step behaviour is 66.2%.

According to above-mentioned result of study, determine that paeonol extraction process condition is: Cortex Moutan is collected the distillate of 10 times of amounts of Cortex Moutan with the water distillation of 15 times of amounts, in 0～10 ℃ of following cold preservation 8 hours, filter, get paeonol crystallization I, filtrate adds the sodium chloride of Cortex Moutan weight 5%, and redistillation is collected the distillate of 3 times of amounts of Cortex Moutan, in 0～10 ℃ of following cold preservation 8 hours, filter, get the paeonol crystal II, merge paeonol crystallization I and II, in 40 ℃ of cold drying, promptly get paeonol.

2, decocting boils Study on Conditions

(1) moisture content test

Take by weighing Radix Bupleuri, Radix Salviae Miltiorrhizae, Radix Rehmanniae Preparata, the Radix Angelicae Dahuricae, Radix Glycyrrhizae by prescription Billy and add ten times in water, soaked overnight is all soaked into medical material, and the unabsorbed water liquid of filtering is tried to achieve water absorption rate and is about 210%.

(2) decocting condition screening

The principal element that amount of water, decocting time, decoction number of times extract for influence is so need it is screened.Adopt four factors, three levels to carry out orthogonal test research, the screening optimum process condition.

Extraction ratio with danshensu is an evaluation index, adopts orthogonal test that amount of water (A), decocting time (B), decoction number of times (C) are carried out screening study.Take by weighing Radix Bupleuri 20g, Radix Salviae Miltiorrhizae 30g, Radix Rehmanniae Preparata 30g, Radix Angelicae Dahuricae 20g, Cortex Moutan 20g, Radix Glycyrrhizae 6g respectively, totally 9 parts, press L ₉, the design of (3) orthogonal test table 3 and table 4 decocts with water, filters, and merging filtrate, mixing, measurement volumes, taking liquid is an amount of, presses the preparation method of need testing solution and handles, and gets the 1-9 need testing solution.The concentration of danshensu in the working sample calculates the total amount of danshensu in the decocting liquid.

A. material and instrument

Waters liquid chromatographic system: comprise Waters 1525 pumps; Waters 2487 dual wavelength UV-detector; The Waters column oven; Breeze liquid chromatograph work station.

Danshensu sodium: the assay reference substance, available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number: 855-200102; Methanol is chromatographically pure, and water is that redistilled water is through 0.45 μ m membrane filtration.All the other reagent are analytical pure.

B. method and result

Chromatographic condition mobile phase: methanol-0.5% glacial acetic acid (20: 80); Chromatographic column: LunaC ₁₈(2) 4.6 * 150mm, 5 μ m; Detect wavelength: 280nm; Flow velocity: 0.6ml/min; Column temperature: 30 ℃.

It is an amount of that the preparation precision of reference substance solution takes by weighing the danshensu sodium reference substance, is mixed with the solution that every 1ml contains 0.0772mg (amounting to danshensu 0.06755mg/ml) with mobile phase, promptly.

The accurate decocting liquid 20ml that draws of the preparation of need testing solution puts in the evaporating dish, evaporate to dryness, and residue is transferred in the 25ml measuring bottle with methanol, filter, get subsequent filtrate 10ml evaporate to dryness, residue dissolves with mobile phase and is transferred in the 10ml measuring bottle, add mobile phase to scale, shake up, promptly.

Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure peak area, calculate, promptly.

The result: seeing Table 3, table 4 and table 5, is evaluation index with the total amount (mg) of danshensu in the decocting liquid.Show that by the size of the extreme difference R value in the table 4 each factor effect primary and secondary is B＞C＞A, with A ₃B ₃C ₃For good.By The results of analysis of variance as can be known, A factor there was no significant difference, B and C factor have utmost point significant difference, with A ₁B ₃C ₃For good, consider the production practical situation, from saving water source and energy consumption, shorten the production cycle, the angle that reduces production costs is set out, and determines that actual production technology is A ₁B ₂C ₃

(3) demonstration test

Take by weighing with a collection of medical material respectively by A ₁B ₃C ₃With A ₁B ₂C ₃Test, repeat 3 times, the results are shown in Table 6.By the visible alternative plan A of table 6 ₁B ₂C ₃It is about 5% that the total amount of extracting danshensu only reduces, and reduces 1 time but decoct number of times, and actual from producing, this should be preferred plan.Promptly decoct 2 times, add 10 times in water the 1st time, add 8 times in water, each 2 hours the 2nd time.

Table 3 decocting boils the design of orthogonal test gauge outfit

Table 4 Orthogonal Experiment and Design table and result

Table 5 orthogonal test analysis of variance table

F _0.01(2，4)＝18.0

Table 6 demonstration test result

Experimental example 2-7 all adopts following reagent thing, positive drug, animal, reagent and the instrument of being subjected to

1, is subjected to reagent thing and positive drug

The capsule content that pharmaceutical composition of the present invention: embodiment 1 prepares, pale brown color medicated powder, no special odor, 1g medicated powder is equivalent to crude drug in whole 4.26g.Yi Jinhai researcher provides by the Traditional Chinese Medicine Research Institute, Sichuan Province, lot number 030604.

LIUWEI DIHUANG WAN: the accurate word Z41022128 of traditional Chinese medicines.Specification: every ball is equivalent to crude drug 3g, every bottle 200 ball.The Henan Province Wanxi Pharmacy Stock Co., Ltd produces.Lot number 030605.

VITAMIN C TABLET: the accurate word H50020036 of traditional Chinese medicines.Specification: 0.1g * 100 slice, Xinan Pharmaceutical Co., Ltd. produces, lot number 030207.

Synestrin tablets: the accurate word (1981) of medicine is defended No. 001135 in Tianjin.Specification: 0.5mg * 100 slice, Tianjin Lisheng Pharmaceutical Co., Ltd. produces, lot number 0208015.

The medroxyprogesterone acetate sheet: the accurate word (1996) of medicine is defended No. 121701 in Zhejiang.Specification: 2mg * 100 slice, Zhejiang Province XianJu Pharmacy stock Co., Ltd, lot number: 021217.

PAIDU YANGYAN JIAONANG: the accurate word Z53020685 of traditional Chinese medicines.Specification: 0.4g/ grain, 10 of every plates, 6 plates/box.Yunnan Panlongyunhai Pharmaceutical Co., Ltd. produces, lot number 030546.

Alloxan: ALLDXAN5.6DIOXYURACTI.Specification: 25g/ bottle.SIGMA company provides, lot:12K1460.

Radix Salviae Miltiorrhizae drop pill: (95) are defended the accurate word Z-01 of medicine number.Specification: 25mg * 100. Tianjin Tasly Pharmaceutical Co., Ltd produces, lot number 20020807.

Adrenalin hydrochloride injection: the accurate word H50020015 of traditional Chinese medicines.Specification: 1ml: 1mg, 1mg/ prop up * 10/box.Xinan Pharmaceutical Co., Ltd. produces, lot number 030401.

2, animal and cell strain

The strain of mice B-16 melanoma cells is provided by the refreshing group of China;

Km (Kunming kind) mice, one-level is qualified, and the real kinoplaszm in river 2002-33 number is provided by the Traditional Chinese Medicine Research Institute, Sichuan Province Experimental Animal Center;

The SD rat, one-level is qualified, and the real kinoplaszm in river 2002-32 number is provided by the Traditional Chinese Medicine Research Institute, Sichuan Province Experimental Animal Center.

Propionibacterium acne CMCC65102, micrococcus luteus CMCC28001 preserve the Quality Control bacterial strain from Sichuan Province's epidemic prevention station.

3, instrument and reagent

Microplate reader (Bio-Rad, Model3550); Centrifuge (Beijing Medical Centrifugal Machine Factory), LDZ5-2); Inverted microscope (Nikon DEAPHOT Fx-35DX); Carbon dioxide cell incubator (BNA-311, ESPEC); Day island proper Tianjin UV-730 automatic biochemical analyzer; Day island proper Tianjin EB-3200D precise electronic balance (precision 0.01g); The JA1003A of Shanghai Jingtian Electronic Instrument Co., Ltd. type electronic balance (precision 1mg); The 80-2 of Shanghai Surgical Operation Equipment Factory centrifugation device; The continuous adjustable quantitative pipettor of the vigorous Finnpipette of Finland's thunder 10-100,20-200,100-1000 μ l and 1-5ml adjustable pipette; Quartz ultraviolet germicidal lamp, the Yangzhou health equipment company limited of leading to; The electronic refiner of DY89-1 type, Ningbo new peak section instrument institute; The R80 of Beijing Steellex Scientific Instrument Company hemorheology instrument; The full-automatic pathology image of the non-clinical safety evaluation of the XYTX-2000 of Traditional Chinese Medicine Research Institute, Sichuan Province Sichuan University medicine quantified system analysis.

SOD reagent: Nanjing is built up bio-engineering research and is provided, lot number 20031225.

MDA reagent: Nanjing is built up bio-engineering research and is provided, lot number 20031226.

Sodium sulfide: 500g/ bottle, chemical pure, one-tenth person chemical reagent factory, lot number 980923.

Normal saline: 0.9% sodium chloride injection, 500ml:4.5g.The accurate word H51021158 of traditional Chinese medicines, Sichuan Kelun Large Pharmaceutical Factory Co. Ltd, lot number 20030921-18.

Formalin: 200ml/ bottle, analytical pure, Kingsoft, Chengdu chemical reagent factory, lot number 030820.

Alloxan: ALLDXAN 5.6-DIOXYURACTI. specification: 1ml: 1mg, 1ml/ prop up * 10/box.Xinan Pharmaceutical Co., Ltd. produces, lot number 030401.

