CN100467015C

CN100467015C - A pharmaceutical composition for treating kidney-qi deficiency syndrome and preparation method thereof

Info

Publication number: CN100467015C
Application number: CNB2005100752625A
Authority: CN
Inventors: 张友生; 张思宁
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-06-09
Filing date: 2005-06-09
Publication date: 2009-03-11
Anticipated expiration: 2025-06-09
Also published as: CN1876089A

Abstract

The invention relates to a pharmaceutical composition for treating kidney deficiency and quality control method thereof, wherein the pharmaceutical composition can be prepared into granular formulation. According to the granule quality control method of the invention, cnidium fruit, psoralea fruit, polygonum multiflorum thumb and licorice root are undergone through specific thin layer discrimination, and cnidium is subject to content determination.

Description

A kind of pharmaceutical composition for the treatment of syndrome of deficiency of kidney-QI and preparation method thereof

Technical field

The present invention relates to a kind of pharmaceutical composition, particularly a kind of pharmaceutical composition for the treatment of syndrome of deficiency of kidney-QI and preparation method thereof.

Background technology

Sexual impotence, seminal emission, waist and leg ache, lassitude, nocturia, fear of cold is afraid of cold, women's menorrhagia, clear and thin leucorrhea, above-mentioned performance belongs to " syndrome of deficiency of kidney-QI " and belongs to the traditional Chinese medical science " deficiency of five viscera " category.Its generation, how because of the natural endowment weakness, body constitution is not strong, because of void causes labor, or due to illness causes void, and is with the passing of time not multiple, becomes asthenia; Or, undermine the five internal organs because of bothering excessively, and all can form deficiently, primary disease is the frequently-occurring disease of middle-aged and elderly people, commonly encountered diseases, the current because development of aged tendency of population trend, the primary disease sickness rate is soaring year by year, provides the medicine of new determined curative effect to be necessary.

Summary of the invention

The object of the invention is to provide a kind of pharmaceutical composition, and another object of the present invention is to provide the method for quality control of this medicament composition granule agent.

The present invention seeks to be achieved through the following technical solutions.

Pharmaceutical composition of the present invention is made by following bulk drugs:

Fructus Cnidii 20-40 weight portion Rhizoma Chuanxiong 20-40 weight portion Semen Cuscutae 50-80 weight portion

Fructus Psoraleae 20-40 weight portion Poria 20-40 weight portion Radix Ginseng Rubra 15-25 weight portion

Fructus Foeniculi 10-20 weight portion Fructus Schisandrae Chinensis 30-40 weight portion Fructus Rosae Laevigatae 60-120 weight portion

Rhizoma Atractylodis Macrocephalae 10-20 weight portion Radix Angelicae Sinensis 40-60 weight portion Fructus Rubi 25-40 weight portion

Radix Polygoni Multiflori Preparata 60-90 weight portion Semen Plantaginis 10-20 weight portion Radix Rehmanniae Preparata 60-120 weight portion

Fructus Lycii 50-80 weight portion Rhizoma Dioscoreae 40-60 weight portion Herba Epimedii 60-120 weight portion

Semen Trigonellae 60-120 weight portion Radix Astragali 40-60 weight portion Herba Cistanches 40-60 weight portion

Radix Glycyrrhizae Preparata 10-20 weight portion.

Be preferably as follows bulk drugs:

Fructus Cnidii 28 weight portion Rhizoma Chuanxiongs 28.3 weight portion Semen Cuscutae 66 weight portions

Fructus Psoraleae 28.5 weight portion Poria 30 weight portion Radix Ginseng Rubra 20 weight portions

Fructus Foeniculi 14.4 weight portion Fructus Schisandrae Chinensis 36 weight portion Fructus Rosae Laevigatae 94.6 weight portions

The Rhizoma Atractylodis Macrocephalae 14.2 weight portion Radix Angelicae Sinensis 46.8 weight portion Fructus Rubies 32.9 weight portions

Radix Polygoni Multiflori Preparata 74.4 weight portion Semen Plantaginiss 16.5 weight portion Radix Rehmanniae Preparata 94 weight portions

Fructus Lycii 66 weight portion Rhizoma Dioscoreaes 46.3 weight portion Herba Epimedii 94.6 weight portions

The Semen Trigonellae 94 weight portion Radixs Astragali 51.4 weight portion Herba Cistanches 47.3 weight portions

Radix Glycyrrhizae Preparata 14.2 weight portions.