The heparin sodium powder: the 1g/ bottle, tire 〉=150 μ/mg moisture content＜4%.Shanghai uncle bio tech ltd difficult to understand, lot number 03070.

RPMI-1640 (GIBCO company)

Hyclone (HyClone company)

Calf serum (HyClone company);

L DOPA (U.S. Slpa company product, lot number D9628);

The magnificent biotech firm in trypsin Shanghai product, lot number gets 2186);

MTT, SDS, ConA, agar are Sigma company.

Other has glutamine, penicillin, gentamycin, Tris Base and other homemade analytical reagent.

Experimental example 2 pharmaceutical compositions of the present invention are to B16 melanocyte strain cell proliferation, cruel propylhomoserin prunus mume (sieb.) sieb.et zucc. activity and the synthetic influence of melanin

1, culture fluid preparation

(1) RPMI-1640 culture fluid (1000ml): RPMI-1640 dry powder is dissolved in the 300ml tri-distilled water, washes packaging bag with water 2 times, pour merging into, stir, add tri-distilled water to nearly 1000ml place: add NaHCO ₃2g, replenishing glutamine to final concentration is 3mmol/L, adds phenol red (final concentration is 5.3mg/L); Add antibiotic a penicillin (100U/ml) b gentamycin (50U/ml); Be settled to 1000ml; Regulate PH to 7.2 with NaOH or HCL.Filtration sterilization, 4 ℃ of preservations.The RPMI-1640 culture fluid 2ml that absorption prepares goes in the glass Tissue Culture Flask of 25ml, at 37 ℃, 5%CO ₂With overnight incubation under the complete damp condition, inverted microscope is observed down not microbiological contamination.The culture fluid of microbiological contamination can not experimentize.

The RMPI-1640 culture fluid (100ml) of (2) 10% calf serums: get the RPMI-1640 culture fluid 90ml for preparing, add the 10ml calf serum, regulate PH to 7.2 with NaOH or HCI.Filtration sterilization, 4 ℃ of preservations.The RPMI-1640 culture fluid 2ml that contains 10% calf serum that absorption prepares goes in the glass Tissue Culture Flask of 25ml, at 37 ℃, 5%CO ₂With overnight incubation under the complete damp condition, inverted microscope is observed down not microbiological contamination.The culture fluid of microbiological contamination can not experimentize.

The RMPI-1640 culture fluid (100ml) of (3) 10% hyclones: get the RPMI-1640 culture fluid 80ml for preparing, add 10ml hyclone and 10mlHeper ' s (dense crossing is 5mM eventually); Adding 2 mercapto ethanol (2-Me) to final concentration is that 50 μ mol/L regulate PH to 7.2 with NaOH or HCI.Filtration sterilization, 4 ℃ of preservations.The RPMI-1640 culture fluid 2ml that contains 10% hyclone that absorption prepares goes in the glass Tissue Culture Flask of 25ml, at 37 ℃, 5%CO ₂With overnight incubation under the complete damp condition, observe whether microbiological contamination under the inverted microscope.The culture fluid of microbiological contamination can not experimentize.

2, Digestive system preparation

(1) mother solution preparation: with the trypsin solution of no calcium magnesium Hanks liquid preparation 0.25%.Filtration sterilization, 4 ℃ of preservations.With the EDTA of PBS preparation 1%, 10 pounds of autoclavings 15 minutes, 4 ℃ of preservations.

(2) use the liquid preparation: get the EDTA of 0.4ml 1%, 4ml 0.25 trypsin adds aseptic PBS to 2ml, 4 ℃ of preservations.

3, inorganic salt buffer preparation

(1) PBS solution (1000ml): 8gNaCI (137mmol/L), 0.2gKCI (2.7mmol/L), 1.15gNa ₂HPO ₄7H ₂O (4.3mmol/L), 0.2gKH ₂PO ₄(1.4mmol/L) be dissolved in the 1L distilled water, regulate PH to 7.2.Autoclaving, 4 ℃ of preservations are standby.

(2) 0.83%Tris-NH ₄Cl solution (500ml): 3.0275gTris (0.05mol/L) is dissolved in the 250ml distilled water.Two kinds of solution mix, and add in the distilled water; 4.15gnNH ₄Cl (0.83%) is dissolved in the 250ml distilled water.Two kinds of solution mix, and add to be settled to 500ml in the distilled water.Autoclaving, 4 ℃ of preservations are standby.

4, to the observation of B16 cell inhibitory effect effect:

The take the logarithm B16 cell of trophophase, 0.25% trypsinization, RMPI-1640 culture fluid are collected digestion back cell, the centrifugal 3-5min of 4000r/m.Discard the supernatant.RMPI-1640 culture fluid with 10% calf serum dispels cell, is 5 * 104/ml with its dilution.Get 0.1ml cell suspension and add 96 porocyte culture plates, be placed on 37 ℃, 5%CO ₂Cultivate in the CO2 gas incubator.

Pharmaceutical composition of the present invention is configured to the mother solution of 200mg/ml with normal saline, 121 ℃ of sterilization 20min.With the pharmaceutical composition mother solution of the present invention of 200mg/ml with final concentration: 0,25,50,75,100,150,200,250,300,350,400,450,500,750,1000,1500,2000,2500,3000,3500,4000,4500,5000 μ g/ml (totally 23 gradients) add in the cell culture system of adherent growth 4h, every hole final volume is 200 μ g, each concentration is established 4 multiple holes, averages.At 37 ℃, 5%CO ₂Cultivated 72 hours in the CO2 gas incubator.Finish preceding 5 hours every holes in cell culture and discard culture supernatant 100 μ l, add the dense MTT10 μ l (with normal saline preparation, filtration sterilization) that crosses to 5mg/ml.After cultivate finishing, every hole adds 10%SDS (with the HCI configuration of 0.01M) the 100 μ l mixing that vibrates, 37 ℃ following 4 hours, read the 0D value with microplate reader at the 570nm place.Make the B16 cell be subjected to pharmaceutical composition of the present invention suppress with drug regimen substrate concentration of the present invention between curve, dense the crossing of effective inhibition of judgement pharmaceutical composition of the present invention.

5, pharmaceutical composition of the present invention is to the influence of tyrosinase activity

The take the logarithm B16 cell of trophophase, 0.25% trypsinization, RMPI-1640 culture fluid are collected digestion back cell, the centrifugal 3-5min of 4000r/m.Discard the supernatant.RMPI-1640 culture fluid with 10% calf serum dispels cell, is 8 * 104/ml with its dilution.Get 0.1ml cell suspension and add 96 porocyte culture plates.Be placed on 37 ℃, 5%CO ₂Cultivate in the saturation vapour CO2 gas incubator.

With the pharmaceutical composition of the present invention of 200ml/ml and PAIDU YANGYAN JIAONANG mother solution respectively with final concentration: 0,25,50,75,100,150,200,250,300,350,400,450 μ g/ml (totally 12 gradients) add in the cell culture system of adherent growth 4h, every hole final volume is 200 μ g, and each concentration is established 4 multiple holes.At 37 ℃, 5%CO ₂Cultivated 48 hours in the CO2 gas incubator.Discard the supernatant, the PBS washed cell of usefulness PH7.4 2 times, every hole adds 50 μ l, 1%TritonX-100.96 porocyte culture plates are put into-30 ℃ of refrigerators rapidly, take out behind the 30min, destroy cell thereby thaw naturally under the room temperature.37 ℃ of pre-temperature backs add 1%L-DOPA solution (10 μ l/ hole), and 37 ℃ of reaction 2h measure 490nm trap value, to cultivate the negative control wells of its liquid.

6, pharmaceutical composition of the present invention is to the influence of melanin generation

The take the logarithm B16 cell of trophophase, 0.25% trypsinization, RMPI-1640 culture fluid are collected digestion back cell, the centrifugal 3-5min of 4000r/m.Discard the supernatant.RMPI-1640 culture fluid with 10% calf serum dispels cell, is 1 * 105/ml with its dilution.Get 1ml cell suspension and add 24 porocyte culture plates.At 37 ℃, 5%CO ₂Cultivate in the CO2 gas incubator.

Pharmaceutical composition of the present invention and the PAIDU YANGYAN JIAONANG mother solution of 200mg/ml are diluted to 2000 μ g/ml, 200 μ g/ml working solutions with the RMPI-1640 culture fluid respectively, with final concentration 0-300 μ g/ml (totally 5 gradients, be respectively: 0,50,100,200,300 μ g/ml) add in 4 hours the cell culture system of adherent growth, every hole final volume is 200 μ g, each concentration is established 4 multiple holes, at 37 ℃, 5%CO ₂Cultivated 96 hours in the CO2 gas incubator.0.25% trypsin digestion and cell, every hole are got 1 * 105 cell centrifugation and are discarded the supernatant, add the NaOH of 1ml 1mol/L, shake 5 minutes.Put microplate reader and survey the A value, wavelength is 490nm.

7, result of the test

(1) inhibiting observation is grown in training to the B16 cell

By Fig. 1 and table 7 as seen, measure the influence of pharmaceutical composition of the present invention, compared the obvious suppression effect with blank B-16 melanoma cell propagation with mtt assay.When extremely hanging down dense crossing, pharmaceutical composition of the present invention can stimulate the propagation of B16 cell; But when its concentration during greater than 50 μ g/ml, increase with concentration, then present inhibitory action, optimal inhibition concentration is between the 400 μ g/ml-500 μ g/ml; When pharmaceutical composition of the present invention is dense when crossing greater than 500 μ g/ml, can not increase and increase along with concentration again the effect of B-16 melanoma cell inhibition of proliferation, it suppresses effect and reaches maximum.

The influence of table 7 pharmaceutical composition vitro inhibition of the present invention B-16 propagation (n-4, x ± S)

(2) pharmaceutical composition of the present invention is to the influence of tyrosinase activity

Shown in table 8 and accompanying drawing 2-accompanying drawing 3, mice B-16 melanoma cell is through pharmaceutical composition effect of the present invention after 48 hours, low concentration pharmaceutical composition of the present invention (＜100 μ g/ml) has certain promotion to tyrosinase activity, but increase with concentration, then tyrosinase activity is had the obvious suppression effect.Drug regimen substrate concentration of the present invention can effectively suppress the tyrosinase activity of B-16 melanoma cell strain, and this effect is better than the effect with the concentration PAIDU YANGYAN JIAONANG in the scope of 150-450 μ g/ml.

Table 8 pharmaceutical composition of the present invention is to tyrosinase activity influence (n-4, x ± s)

(3) pharmaceutical composition of the present invention is to the influence of melanin generation

By table 9 and accompanying drawing 3 as seen, pharmaceutical composition of the present invention to mice B-16 melanoma cell effect 4d after, measure melanin content, below 50 μ g/ml, increase with drug level, melanin content constantly increases; During greater than 50 μ g/ml concentration, increase with dense crossing, pharmaceutical composition of the present invention can obviously suppress melanic generation, and when drug regimen substrate concentration of the present invention was 100 μ g/ml, its inhibition that melanin is generated reached maximum; Between 100-300 μ g/ml, melanic inhibition is tended to be steady explanation when the higher concentration, pharmaceutical composition of the present invention has an obvious inhibitory action to melanin is synthetic

The influence that table 9 pharmaceutical composition of the present invention generates melanin (n-4, x ± s)

The influence that experimental example 3 pharmaceutical compositions of the present invention form to the mouse skin chloasma hormone disturbance

1, formative method:

Every day is to mouse peritoneal injection (ip) diethylstilbestrol tablet and medroxyprogesterone acetate sheet mixed solution 0.1ml/10g (diethylstilbestrol concentration 4mg/dl, medroxyprogesterone acetate concentration 20mg/dl), that is: diethylstilbestrol 0.4mg/kg+ medroxyprogesterone acetate 2mg/kg).Every day 1 time, continuous 30 days.

Grouping and administration: 78 of Kunming kind female mices, body weight 25-30g is divided into 7 groups at random by body weight, and every group of 11-12 is only.If normal control (negative control), model contrast, are subjected to 3 dosage groups of reagent thing and 2 of positive drug matched groups (LIUWEI DIHUANG WAN and vitamin C).Except that the normal control group (distilled water of ip respective volume), all the other animals are injected (ip) synestrin tablets and the moulding of medroxyprogesterone acetate sheet mixed solution to mouse peritoneal equal every day.Beginning on the 1st in moulding, be subjected to 3 dosage groups of reagent thing (2.5,5,10g crude drug in whole/kg, liquor strength is respectively 25,50,100g crude drug in whole/dl, be equivalent to 6,12,24 times of clinical every day of recommended dose respectively), 2 (LIUWEI DIHUANG WAN 3g/kg of positive controls, liquor strength 30g/dl) and vitamin C (0.9g/kg, liquor strength is 9g/dl), it is 0.1ml/10g that the beginning gastric infusion is irritated the stomach volume.Normal control group and model control group gavage the distilled water of respective volume.Irritate stomach every day 1 time, successive administration 30 days.

Observation index: mice is put to death in the dislocation of 1hr cervical vertebra after the last administration, each is organized 5 mices of picked at random and strips skin of back in one, 10% formalin solution is fixed, the routine paraffin wax embedded section, the SP of immunohistochemistry technology method (the strepto-avidin staining kit of peroxidase labelling) labelling dermal melanin.5 different visuals field of every pathology section examination, after finding positive target (presenting the mark colour response in skin surface and the adnexa epithelial cell endochylema), with the full-automatic pathology of the non-clinical safety evaluation of the XYTX-2000 medicine of Traditional Chinese Medicine Research Institute, Sichuan Province Sichuan University joint research and development image quantified system analysis target image is carried out quantitative analysis, draw the average area, target of 5 visuals field of every section and every group of positive target of 5 boneblack pigments and the ratio (surface density) of statistics area.With the t method of inspection numerical value between each group is carried out statistical procedures, inspection level α-0.05.

2, result

By table 10 as seen, pharmaceutical composition treated animal serum MDA of the present invention produces and reduces, and shows chloasma model mice serum lipid peroxide is formed with certain inhibitory action.By table 11 and accompanying drawing 4 as seen, in the pharmaceutical composition of the present invention institute amount of reagent scope in the experiment of estrogen and progestogen induced mice chloasma, average area, the target of the positive target of skin immunization histochemical stain melanin all are lower than model group with the ratio (surface density) of statistics area, statistical discrepancy highly significant (p＜0.05-0.01), point out pharmaceutical composition of the present invention inhibited to the formation of mice chloasma laboratory animal dermal melanin.

Table 10 pharmaceutical composition of the present invention is to the influence of mice chloasma laboratory animal SOD in serum, MDA (x ± s)

Compare (t check or t ' check) * P＞0.05 with model control group; * P＜0.05; * * P＜0.01

The influence that table 11 pharmaceutical composition of the present invention forms mice chloasma laboratory animal dermal melanin (x ± s)

Experimental example 4 pharmaceutical compositions of the present invention cause the influence of mouse skin damage to ultraviolet radiation

1, method

80 of Kunming kind female mices, body weight 18-22g is divided into 7 groups at random by body weight, and every group of 11-12 is only.If normal control (negative control), model control group are subjected to 3 dosage groups of reagent thing (2.5,5,10g crude drug in whole/kg, concentration is respectively 25,50,100g crude drug in whole/dl, is equivalent to 6,12,24 times of clinical every day of recommended dose respectively).2 positive controls (PAIDU YANGYAN JIAONANG 0.8g/kg, vitamin C 0.9g/kg).Medicine group gastric infusion every day 1 time, continuous 5 days.Irritating the stomach volume is 0.1mg/10g.Normal control group and model control group gavage the distilled water of respective volume.

Gastric infusion the 2nd day is with 8% Na ₂S loses hair or feathers to mouse back, and the 3rd day gastric infusion of administration be after 40 minutes, mice is put under the uviol lamp (30W, with back part of animal apart from 25cm) irradiation 2 hours.Respectively at irradiation back 2hr, 24hr, 48hr animal is taken out perusal irradiated site erythema, edema situation (the perusal standards of grading see Table 12).Testing after the last administration in the 5th day the 1hr eyeball excise gets blood system and detects SOD, MDA from serum.Mice is put to death in the cervical vertebra dislocation, gets irradiated site skin in the broad liquid of 10% formaldehyde, and HE dyeing is done tissue pathological slice and observed (the histopathology standards of grading see Table 13).Sxemiquantitative data rank tests such as erythema, edema situation and histopathology scoring as a result, SOD, MDA relatively have there was no significant difference, inspection level α=0.05 between each group with the t check.

Table 12 skin ultraviolet injury macroscopic score table

Table 13 skin ultraviolet injury histopathology grade form

2, result of the test

By table 14 as seen, compare with model control group, pharmaceutical composition group SOD activity of the present invention raises to some extent, MDA has to a certain degree, and generation reduces, but statistical discrepancy is remarkable (p＞0.05) not, shows that pharmaceutical composition of the present invention is formed with certain inhibitory action trend to Mutations in Skin of Different Ultraviolet damage model mice serum lipid peroxide.

By table 15 as seen, perusal, pharmaceutical composition of the present invention can alleviate erythema, the edema that ultraviolet causes mouse skin, and skin ultraviolet injury macroscopic score value significantly is lower than model control group (p＜0.05).

By table 16 as seen, the ultra-vioket radiation line can cause the mouse skin histopathology and take place obviously to change, and mainly shows as degeneration, the necrosis of skin surface epithelium, and cell infiltration can be accompanied obvious congestion and edema.Pharmaceutical composition of the present invention is to the effect that all has clear improvement of above-mentioned pathological changes, and mouse skin damaged tissue histological scores value significantly is lower than matched group (p＜0.05-0.01).

Table 14 pharmaceutical composition of the present invention causes the influence (x ± s) of mouse skin damage SOD in serum, MDA to ultraviolet

Table 15 pharmaceutical composition of the present invention causes mouse skin damage macroscopic score value (x ± s) to ultraviolet

Table 16 pharmaceutical composition of the present invention causes mouse skin damaged tissue histological scores value (x ± s) to ultraviolet

Compare (t check or t ' check) * P＞0.05 with model control group; Water * P＜0.05; * * P＜0.01

Experimental example 5 pharmaceutical compositions of the present invention are to propionibacterium acnes, the experiment of micrococcal inhibitory action

1, medicine preparation

Pharmaceutical composition of the present invention: accurately take by weighing former medicated powder 2.00g with electronic balance with weighting procedure, add sterile distilled water 29.3ml dissolving, concentration is 64mg/ml, and 100 ℃ are boiled and killed assorted bacterium in 5 minutes.Get 0.5ml during experiment with aseptic meat soup (propionibacterium acne the is used anaerobism meat soup) dilution of opposing doubly, make that concentration series is 32,16,8,4,2,1,0.5,0.25mg/ml.