Or:

Fructus Cnidii 25 weight portion Rhizoma Chuanxiongs 35 weight portion Semen Cuscutae 60 weight portions

Fructus Psoraleae 35 weight portion Poria 25 weight portion Radix Ginseng Rubra 23 weight portions

Fructus Foeniculi 12 weight portion Fructus Schisandrae Chinensis 38 weight portion Fructus Rosae Laevigatae 70 weight portions

The Rhizoma Atractylodis Macrocephalae 27 weight portion Radix Angelicae Sinensis 42 weight portion Fructus Rubies 38 weight portions

Radix Polygoni Multiflori Preparata 65 weight portion Semen Plantaginiss 18 weight portion Radix Rehmanniae Preparata 70 weight portions

Fructus Lycii 75 weight portion Rhizoma Dioscoreaes 42 weight portion Herba Epimedii 110 weight portions

The Semen Trigonellae 70 weight portion Radixs Astragali 55 weight portion Herba Cistanches 42 weight portions

Radix Glycyrrhizae Preparata 18 weight portions.

Or:

Fructus Cnidii 35 weight portion Rhizoma Chuanxiongs 25 weight portion Semen Cuscutae 75 weight portions

Fructus Psoraleae 25 weight portion Poria 35 weight portion Radix Ginseng Rubra 17 weight portions

Fructus Foeniculi 18 weight portion Fructus Schisandrae Chinensis 32 weight portion Fructus Rosae Laevigatae 110 weight portions

The Rhizoma Atractylodis Macrocephalae 12 weight portion Radix Angelicae Sinensis 55 weight portion Fructus Rubies 27 weight portions

Radix Polygoni Multiflori Preparata 80 weight portion Semen Plantaginiss 12 weight portion Radix Rehmanniae Preparata 110 weight portions

Fructus Lycii 55 weight portion Rhizoma Dioscoreaes 56 weight portion Herba Epimedii 70 weight portions

The Semen Trigonellae 110 weight portion Radixs Astragali 45 weight portion Herba Cistanches 55 weight portions

Radix Glycyrrhizae Preparata 12 weight portions.

Preparation of drug combination method of the present invention is:

More than 22 the flavor crude drug, Fructus Cnidii, Herba Epimedii, Radix Angelicae Sinensis, Rhizoma Chuanxiong, Fructus Foeniculi are ground into coarse powder, according to the percolation under Chinese Pharmacopoeia version in 2000 appendix I O fluid extract and the extractum item, make solvent with 60-80% ethanol, flood after 48 hours, slowly percolation, collect percolate, reclaim ethanol, filter filtrate for later use; Red ginseng powder is broken into coarse powder, makes solvent, flood after 8 hours with 10-30% ethanol, backflow 1-2 time, each 1-2 hour, merge backflow, reclaim ethanol, filter filtrate for later use; Get ten Six-elements such as all the other Fructus Rubies,, decoct with water 1-2 time, each 1-2 hour with the Radix Ginseng Rubra medicinal residues, merge decoction liquor, filter, it is about 1.10 that filtrate is concentrated into relative density, puts cold, add ethanol, make that to contain the alcohol amount be 70%, stir evenly, cooling is left standstill, and gets supernatant and reclaims ethanol, merges above-mentioned filtrate, be evaporated to relative density and be 1.30～1.33 clear paste, add cane sugar powder, make granule, drying is distributed into bag, promptly gets medicament composition granule agent of the present invention.