Benzylpenicillin: Harbin Pharmaceutical General Factory produces, lot number B0202004, every bottle 800,000 unit, add the 8.0ml dissolved in distilled water, concentration is 100,000 units/ml, get 0.3ml and add the 27ml distilled water diluting, concentration is 10,000 units/ml, gets 0.3ml and adds the 2.7ml distilled water diluting, and concentration is 1000 units/ml, get 0.3ml and add the 2.7ml distilled water diluting, concentration is 100 units/ml, gets 1.0ml and adds the dilution of 6.8ml meat soup, and concentration is 12.8 units/ml, get 0.5ml then with the dilution of opposing doubly of aseptic meat soup, concentration series is 6.4,3.2,1.6,0.8,0.4,0.2,0.1,0.05,0.025 unit/ml.Survey periodic positive drug contrast as propionibacterium acne and micrococcus luteus antibacterial activity.

Polymyxin B: Amresco company product, Huamei Bio-Engrg Co.,'s packing, activity＞6000u/mg (in 6000u/mg), accurately take by weighing polymyxin B 50mg, add the 5.0ml dissolved in distilled water, concentration is 60000u/ml, get 0.3ml and add the 2.7ml distilled water diluting, concentration is 6000u/ml, gets 0.3ml and adds the 2.7ml distilled water diluting, and concentration is 600u/ml, get 0.3ml and add the dilution of 2.5ml meat soup, concentration is 64u/ml, gets 0.5ml then with aseptic meat soup two-fold dilution, and concentration series is 32,16,8,4,2,1,0.5,0.25,0.125u/ml

2, the preparation of test organisms liquid

Test organisms is inoculated dried plain agar flat board or anaerobism flat board by nutritional need, cultivate 18～24 hours (corynebacterium parvum was cultivated 48 hours) for 37 ℃, scrape the lawn that takes a morsel and be emulsifiable in meat soup (propionibacterium acne is used anaerobism meat soup), than its concentration of turbid correction, do 100,000 times of dilutions (propionibacterium acne is cooked 10,000 times of dilutions) again, make final concentration be equivalent to 104～105cfu/ml meat soup.

3, test method

MIC measures: adopt test tube liquid doubling dilution, the every pipe dosage of test organisms liquid 0.5ml is put 37 ℃ of cultivations behind the mixing, observed result next day (propionibacterium acne is cultivated observed result after 48 hours).After adding bacterium, investigational agent and positive control drug all are equivalent to 1 times of redilution.Compare with bacterium liquid meat soup, the least concentration of bacteria growing inhibiting is MIC.

MBC measures: adopt dull and stereotyped viable bacteria counting method.Promptly measure MIC earlier, successively the end is seen that again bacterial growth respectively manages culture and draw 0.1ml respectively and be inoculated in flat board by nutritional need, cultivated 18～24 hours for 37 ℃ again, the lowest concentration of drug of clump count on the flat board＜5 is minimal bactericidal concentration.

Control experiment: do contrast of bacterium liquid meat soup and investigational agent blank simultaneously.

4, experimental result

Table 17 pharmaceutical composition of the present invention is to propionibacterium acnes, micrococcal inhibitory action

By table 17 as seen, pharmaceutical composition of the present invention all has propionibacterium acne, micrococcus luteus inhibition and killing action is obviously all arranged.

Experimental example 6 pharmaceutical compositions of the present invention are to epinephrine and the hemorheological influence experiment of frozen water hyperamization stasis of blood rat model

1, method

Get 64 of SD female rats, body weight 240～300g is divided into 6 groups by body weight with famine, and every group of 10-11 only.If normal control (negative control), model control group, be subjected to 3 dosage groups of reagent thing (2,4,8g crude drug in whole/kg, be equivalent to 4.8,9.6,19.2 times of clinical every day of recommended dose respectively), positive drug Radix Salviae Miltiorrhizae drop pill matched group (dosage is 0.375g/kg).Adopt not isoconcentration medicinal liquid gastric infusion (matched group is given distilled water) of isometric(al), three dose concentrations of pharmaceutical composition of the present invention are respectively for concentration is respectively 20,40,80g (crude drug in whole)/dl, Radix Salviae Miltiorrhizae drop pill concentration is 3.75g/dl, and volume is the 1ml/100g body weight.Irritate stomach every day 1 time, continuous 10 days.Normal control group and model control group gavage the distilled water of equivalent.

Except that the normal control group, 1hr model control group and respectively be subjected to reagent thing group subcutaneous injection epinephrine (0.1g/dl) 0.08ml/100g totally 2 times (asking) after the last administration every 4h, 2hr is soaked in the frozen water 5 minutes with rat after injecting epinephrine for the first time.Dispose the back fasting and can't help water, femoral artery is got blood (heparin sodium anticoagulant) behind the 20hr, and blood is flowed in the 20ml beaker that is added with a small amount of heparin sodium naturally, and jolting makes it anticoagulant gently.Use LG-R-80 type blood viscosity instrument mensuration whole blood viscosity (shear rate 200, shear rate: 100, shear rate 20, shear rate 10) and plasma viscosity, result to compare each group difference significance, inspection level a=0.05 with the t check.

2, result:

By table 18 as seen, pharmaceutical composition whole blood 200 of the present invention (1/s) shear rate, plasma viscosity are lower than model control group (P＜0.05～0.01), show that pharmaceutical composition of the present invention causes people Mus blood stasis model hemorheological property to epinephrine and frozen water certain improvement effect is arranged.

Table 18 pharmaceutical composition of the present invention is to the influence of rat blood viscosity number (X ± S)

Compare (t check or t ' check) with model control group: * P＞0.05; * P＜0.05; * * P＜0.01

Experimental example 7 pharmaceutical compositions of the present invention are to the influence of mice serum and the formation of hepatic tissue lipid peroxide

1, method:

Get 63 of Kunming mouses, male, body weight 18～22g is divided into 6 groups at random by body weight, and every group of 10-11 only.If negative control (solvent control), model control group, be subjected to 3 dosage groups of reagent thing (2.5,5,10g crude drug in whole/kg, concentration is respectively 25,50,100g crude drug in whole/dl, be equivalent to 5,10,20 times of clinical every day of recommended dose respectively), positive drug vitamin C matched group (concentration 9g/dl), dosage is 0.9g/kg).

Adopt the isometric(al) gastric infusion, volume is 0.1ml/10g.Every day 1 time, continuous 10 days, normal control group and model control group were given the distilled water of respective volume.

Administration the 6th day, mice fasting are after 16 hours, and administration group and model control group are pressed 75mg/kg dosage tail vein injection shot alloxan saline solution, the normal saline of normal control group tail vein injection respective volume.Eyeball of mouse is extractd and is got blood behind the last administration 1hr, and the centrifugal serum of leaving and taking detects SOD in serum, MDA.Mice is put to death in the cervical vertebra dislocation, gets liver 0.5g, the homogenate of 4.5ml cold saline, and the centrifuging and taking supernatant detects hepatic tissue SOD, MDA.There was no significant difference is arranged, inspection level α=0.05 with SOD, MDA between t check comparable group.

2, result

By table 19 as seen, in the pharmaceutical composition of the present invention institute amount of reagent scope alloxan is caused in the experiment of mice lipid peroxide, can significantly reduce the formation of serum and hepatic tissue lipid peroxide.

Table 19 pharmaceutical composition of the present invention causes the influence (X ± S) of mice lipid peroxide to alloxan

Experimental example 8 pharmaceutical composition toxicity tests of the present invention

1, is subjected to the reagent thing

Pharmaceutical composition of the present invention: the embodiment of the invention 1 described medicament composition capsule content, pale brown toner end, 1g medicated powder is equivalent to crude drug in whole 4.26g.Become body weight for humans 60kg to calculate by the women, pharmaceutical composition recommended oral dose every day of the present invention is equivalent to 0.42g (crude drug in whole)/kg body weight.Lot number 20030604 is provided by medicine chamber, Traditional Chinese Medicine Research Institute, Sichuan Province Yi Jinhai researcher.

Get its content 28.17g during experiment, grind the back adding distil water and be made into 60ml suspendible medicinal liquid, promptly get its Cmax 200% suspendible medicinal liquid (100ml suspendible medicinal liquid is equivalent to crude drug in whole 200g crude drug in whole approximately, and its viscosity is to exceed by No. 16 stomach pins) and be for experiment.

2, laboratory animal

The Kunming kind is female, 20 of mices, and one-level is qualified, and (quality certification number: the real kinoplaszm in river 2002-33 number), body weight 18-22g is provided by the Traditional Chinese Medicine Research Institute, Sichuan Province Experimental Animal Center.

3, experimental technique

Mice fasting 12hr, can't help water, (100ml suspendible medicinal liquid is equivalent to crude drug in whole 200g crude drug in whole to capsule 's content Cmax 200% suspendible medicinal liquid approximately that can accept with animal, its viscosity is to exceed by No. 16 stomach pins) and the dosage of maximum volume (0.4ml/10g B.W.) 1 day in 2 times (6hr at interval) irritate stomach animals, observed 7 days continuously, the reaction of record animal toxicity, repetition measurement body weight behind the 7d, not produce dead maximal dose is the maximum dosage-feeding of an oral administration, and extrapolates the multiple that this dosage is equivalent to clinical every day of recommended drug amount.

4, result of the test

Irritate stomach mices (6hr at interval) 2 times in pharmaceutical composition Cmax 200% suspendible medicinal liquid of the present invention (100ml suspendible medicinal liquid is equivalent to crude drug in whole 200g crude drug in whole approximately, and its viscosity is to exceed by No. 16 stomach pins) and the maximum volume (0.4ml/10g B.W.) 1 day.After each administration after several minutes animal spontaneous activity occurs and obviously reduce, individual animal is performances (table 20) such as cold and detached, the blepharoptosis of reaction, loose stool to external world as seen, above-mentioned symptom alleviates gradually and disappears behind about 3-6hr.Observed 7 days continuously later on, the outward appearance of animal, behavioral activity, the mental status, defecation and color thereof, quilt hair, the colour of skin, breathing, nose, eye, oral secretion there is no unusually, and the mice body weight is normal growth state (table 21) before and after the test.Eye anatomy is not seen obvious pathological change during off-test.So it is 160g (crude drug in whole)/kg that pharmaceutical composition of the present invention a day is irritated the maximum dosage-feeding of stomach mice, this dosage is equivalent to 381 times of clinical every day of recommended amounts approximately.

5, conclusion (of pressure testing)

Pharmaceutical composition of the present invention is given in the female mice 1 day and to be irritated stomach maximum dosage-feeding 160g (crude drug in whole)/kg 2 times, and this dosage is given 381 times that are equivalent to clinical every day of recommended amounts.

The interior as seen animal spontaneous activity of 3-6hr obviously reduces after the administration, and performances such as seen individual animal reacts indifferently to external world, blepharoptosis, loose stool are due to supposition and filling stomach volume are excessive.Observed 7 days continuously later on, the outward appearance of animal, behavioral activity, the mental status, defecation and color thereof, quilt hair, the colour of skin, breathing, nose, eye, oral secretion etc. there is no unusual, and eye anatomy is not seen obvious pathological change during off-test.The toxicity performance judges that pharmaceutical composition of the present invention belongs to no overt toxicity medicine behind acute toxicity institute's amount of reagent of pharmaceutical composition of the present invention and the absolute value medicine.

Table 20 pharmaceutical composition acute toxicity test in mice of the present invention symptom spectrum

Body weight change before and after the table 21 pharmaceutical composition acute toxicity test in mice of the present invention (X ± S)

The experiment of experimental example 9 content of Danshensu assay methods

1, instrument and reagent: high performance liquid chromatograph comprises the Warers1525 pump, 2487 dual pathways UV-detector, Warers column oven, Breeze liquid chromatograph work station, Brasson ultrasonic cleaning instrument.The danshensu sodium reference substance is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, for assay usefulness, lot number 855-200102.Methanol is U.S. Tedia chromatographically pure, and water is ultra-pure water, and other reagent are analytical pure.

2, chromatographic condition: chromatographic column: Phenomenex Luna C ₁₈(2), 4.6mm * 150mm, 5 μ m; Column temperature: 30 ℃; Mobile phase: methanol-0.5% glacial acetic acid (20: 80); Detect wavelength: 180nm; Flow velocity: 0.6ml/min.

3, the preparation of sample solution

The preparation of reference substance solution: precision takes by weighing danshensu sodium reference substance 9.04mg, adds mobile phase and is mixed with the solution that every ml contains 0.0904mg (amounting to danshensu 0.0791mg), promptly.

The preparation of need testing solution: get the medicament composition capsule agent content of the present invention of the embodiment of the invention 1 preparation, porphyrize, precision takes by weighing 0.5g, put in the 10ml triangular flask, precision adds methanol 50ml, supersound process 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 10ml, put in the 50ml round-bottomed flask, reclaim solvent to doing, residue adds the mobile phase dissolving and changes in the 10ml measuring bottle, add mobile phase to scale, shake up, promptly.

The preparation of negative control product solution: press the preparation method of need testing solution, make negative control product solution (lacking Radix Salviae Miltiorrhizae).

4, linear relationship is investigated

Accurate respectively reference substance solution (0.0791mg/ml) 2.0,4.0,6.0,8.0, the 10.0ul of drawing injects chromatograph of liquid, measures peak area.With the sample size is abscissa, and peak area is a vertical coordinate, sets up working curve, the results are shown in Table 22 and accompanying drawing 5.

The standard curve of table 22 danshensu

Calculate regression equation: Y=1026500.6X-26498.7

Show that danshensu has good linear relationship in 0.1582-0.791 μ g scope.

5, the interferential investigation of negative control product

Draw each 10 μ l of reference substance solution 5 μ l, need testing solution and negative control product solution respectively.Inject liquid, chromatography the results are shown in accompanying drawing 6.Conclusion: danshensu reaches baseline separation in the need testing solution, and negative control product solution is noiseless, baseline stability, and method is feasible.

6, stability test

The accurate need testing solution 10 μ l that draw, at regular intervals sample introduction once, totally 6 times, measure the peak area of danshensu, RSD is 0.80%, shows that measurement result is stable in the test sample 8 hours, the results are shown in Table 23 and accompanying drawing 7.

Table 23 stability test

7, precision test

The accurate need testing solution 10 μ l that draw inject chromatograph of liquid, measure the peak area of danshensu, repeat 5 times, the results are shown in Table 24 and accompanying drawing 8.

The experiment of table 24 precision

8, repeatability test

According to the down operation of assay item, to same batch sample (lot number: 01) repeatedly measure, calculate relative standard deviation, the results are shown in Table 25 and accompanying drawing 9.

The test of table 25 repeatability

9, average recovery test

Get the pharmaceutical composition of the present invention (lot number: 01 of known content of Danshensu, 3.78mg/g) about 0.25g, the accurate title, decide, and accurately adds a certain amount of danshensu sodium reference substance solution (amounting to danshensu 0.762mg/ml) and measure content, be calculated as follows the response rate, the results are shown in Table 26.

The test of table 26 average recovery

10, the assay of danshensu in the pharmaceutical composition of the present invention

According to the operation of the content assaying method of quality standard text danshensu, measured the content of danshensu in 6 batches of pharmaceutical compositions of the present invention, the results are shown in Table 27 and accompanying drawing 10.

The assay of table 27 pharmaceutical composition danshensu of the present invention

According to the measurement result of 12 data of above-mentioned 6 batch samples, with average content 2.01 (mg/ grain) 20% lower limit as this product content of floating downward, promptly 2.01 * 80%=1.61 (mg/ grain) contains Radix Salviae Miltiorrhizae with danshensu (C so order every of this product temporarily ₉H ₁₀O ₅) meter, must not be less than 1.6mg.

The experiment of experimental example 10 content detection of paeonol methods

1, instrument and reagent: Waters liquid chromatographic system (comprising 1525 pumps, 2487 dual wavelength UV-detector Waters column ovens, Breeze liquid chromatograph work station); The paeonol reference substance is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number 0708-9704, and assay is used; Methanol is U.S. Tedia chromatographically pure, and water is ultra-pure water, and other reagent are analytical pure.

2, chromatographic condition

Chromatographic column: Phenomenex Luna C ₁₈(2), 4.6mm * 150mm, 5 μ m: column temperature: 35 ℃; Mobile phase: methanol-water-glacial acetic acid (60: 40: 1) detects wavelength: 274nm; Flow velocity: 0.6ml/min.

3, the preparation of sample solution

The preparation of reference substance solution: precision takes by weighing paeonol reference substance 5.93mg, adds the first ferment and makes the solution that concentration is 0.01186mg/ml, promptly.

The preparation of need testing solution: get the medicament composition capsule agent content of the present invention that the embodiment of the invention 1 prepares, porphyrize, precision takes by weighing 0.25g, put in the 100ml triangular flask, precision adds methanol 50ml, supersound process 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 1ml, put in the 10ml measuring bottle, add methanol to scale, shake up, promptly.

The preparation of negative control product solution: press the preparation method of need testing solution, make negative control product solution (lacking Cortex Moutan).

4, linear relationship is investigated

Accurate reference substance solution 2,4,6,8, the 10 μ l that draw inject chromatograph of liquid, are abscissa with the sample size, and peak area is a vertical coordinate, sets up working curve, the results are shown in Table 28 and accompanying drawing 11.

The standard curve of table 28 paeonol

a＝8517255.5.b＝-3447.9r＝0.9998

Calculate regression equation: Y=8517255.5X-3447.9

Show that paeonol has good linear relationship in 0.02372-0.1186 μ g scope

5, the interferential investigation of negative control product

Draw each 10 μ l of reference substance solution, need testing solution and negative control product solution respectively, inject chromatograph of liquid, the results are shown in accompanying drawing 12.Conclusion: paeonol reaches baseline separation in the need testing solution, and negative control product solution is noiseless, baseline stability, and method is feasible.

6, stability test

The accurate need testing solution 10 μ l that draw, at regular intervals sample introduction once, totally 6 times, measure the peak area of paeonol, RSD is 1.64%, shows that measurement result is stable in the need testing solution 24 hours, the results are shown in Table 29 and accompanying drawing 13.

Table 29 stability test

7, precision test

The accurate need testing solution 10 μ l that draw inject chromatograph of liquid, measure the peak area of paeonol, repeat 5 times, the results are shown in Table 30 and accompanying drawing 14.

The test of table 30 precision

8, repeatability test

According to the down operation of assay item, to same batch sample (lot number: 01) repeatedly measure, calculate relative standard deviation, the results are shown in Table 31 and accompanying drawing 15.

The test of table 31 repeatability

9, average recovery test

Get known paeonol content pharmaceutical composition of the present invention (lot number: 01,9.31mg/g) the about 125mg of content, accurate claim fixed, accurately add a certain amount of paeonol reference substance solution (1.22mg/ml), measure content, be calculated as follows the response rate, the results are shown in Table 32.

The test of table 32 average recovery

10, the assay of paeonol in the pharmaceutical composition of the present invention

According to the operation of the content assaying method of quality standard text paeonol, measured the content of paeonol in 6 batches of pharmaceutical compositions of the present invention, the results are shown in Table 33 and accompanying drawing 16.