The method of quality control of granule of the present invention comprises one or more in following discriminating and/or the content assaying method:

Differentiate: A, get this product 20g, add water 30ml heating and make dissolving, put coldly, add ethyl acetate extraction 2 times, each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the osthole reference substance, adds methanol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, benzene-ethyl acetate with 25-35:1 is developing solvent, launch, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

B, get psoralen, isopsoralen reference substance, add ethyl acetate and make the mixed solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin layer chromatography (an appendix V1 of Chinese Pharmacopoeia version in 2000 B), draw the need testing solution 10 μ l that differentiate under the A item, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the Sodium Tvlose, be developing solvent with petroleum ether-ethyl acetate of 3-5:1, wherein the petroleum ether temperature is 60～90 ℃, launch, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show two identical blue and white fluorescence speckles.

C, get Radix Polygoni Multiflori control medicinal material 1g, add water 30ml, reflux 30 minutes filters, and filtrate adds ethyl acetate extraction 2 times, and each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methanol 1ml makes dissolving, in contrast medical material solution; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw need testing solution and each 10 μ l of control medicinal material solution of differentiating under the A item, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 1% sodium hydroxide, placing stratified upper strata liquid with 10-20:1-3:1 toluene-ethyl acetate-formic acid is developing solvent, launch, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical fluorescent orange speckle; Put in the ammonia steam smoked after, speckle becomes redness.

D, get and differentiate the aqueous solution behind the ethyl acetate extraction under the A item, with water saturated n-butanol extraction 2 times, 30ml at every turn, merge n-butyl alcohol liquid, add water 20ml washing, evaporate to dryness, residue adds methanol 1ml makes dissolving, adds neutral alumina 1g, mixes thoroughly, water bath method is contained on the neutral alumina pillar of 100～200 orders, 3g, internal diameter 10mm, with 40% methanol 50ml eluting, collect eluent, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Extracting liquorice 0.2g adds water 30ml in addition, and reflux 30 minutes filters, and the n-butyl alcohol that filtrate water is saturated shines medical material solution in pairs with legal system; Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw need testing solution 10 μ l, control medicinal material solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 1% sodium hydroxide, with 10-20:1:1:1-3 ethyl acetate-formic acid-glacial acetic acid-water is developing solvent, launch, take 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 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.

Developing solvent benzene among the above-mentioned discrimination method A-ethyl acetate preferred proportion 30:1; Developing solvent petroleum ether among the discrimination method B-ethyl acetate preferred proportion 4:1; Developing solvent toluene-ethyl acetate among the discrimination method C-formic acid preferred proportion 15:2:1; Developing solvent ethyl acetate-formic acid among the discrimination method D-glacial acetic acid preferred proportion 15:1:1:2;

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

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With the 60-70:30-40 acetonitrile-water is mobile phase; The detection wavelength is 322nm, and number of theoretical plate calculates by the osthole peak and is not less than 3000;

It is an amount of that precision takes by weighing the osthole reference substance, adds methanol and make the solution that every 1ml contains 4ug, promptly gets reference substance solution;

Get content under this product content uniformity item, porphyrize is got 2g, and accurate the title decides, and puts in the apparatus,Soxhlet's, it is an amount of to add ethanol, and reflux was put cold in 8 hours, move in the evaporating dish, evaporate to dryness, residue add methanol make the dissolving and move in the 5ml measuring bottle, add methanol to scale, shake up, filter, promptly get need testing solution;

Accurate respectively reference substance solution and each 10ul of need testing solution of drawing injects chromatograph of liquid, measures, promptly;

This product contains Fructus Cnidii by osthole (C for every bag ₁₅H ₁₆O ₃) meter, must not be less than 0.10mg.

The preferred proportion of mobile phase acetonitrile-water is 65:35 in the above-mentioned content assaying method.

Pharmaceutical composition of the present invention and granular preparation thereof have YIN and YANG balance regulating, temperature male wind-supplying kidney, and the controlling nocturnal emission with astringent drugs of calming the nerves, the function of strengthening the body resistance, the clinical sexual impotence that is used for, seminal emission, waist and leg ache, lassitude, nocturia, fear of cold is afraid of cold; Women's menorrhagia, all diseases of clear and thin leucorrhea.