The assay of table 33 pharmaceutical composition paeonol of the present invention

According to the content assaying method of pharmaceutical composition of the present invention, six batch samples are measured, the results are shown in Table 30.Technical study shows: paeonol is about 65%-75% to the rate of transform of finished product pharmaceutical composition of the present invention in the Cortex Moutan medical material; Paeonol content must not be less than 1.2% in the current edition pharmacopeia regulation Cortex Moutan.The preparation prescription of pharmaceutical composition according to the present invention, calculate with the lower bound of paeonol content in the Cortex Moutan and the lower limit of the rate of transform thereof, the every capsules of this product contains paeonol and should be: 1.2% * 333.3g * 1000=2.6mg of 65% ÷, so tentative every of this product contains Cortex Moutan with paeonol (C ₁₉H ₁₀O ₃) meter, must not be less than 2.6mg.

Following embodiment all can realize the described effect of above-mentioned experimental example.

The specific embodiment

Embodiment 1: medicament composition capsule agent of the present invention

Radix Bupleuri 333.3g Radix Salviae Miltiorrhizae 500g Radix Rehmanniae Preparata 500g

Radix Angelicae Dahuricae 333.3g Cortex Moutan 333.3g Radix Glycyrrhizae 100g.

Get crude drug, Cortex Moutan distills with the water of 5000ml, collects the distillate of 3333ml, in 0～10 ℃ of following cold preservation 8 hours, filter, get paeonol crystallization I, filtrate adds the sodium chloride of Cortex Moutan weight 5%, redistillation, the distillate of collection 1000ml, in 0～10 ℃ of following cold preservation 8 hours, filter, get the paeonol crystal II, merge paeonol crystallization I and II,, promptly get paeonol in 40 ℃ of cold drying, standby, filtrate A device in addition collects medicinal residues and all the other Radix Bupleuri, Radix Salviae Miltiorrhizae, Radix Rehmanniae Preparata, the Radix Angelicae Dahuricae and Radix Glycyrrhizae five tastes crude drug, decoct with water secondary, add for the first time water 10 times of weight, add water 8 times of weight the 2nd time, the each decoction 2 hours, filter, merging filtrate gets liquor B, mixes filtrate A and B, be concentrated into 60～65 ℃ of following relative densities and be 1.20～1.25 thick paste, drying is pulverized, and crosses 60 mesh sieves, get extract powder, standby; Get above-mentioned paeonol porphyrize, with the magnesium stearate mix homogeneously that accounts for extract powder weight 0.5%, add in the above-mentioned extract powder, mixing adds conventional adjuvant, makes 1000 of capsules according to common process, and every capsules 0.5g is oral, one time 4,3 times on the one.

Embodiment 2: pharmaceutical composition tablet of the present invention

Radix Bupleuri 310g Radix Salviae Miltiorrhizae 750g Radix Rehmanniae Preparata 350g

Radix Angelicae Dahuricae 390g Cortex Moutan 310g Radix Glycyrrhizae 130g.

Get crude drug, Cortex Moutan distills with the water of 4650ml, collects the distillate of 3100ml, in 0～10 ℃ of following cold preservation 8 hours, filter, get paeonol crystallization I, filtrate adds the sodium chloride of Cortex Moutan weight 5%, redistillation, the distillate of collection 930ml, in 0～10 ℃ of following cold preservation 8 hours, filter, get the paeonol crystal II, merge paeonol crystallization I and II,, promptly get paeonol in 40 ℃ of cold drying, standby, filtrate A device in addition collects medicinal residues and all the other Radix Bupleuri, Radix Salviae Miltiorrhizae, Radix Rehmanniae Preparata, the Radix Angelicae Dahuricae and Radix Glycyrrhizae five tastes crude drug, decoct with water secondary, add for the first time water 10 times of weight, add water 8 times of weight the 2nd time, the each decoction 2 hours, filter, merging filtrate gets liquor B, mixes filtrate A and B, be concentrated into 60～65 ℃ of following relative densities and be 1.20～1.25 thick paste, drying is pulverized, and crosses 60 mesh sieves, get extract powder, standby; Get above-mentioned paeonol porphyrize, with the magnesium stearate mix homogeneously that accounts for extract powder weight 0.5%, add in the above-mentioned extract powder, mixing adds conventional adjuvant, makes tablet according to common process.

Embodiment 3: medicament composition granule agent of the present invention

Radix Bupleuri 390g Radix Salviae Miltiorrhizae 350g Radix Rehmanniae Preparata 750g

Radix Angelicae Dahuricae 310g Cortex Moutan 390g Radix Glycyrrhizae 70g.

Get crude drug, Cortex Moutan distills with the water of 5850ml, collects the distillate of 3900ml, in 0～10 ℃ of following cold preservation 8 hours, filter, get paeonol crystallization I, filtrate adds the sodium chloride of Cortex Moutan weight 5%, redistillation, the distillate of collection 1170ml, in 0～10 ℃ of following cold preservation 8 hours, filter, get the paeonol crystal II, merge paeonol crystallization I and II,, promptly get paeonol in 40 ℃ of cold drying, standby, filtrate A device in addition collects medicinal residues and all the other Radix Bupleuri, Radix Salviae Miltiorrhizae, Radix Rehmanniae Preparata, the Radix Angelicae Dahuricae and Radix Glycyrrhizae five tastes crude drug, decoct with water secondary, add for the first time water 10 times of weight, add water 8 times of weight the 2nd time, the each decoction 2 hours, filter, merging filtrate gets liquor B, mixes filtrate A and B, be concentrated into 60～65 ℃ of following relative densities and be 1.20～1.25 thick paste, drying is pulverized, and crosses 60 mesh sieves, get extract powder, standby; Get above-mentioned paeonol porphyrize, with the magnesium stearate mix homogeneously that accounts for extract powder weight 0.5%, add in the above-mentioned extract powder, mixing adds conventional adjuvant, makes granule according to common process.

Embodiment 4: medicinal composition powders of the present invention

Radix Angelicae Dahuricae 333.3g Cortex Moutan 333.3g Radix Glycyrrhizae 100g.

Get crude drug, add conventional adjuvant, make powder according to common process.

Embodiment 5: pharmaceutical composition pill of the present invention

Radix Angelicae Dahuricae 390g Cortex Moutan 310g Radix Glycyrrhizae 130g.

Get crude drug, Cortex Moutan distills with the water of 5580ml, collects the distillate of 1860ml, in 2 ℃ of following cold preservations 10 hours, filter, get paeonol crystallization I, filtrate adds the sodium chloride of Cortex Moutan weight 7%, redistillation, the distillate of collection 620ml, in 2 ℃ of following cold preservations 10 hours, filter, get the paeonol crystal II, merge paeonol crystallization I and II,, promptly get paeonol in 45 ℃ of cold drying, standby, filtrate A device in addition collects medicinal residues and all the other Radix Bupleuri, Radix Salviae Miltiorrhizae, Radix Rehmanniae Preparata, the Radix Angelicae Dahuricae and Radix Glycyrrhizae five tastes crude drug, decoct with water 1 time, add water 6 times of weight, decocted 3 hours, filter, merging filtrate gets liquor B, mix filtrate A and B, be concentrated into 65 ℃ of following relative densities and be 1.10～1.20 thick paste, drying, pulverize, cross 60 mesh sieves, get extract powder, standby; Get above-mentioned paeonol porphyrize, with the magnesium stearate mix homogeneously that accounts for extract powder weight 1%, add in the above-mentioned extract powder, mixing adds conventional adjuvant, makes pill according to common process.

Embodiment 6: drug composition oral liquid of the present invention

Radix Angelicae Dahuricae 310g Cortex Moutan 390g Radix Glycyrrhizae 70g.

Get crude drug, Cortex Moutan distills with the water of 4680ml, collects the distillate of 4680ml, in 8 ℃ of following cold preservations 8 hours, filter, get paeonol crystallization I, filtrate adds the sodium chloride of Cortex Moutan weight 4%, redistillation, the distillate of collection 1560ml, in 8 ℃ of following cold preservations 10 hours, filter, get the paeonol crystal II, merge paeonol crystallization I and II,, promptly get paeonol in 35 ℃ of cold drying, standby, filtrate A device in addition collects medicinal residues and all the other Radix Bupleuri, Radix Salviae Miltiorrhizae, Radix Rehmanniae Preparata, the Radix Angelicae Dahuricae and Radix Glycyrrhizae five tastes crude drug, decoct with water 3 times, add water 10 times of weight at every turn, decocted 1 hour at every turn, filter, merging filtrate gets liquor B, mix filtrate A and B, be concentrated into 55 ℃ of following relative densities and be 1.25～1.35 thick paste, drying, pulverize, cross 60 mesh sieves, get extract powder, standby; Get above-mentioned paeonol porphyrize, with the magnesium stearate mix homogeneously that accounts for extract powder weight 1.5%, add in the above-mentioned extract powder, mixing, add conventional adjuvant, make clinical pharmaceutical dosage form: granule, pill, powder, capsule, tablet, slow releasing agent, oral liquid or lyophilized injectable powder according to common process.

Embodiment 7: pharmaceutical composition slow releasing preparation of the present invention

Radix Angelicae Dahuricae 333.3g Cortex Moutan 333.3g Radix Glycyrrhizae 100g.

Get crude drug, add conventional adjuvant, make slow releasing preparation according to common process.

Embodiment 8: medicinal composition freezing-dried powder injection of the present invention

Radix Angelicae Dahuricae 333.3g Cortex Moutan 333.3g Radix Glycyrrhizae 100g.