Granule method of quality control of the present invention has been revised and enlarged the specificity thin layer discrimination method of Fructus Cnidii, Fructus Psoraleae, Radix Polygoni Multiflori and Radix Glycyrrhizae and the content assaying method of Fructus Cnidii, controlled product quality better, through experimental study method of quality control stability provided by the present invention and favorable reproducibility, steady quality.The effect of Fructus Cnidii tool warming the kidney to invigorate YANG is the principal agent in the side.Adopt high performance liquid chromatography, measure the content of the contained osthole of Fructus Cnidii, as the content assaying method of this product.Chromatographic condition: with the octadecylsilane chemically bonded silica is filler: with acetonitrile-water (65:35) is mobile phase: the detection wavelength is 322nm; Average recovery rate is 95.17%; The average deviation is 1.10%.

Experimental example 1 granule therapy deficiency of five viscera of the present invention (syndrome of deficiency of kidney-QI) 30 routine final reports

Medicine material composition, effect and cure mainly according to the present invention select deficiency of five viscera (syndrome of deficiency of kidney-QI) for observing syndrome, it have been carried out the clinical trial observation, so that this medicine clinical efficacy and safety are revalued.Now that the result of the test final report is as follows.

The clinical trial case comes from outpatient service and partial hospitalization patient.Observe deficiency of five viscera syndrome of deficiency of kidney-QI patient 30 examples altogether, all meet the case choice criteria.

One, clinical curative effect analysis

1, tcm syndrome curative effect: clinical cure 9 examples (accounting for 30%), produce effects 11 examples (accounting for 36.7%), effective 7 examples (accounting for 23.3%), invalid 3 examples (accounting for 10%), total effective rate 90%.

2, tcm symptom curative effect sees Table 1, table 2

Traditional Chinese medical science primary symptom curative effect relatively before and after table 1 treatment

Through the Ridit check, traditional Chinese medical science primary symptom is improved relatively before and after the treatment, and remarkable significant difference P＜0.05 is arranged.

The traditional Chinese medical science time disease curative effect relatively before and after table 2 treatment

Through the Ridit check, the traditional Chinese medical science time disease is improved relatively before and after the treatment, and remarkable significant difference P＜0.05 is arranged.

3, tongue, pulse condition improve, and see Table 3

Table 3 tongue, pulse condition improve relatively

Through X ²Check, treatment back tongue, pulse condition improve relatively, do not have remarkable significant difference P〉0.05.

4, the state of an illness and curative effect see Table 4

Table 4 state of an illness and curative effect correlation analysis

Through the Ridit check, the different state of an illness curative effects of test group relatively do not have remarkable significant difference P〉0.05.

5, the course of disease and curative effect see Table 5

The table 5 two-stage course of disease and curative effect correlation analysis

Through the Ridit check, the different course of disease curative effects of test group relatively do not have remarkable significant difference P〉0.05.

6, age and curative effect see Table 6

Table 6 age and curative effect correlation analysis

Through the Ridit check, all ages and classes section curative effect does not relatively have remarkable significant difference P〉0.05.

7, sex and curative effect see Table 7

Table 7 sex and curative effect correlation analysis

Sex	The example number	Clinical cure	Produce effects	Effectively	Invalid	Total effective rate %
Sex	The example number	Clinical cure	Produce effects	Effectively	Invalid	Total effective rate %	The man	19	6	7	5	1	94.74
The woman	11	3	4	2	2	81.82	The man	19	6	7	5	1	94.74

Through the Ridit check, the different sexes curative effect does not relatively have remarkable significant difference P〉0.05.

Two, safety is observed

1, the security inspection result sees Table 8

Table 8 security inspection result

2, untoward reaction

Whole skin mucosa, gastrointestinal tract and other untoward reaction are not found in clinical observation this time.

End clinical setting: clinical observation this time, there is not the test of termination case.

Elimination test case situation: clinical observation this time, no elimination test case.

Three, conclusion

Through to 30 routine deficiency of five viscera syndrome of deficiency of kidney-QI patients' clinical trial, the result shows: do not find the untoward reaction due to the trial drug in the process of the test, and blood, urine, just routine test before and after the test, toxic and side effects is not found in liver, renal function and Electrocardioscopy.

Curative effect to the deficiency of five viscera syndrome of deficiency of kidney-QI: granule cure-remarkable-effectiveness rate 66.7% of the present invention, effective percentage 23.3%, total effective rate 90.00%.Results suggest granule of the present invention is clinical safe in utilization, and curative effect is reliable.

Correlation analysis shows: test group curative effect and age, sex, the state of an illness, the course of disease do not have dependency.

Conclusion is thought: granule therapy deficiency of five viscera syndrome of deficiency of kidney-QI curative effect of the present invention is reliable, clinical safe in utilization.

The thin layer chromatography of experimental example 2 granule Fructus Cnidiis of the present invention is differentiated

Instrument and reagent: osthole reference substance (calibrating of 0822-200003 Chinese biological goods provides); Tlc silica gel G (chemical pure, Qingdao Marine Chemical Co., Ltd. makes, lot number: 031215); Ethyl acetate etc.: be analytical pure.

1, the preparation of need testing solution: get this product 20g, add water 30ml heating and make dissolving, put coldly, add ethyl acetate extraction 2 times, each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.

2, the preparation of reference substance solution: get the osthole reference substance, add methanol and make the solution that every 1ml contains 0.5mg, in contrast product solution.

3, the preparation of negative control solution: make the negative sample that lacks Fructus Cnidii in prescription ratio and method for making, get 20g, press the preparation method of test sample, make the negative control solution that lacks Fructus Cnidii.

4, thin layer chromatography: according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw need testing solution and negative control solution each 10 μ l, reference substance solution 5 μ l, put respectively in same be silica gel g thin-layer plate (the hands bed board of adhesive with the sodium carboxymethyl cellulose, thick 0.3mm) on, be developing solvent with benzene-ethyl acetate (30:1), room temperature is launched, exhibition is apart from 8cm, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.The negative control chromatograph is noiseless on the relevant position.

The thin layer chromatography of experimental example 3 granule Fructus Psoraleaes of the present invention is differentiated

Instrument and reagent: psoralen reference substance (calibrating of 738-8802 Chinese biological goods provides), isopsoralen reference substance (calibrating of 110739-200309 Chinese biological goods provides); (chemical pure, Qingdao Marine Chemical Co., Ltd. makes tlc silica gel G, lot number: 031215): ethyl acetate etc.: be analytical pure.

2, the preparation of reference substance solution: get psoralen, isopsoralen reference substance, add ethyl acetate and make the mixed solution that every 1ml contains 0.5mg, in contrast product solution.

3, the preparation of negative control solution: make the negative sample that lacks Fructus Psoraleae in prescription ratio and method for making, get 20g, press the preparation method of test sample, make the negative control solution that lacks Fructus Psoraleae.

4, thin layer chromatography: according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw need testing solution and negative control solution each 10 μ l, reference substance solution 5 μ l, put respectively in same be silica gel g thin-layer plate (the hands bed board of adhesive with the sodium carboxymethyl cellulose, thick 0.3mm) on, be developing solvent with petroleum ether (60～90 ℃)-ethyl acetate (4:1), room temperature is launched, exhibition is apart from 8cm, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show two identical blue and white fluorescence speckles.The negative control chromatograph is noiseless on isopsoralen reference substance (top speckle) relevant position, displaing yellow fluorescence speckle on psoralen reference substance (below speckle) relevant position.

With reference to " " Fructus Psoraleae " thin layer of recording of Chinese pharmacopoeia one of version in 2000 differentiates and down use n-hexane-ethyl acetate (4:1) instead and be developing solvent that thin layer collection of illustrative plates display dot separating degree does not have improvement.

The thin layer chromatography of experimental example 4 granule Radix Polygoni Multiflori of the present invention is differentiated

Instrument and reagent: Radix Polygoni Multiflori control medicinal material (calibrating of 934-200106 Chinese biological goods provides), (chemical pure, Qingdao Marine Chemical Co., Ltd. makes tlc silica gel G, lot number: 031215): ethyl acetate etc.: be analytical pure.