Get crude drug, Cortex Moutan distills with the water of 5000ml, collects the distillate of 3333ml, in 0～10 ℃ of following cold preservation 8 hours, filter, get paeonol crystallization I, filtrate adds the sodium chloride of Cortex Moutan weight 5%, redistillation, the distillate of collection 1000ml, in 0～10 ℃ of following cold preservation 8 hours, filter, get the paeonol crystal II, merge paeonol crystallization I and II,, promptly get paeonol in 40 ℃ of cold drying, standby, filtrate A device in addition collects medicinal residues and all the other Radix Bupleuri, Radix Salviae Miltiorrhizae, Radix Rehmanniae Preparata, the Radix Angelicae Dahuricae and Radix Glycyrrhizae five tastes crude drug, decoct with water secondary, add for the first time water 10 times of weight, add water 8 times of weight the 2nd time, the each decoction 2 hours, filter, merging filtrate gets liquor B, mixes filtrate A and B, be concentrated into 60～65 ℃ of following relative densities and be 1.20～1.25 thick paste, drying is pulverized, and crosses 60 mesh sieves, get extract powder, standby; Get above-mentioned paeonol porphyrize, with the magnesium stearate mix homogeneously that accounts for extract powder weight 0.5%, add in the above-mentioned extract powder, mixing adds conventional adjuvant, makes lyophilized injectable powder according to common process.

Embodiment 9: the quality determining method of the medicament composition capsule agent of the present invention of embodiment 1 preparation

Differentiate:

A. get medicament composition capsule agent content 0.5g of the present invention, add water 25ml and make dissolving, filter, filtrate is put in the separatory funnel, extracts 3 times with water saturated n-butyl alcohol jolting, uses 15ml at every turn, merge n-butanol extracting liquid, add isopyknic ammonia solution, shake up, place and make layering, divide and get n-butyl alcohol liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution;

Other gets Radix Bupleuri control medicinal material 1g, adds methanol 20ml, and reflux 30 minutes filters, filtrate evaporate to dryness, residue add water 20ml, and reflux 30 minutes filters, filtrate evaporate to dryness, residue add water 20ml makes dissolving, and reflux 1 hour is put in the separatory funnel, extract 3 times with water saturated n-butyl alcohol jolting, use 15ml at every turn, merge n-butanol extracting liquid, add isopyknic ammonia solution, shake up, place and make layering, divide and get n-butyl alcohol liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, preparation control medicinal material solution;

Test according to thin layer chromatography (appendix VI B), draw above-mentioned two kinds of each 0.005ml of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, so that chloroform-methanol-water=the lower floor's solution in placement below 10 ℃ was developing solvent in 13: 7: 2, launch, take out, dry, spray is with 1% paradime thylaminobenzaldehyde ethanol solution of sulfuric acid, get 0.5-1.5% paradime thylaminobenzaldehyde sulfuric acid solution 1mL, to 10mL, get this solution spray silica gel g thin-layer plate with ethanol dilution, it is clear to be heated to the speckle colour developing at 70 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of same color; Put under the uviol lamp 365nm and inspect, show identical xanchromatic fluorescence speckle;

B. get medicament composition capsule agent content 0.5g of the present invention, add water 30ml and make dissolving, filter, filtrate transfers pH value to 1-2 with dilute hydrochloric acid, puts in the separatory funnel, extract 3 times with the ethyl acetate jolting, each 20ml merges ethyl acetate liquid, and the reclaim under reduced pressure ethyl acetate is to doing, residue adds methanol 2ml makes dissolving, as need testing solution;

Other gets Radix Salviae Miltiorrhizae control medicinal material 0.5g, adds methanol 20ml, and supersound process 30 minutes filters, and filtrate is concentrated into 2ml, in contrast medical material solution;

Test according to thin layer chromatography (appendix VI B), draw above-mentioned two kinds of each 0.005ml of solution, put respectively on the silica gel g thin-layer plate of same usefulness 1% sodium hydroxide solution preparation, with ethyl acetate-formic acid-glacial acetic acid-water=15: 1: 1: 2 was developing solvent, launch, take out, dry, put under the uviol lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical fluorescence speckle;

C. get medicament composition capsule agent content 1g of the present invention, add ethanol 20ml, close plug, jolting 10 minutes filters, and filtrate is concentrated into 1ml, as need testing solution;

Other gets the paeonol reference substance, adds acetone and makes the solution that every 1ml contains 0.005g, in contrast product solution;

According to thin layer chromatography (appendix VIB test), draw above-mentioned two kinds of each 0.010ml of solution, put respectively on same silica gel g thin-layer plate, with cyclohexane extraction-ethyl acetate=3: 1 was developing solvent, launched, and took out, dry, spray is with the acid 5% ferric chloride alcoholic solution of hydrochloric acid, and it is clear to be heated to the speckle colour developing; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical blue brown speckle;

D. get medicament composition capsule agent content 0.5g of the present invention, add water 25ml and make dissolving, filter, filtrate is put in the separatory funnel, extracts 3 times with water saturated n-butyl alcohol jolting, uses 15ml at every turn, merge n-butanol extracting liquid, add isopyknic ammonia solution, shake up, place and make layering, divide and get n-butyl alcohol liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution;

Other gets Radix Angelicae Dahuricae control medicinal material 0.5g, adds methanol 10ml, and supersound process 30 minutes filters, and filtrate is concentrated into 2ml, in contrast medical material solution;

Test according to thin layer chromatography (appendix VI B), draw above-mentioned two kinds of each 0.005ml of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, with 30-60 ℃ of petroleum ether-ethyl acetate=1: 1.1 was developing solvent, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

E. get medicament composition capsule agent content 1g of the present invention, add water 30ml and make dissolving, filter, filtrate is put in the separatory funnel, extract 3 times with water saturated n-butyl alcohol jolting, each 20ml merges n-butyl alcohol liquid, water 20ml washing 2 times, discard water layer, divide and get n-butyl alcohol liquid, evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution;

Extracting liquorice control medicinal material 0.5g adds methanol 4ml in addition, and supersound process 30 minutes is got supernatant medical material solution in contrast;

Test according to thin layer chromatography (appendix VIB), draw above-mentioned two kinds of each 0.005ml of solution, put respectively on the silica gel g thin-layer plate of same usefulness 1% sodium hydroxide solution preparation, with ethyl acetate-formic acid-glacial acetic acid-water=15: 1: 1: 2 was developing solvent, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, inspects under ultra-violet lamp 365nm; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

A. danshensu:

The reference substance solution preparation: get the danshensu sodium reference substance, the accurate title, decide, and adds methanol and make the solution that every 1ml contains 0.00006g, promptly;

The preparation of need testing solution: get medicament composition capsule agent content 0.5g of the present invention, put in the 100ml triangular flask, precision adds methanol 50ml, supersound process 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 10ml, puts in the 50ml round-bottomed flask, reclaims solvent to doing, residue adds the mobile phase dissolving and changes in the 10ml measuring bottle, add mobile phase to scale, shake up, promptly;

Algoscopy: accurate respectively reference substance solution 0.005ml and 0.010ml, the need testing solution 0.010ml of drawing, inject chromatograph of liquid, measure, promptly;

Contain Radix Salviae Miltiorrhizae with danshensu (C in every medicament composition capsule of the present invention ₉H ₁₀O ₅) meter, must not be less than 1.6mg;

B. paeonol:

The preparation of reference substance solution: get the paeonol reference substance, the accurate title, decide, and adds methanol and make the solution that every 1ml contains 0.000005g, promptly;

The preparation of need testing solution: get 1/24 of the daily amount of formulation of pharmaceutical composition of the present invention, put in the 100ml triangular flask, precision adds methanol 50ml, excusing from death was handled 30 minutes, put coldly, claimed to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 1ml, put in the 10ml measuring bottle, add methanol to scale, shake up, promptly;

Algoscopy: accurate respectively reference substance solution and each 0.010ml of need testing solution of drawing, injecting chromatograph is measured, promptly;

Contain Cortex Moutan with paeonol (C in every medicament composition capsule of the present invention ₉H ₁₀O ₃) meter, must not be less than 2.6mg.

Embodiment 10: the quality determining method of the medicament composition capsule agent of the present invention of embodiment 1 preparation

Test according to thin layer chromatography (appendix VIB), draw above-mentioned two kinds of each 0.005ml of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, so that chloroform-methanol-water=the lower floor's solution in placement below 10 ℃ was developing solvent in 13: 7: 2, launch, take out, dry, spray is with 1% paradime thylaminobenzaldehyde ethanol solution of sulfuric acid, get 0.5-1.5% paradime thylaminobenzaldehyde sulfuric acid solution 1mL, to 10mL, get this solution spray silica gel g thin-layer plate with ethanol dilution, it is clear to be heated to the speckle colour developing at 70 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of same color; Put under the uviol lamp 365nm and inspect, show identical xanchromatic fluorescence speckle;

According to thin layer chromatography (appendix VI B test), draw above-mentioned two kinds of each 0.010ml of solution, put respectively on same silica gel g thin-layer plate, with cyclohexane extraction-ethyl acetate=3: 1 was developing solvent, launched, and took out, dry, spray is with the acid 5% ferric chloride alcoholic solution of hydrochloric acid, and it is clear to be heated to the speckle colour developing; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical blue brown speckle;

Test according to thin layer chromatography (appendix VIB), draw above-mentioned two kinds of each 0.005ml of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, with 30-60 ℃ of petroleum ether-ethyl acetate=1: 1.1 was developing solvent, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

Test according to thin layer chromatography (appendix VI B), draw above-mentioned two kinds of each 0.005ml of solution, put respectively on the silica gel g thin-layer plate of same usefulness 1% sodium hydroxide solution preparation, with ethyl acetate-formic acid-glacial acetic acid-water=15: 1: 1: 2 was developing solvent, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, inspects under ultra-violet lamp 365nm; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.

Embodiment 11: the quality determining method of the medicament composition capsule agent of the present invention of embodiment 1 preparation

A. danshensu:

B. paeonol:

Embodiment 12: the quality determining method of the medicament composition capsule agent of the present invention of embodiment 1 preparation

Differentiate:

B. get medicament composition capsule agent content 1g of the present invention, add ethanol 20ml, close plug, jolting 10 minutes filters, and filtrate is concentrated into 1ml, as need testing solution;

A. danshensu:

Contain Radix Salviae Miltiorrhizae with danshensu (C in every medicament composition capsule of the present invention ₉H ₁₀O ₅) meter, must not be less than 1.6mg.

Embodiment 13: the quality determining method of the pharmaceutical composition tablet of the present invention of embodiment 2 preparations

Differentiate:

A. get 1/12 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 30ml and make dissolving, filter, filtrate transfers pH value to 1-2 with dilute hydrochloric acid, puts in the separatory funnel, extract 3 times with the ethyl acetate jolting, each 20ml merges ethyl acetate liquid, and the reclaim under reduced pressure ethyl acetate is to doing, residue adds methanol 2ml makes dissolving, as need testing solution;

B. get 1/6 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 30ml and make dissolving, filter, filtrate is put in the separatory funnel, extract 3 times with water saturated n-butyl alcohol jolting, each 20ml merges n-butyl alcohol liquid, water 20ml washing 2 times, discard water layer, divide and get n-butyl alcohol liquid, evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution;

Paeonol:

Embodiment 14: the quality determining method of the medicament composition granule agent of the present invention of embodiment 3 preparations

Differentiate:

A. get 1/6 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 30ml and make dissolving, filter, filtrate is put in the separatory funnel, extract 3 times with water saturated n-butyl alcohol jolting, each 20ml merges n-butyl alcohol liquid, water 20ml washing 2 times, discard water layer, divide and get n-butyl alcohol liquid, evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution;

A. danshensu:

The preparation of need testing solution: get 1/12 of the daily amount of formulation of pharmaceutical composition of the present invention, put in the 100ml triangular flask, precision adds methanol 50ml, supersound process 30 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, precision is measured subsequent filtrate 10ml, puts in the 50ml round-bottomed flask, reclaims solvent to doing, residue adds the mobile phase dissolving and changes in the 10ml measuring bottle, add mobile phase to scale, shake up, promptly;

B. paeonol:

Embodiment 15: the quality determining method of the medicinal composition powders of the present invention of embodiment 4 preparations

Differentiate:

Test according to thin layer chromatography (appendix VIB), draw above-mentioned two kinds of each 0.005ml of solution, put respectively on the silica gel g thin-layer plate of same usefulness 1% sodium hydroxide solution preparation, with ethyl acetate-formic acid-glacial acetic acid-water=15: 1: 1: 2 was developing solvent, launch, take out, dry, put under the uviol lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical fluorescence speckle;

B. get 1/12 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 25ml and make dissolving, filter, filtrate is put in the separatory funnel, extracts 3 times with water saturated n-butyl alcohol jolting, uses 15ml at every turn, merge n-butanol extracting liquid, add isopyknic ammonia solution, shake up, place and make layering, divide and get n-butyl alcohol liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution;

C. get 1/6 of the daily amount of formulation of pharmaceutical composition of the present invention, add water 30ml and make dissolving, filter, filtrate is put in the separatory funnel, extract 3 times with water saturated n-butyl alcohol jolting, each 20ml merges n-butyl alcohol liquid, water 20ml washing 2 times, discard water layer, divide and get n-butyl alcohol liquid, evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution;

Test according to thin layer chromatography (appendix VI B), draw above-mentioned two kinds of each 0.005ml of solution, put respectively on the silica gel g thin-layer plate of same usefulness 1% sodium hydroxide solution preparation, with ethyl acetate-formic acid-glacial acetic acid-water=15: 1: 1: 2 was developing solvent, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, inspects under ultra-violet lamp 365nm; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

A. danshensu:

Pharmaceutical composition of the present invention every day is with containing Radix Salviae Miltiorrhizae in the amount of formulation with danshensu (C ₉H ₁₀O ₅) meter, must not be less than 19.2mg.

Claims

1. transfer the liver the kidney invigorating for one kind, the pharmaceutical composition of stasis-resolving spot-removing is characterized in that the crude drug of this pharmaceutical composition consists of:

2. pharmaceutical composition as claimed in claim 1 is characterized in that the crude drug of this pharmaceutical composition consists of:

The Radix Angelicae Dahuricae 333.3 weight portion Cortex Moutans 333.3 weight portion Radix Glycyrrhizaes 100 weight portions;

Or

The Radix Angelicae Dahuricae 390 weight portion Cortex Moutans 310 weight portion Radix Glycyrrhizaes 130 weight portions;

Or

3. as the arbitrary described pharmaceutical composition of claim 1-2, it is characterized in that getting the above-mentioned raw materials medicine, add conventional adjuvant, make clinical pharmaceutical dosage form: granule, pill, powder, capsule, tablet, slow releasing agent, oral liquid or lyophilized injectable powder according to common process.

4. as the arbitrary described preparation of drug combination method of claim 1-2, it is characterized in that this method comprises the steps:

Get crude drug, Cortex Moutan is collected the distillate that Cortex Moutan 5-15 doubly measures with the water distillation that 10-20 doubly measures, in 0～10 ℃ of following cold preservation 6-12 hour, filter, get paeonol crystallization I, filtrate adds the sodium chloride of Cortex Moutan weight 3-8%, and redistillation is collected the distillate that Cortex Moutan 2-4 doubly measures, in 0～10 ℃ of following cold preservation 6-12 hour, filter, get filtrate A and paeonol crystal II, merge paeonol crystallization I and II,, promptly get paeonol in 30-50 ℃ of cold drying, standby, filtrate A device in addition collects medicinal residues and all the other Radix Bupleuri, Radix Salviae Miltiorrhizae, Radix Rehmanniae Preparata, the Radix Angelicae Dahuricae and Radix Glycyrrhizae five tastes crude drug, decoct with water 1-3 time, add water 5-12 times of weight at every turn, decocted 1-3 hour at every turn, filter, merging filtrate gets liquor B, mix filtrate A and B, be concentrated into 50～70 ℃ of following relative densities and be 1.10～1.35 thick paste, drying, pulverize, cross 60 mesh sieves, get extract powder, standby; Get above-mentioned paeonol porphyrize, with the magnesium stearate mix homogeneously that accounts for extract powder weight 1-1.5%, add in the above-mentioned extract powder, mixing, add conventional adjuvant, make clinical pharmaceutical dosage form: granule, pill, powder, capsule, tablet, slow releasing agent, oral liquid or lyophilized injectable powder according to common process.

5. preparation of drug combination method as claimed in claim 4 is characterized in that this method comprises the steps:

Get crude drug, Cortex Moutan is collected the distillate of 10 times of amounts of Cortex Moutan with the water distillation of 15 times of amounts, in 0～10 ℃ of following cold preservation 8 hours, filter, get paeonol crystallization I, filtrate adds the sodium chloride of Cortex Moutan weight 5%, and redistillation is collected the distillate of 3 times of amounts of Cortex Moutan, in 0～10 ℃ of following cold preservation 8 hours, filter, get filtrate A and paeonol crystal II, merge paeonol crystallization I and II,, promptly get paeonol in 40 ℃ of cold drying, standby, filtrate A device in addition collects medicinal residues and all the other Radix Bupleuri, Radix Salviae Miltiorrhizae, Radix Rehmanniae Preparata, the Radix Angelicae Dahuricae and Radix Glycyrrhizae five tastes crude drug, decoct with water secondary, add for the first time water 10 times of weight, add water 8 times of weight the 2nd time, the each decoction 2 hours, filter, merging filtrate gets liquor B, mixes filtrate A and B, be concentrated into 60～65 ℃ of following relative densities and be 1.20～1.25 thick paste, drying is pulverized, and crosses 60 mesh sieves, get extract powder, standby; Get above-mentioned paeonol porphyrize, with the magnesium stearate mix homogeneously that accounts for extract powder weight 0.5%, add in the above-mentioned extract powder, mixing, add conventional adjuvant, make clinical pharmaceutical dosage form: granule, pill, powder, capsule, tablet, slow releasing agent, oral liquid or lyophilized injectable powder according to common process.

6. as the quality determining method of the arbitrary described pharmaceutical composition of claim 1-2, it is characterized in that this method comprises one or more in following discriminating and the content assaying method:

Differentiate:

Test according to thin layer chromatography (appendix VI B), draw above-mentioned each 0.003-0.008 parts by volume of two kinds of solution, put respectively on same silica gel g thin-layer plate with the preparation of 0.5-1.5% sodium hydroxide solution, with ethyl acetate-formic acid-glacial acetic acid-water=10-20: 0.5-1.5: 0.5-1.5: 1-3 is developing solvent, launch, take out, dry, put under the uviol lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical fluorescence speckle;

A. danshensu:

B. paeonol:

7. the quality determining method of pharmaceutical composition as claimed in claim 6 is characterized in that this method comprises one or more in following discriminating and the content assaying method:

Differentiate:

A. danshensu:

B. paeonol:

8. transfer liver the kidney invigorating, the application in the medicine of stasis-resolving spot-removing as the arbitrary described pharmaceutical composition of claim 1-3 in preparation.

9. application as claimed in claim 8 is characterized in that wherein said stasis-resolving spot-removing is meant the treatment chloasma.

10. application as claimed in claim 8 is characterized in that wherein said stasis-resolving spot-removing is meant inhibition B16 cell proliferation; Or the tyrosinase activity of inhibition B-16 melanoma cell strain; Or inhibition melanin forms; Or inhibition Mutations in Skin of Different Ultraviolet damage model mice serum lipid peroxide forms; Or suppress and kill propionibacterium acne, micrococcus luteus; Or improve hemorheological property.