2, the preparation of control medicinal material solution: get Radix Polygoni Multiflori control medicinal material 1g, add water 30ml, reflux 30 minutes filters, filtrate adds ethyl acetate extraction 2 times, and each 30ml merges ethyl acetate liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, in contrast medical material solution.

3, the preparation of negative control solution: make the negative sample that lacks Radix Polygoni Multiflori in prescription ratio and method for making, get 20g, press the preparation method of test sample, make the negative control solution that lacks Radix Polygoni Multiflori.

4, thin layer chromatography: according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 10 μ l of above-mentioned three kinds of solution, put respectively in same be silica gel g thin-layer plate (the hands bed board of adhesive with the carboxymethylcellulose sodium solution that contains 1% sodium hydroxide, thick 0.3mm) on, be developing solvent with ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2), room temperature is launched, exhibition is apart from 8cm, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical fluorescent orange speckle; Put in the ammonia steam smoked after, speckle becomes redness.The negative control chromatograph is noiseless on the relevant position.

The thin layer chromatography of experimental example 5 granule Radix Glycyrrhizaes of the present invention is differentiated

Instrument and reagent: 101-1-BS electric heating constant temperature air dry oven: Radix Glycyrrhizae control medicinal material (calibrating of 121303-200301 Chinese biological goods provides); Chromatography neutral alumina (Shanghai the May 4th chemical reagent company limited); (chemical pure, Qingdao Marine Chemical Co., Ltd. makes tlc silica gel G, lot number: 031215): ethyl acetate etc.: be analytical pure.

1, the preparation of need testing solution: get the aqueous solution after Fructus Cnidii is differentiated a following ethyl acetate extraction, with water saturated n-butanol extraction 2 times, 30ml at every turn, merge n-butyl alcohol liquid, add water 20ml washing, evaporate to dryness, residue adds methanol 1ml makes dissolving, adds neutral alumina 1g, mixes thoroughly, water bath method is contained in neutral alumina pillar (100～200 orders, 3g, internal diameter 10mm) on,, collects eluent with 40% methanol 50ml eluting, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.

2, the preparation of control medicinal material solution: extracting liquorice control medicinal material 0.2g, add water 30ml, reflux 30 minutes filters, and the saturated n-butyl alcohol of filtrate water is made control medicinal material solution with the preparation method of need testing solution.

3, the preparation of negative control solution: make the negative sample that lacks Radix Glycyrrhizae in prescription ratio and method for making, get 20g, press the preparation method of test sample, make the negative control solution that lacks Radix Glycyrrhizae.

4, thin layer chromatography: according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw need testing solution and negative control solution each 10 μ l, control medicinal material solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 1% sodium hydroxide, with ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) is developing solvent, launch, take 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 ℃.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.The negative control chromatograph is noiseless on the principal spot relevant position.

The thin layer Study on Identification of experimental example 6 granule Herba Epimedii of the present invention

Instrument and reagent: icariin reference substance (calibrating of 110737-200312 Chinese biological goods provides): (chemical pure, Qingdao Marine Chemical Co., Ltd. makes tlc silica gel G, lot number: 031215): ethyl acetate etc.: be analytical pure.

2, the preparation of reference substance solution: get the icariin reference substance, add methanol and make the solution that every 1ml contains 0.5mg, in contrast product solution.

3, the preparation of negative control solution: make the negative sample that lacks Herba Epimedii in prescription ratio and method for making, get 20g, press the preparation method of test sample, make the negative control solution that lacks Herba Epimedii.

4, thin layer chromatography: according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw need testing solution and negative control solution each 10 μ l, reference substance solution 5 μ l, put respectively in same be silica gel g thin-layer plate (the hands bed board of adhesive with the sodium carboxymethyl cellulose, thick 0.3mm) makes into strips on, with ethyl acetate-butanone-formic acid-water (10:1:1:1) is developing solvent, room temperature is launched, exhibition is apart from 10cm, take out, dry, spray is with the aluminum chloride test solution, and 105 ℃ of heating were put under the ultra-violet lamp (365nm) and inspected after several minutes.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical fluorescence speckle, but that speckle separates is unintelligible." the colored atlas of Chinese pharmacopoeia Chinese medicine thin layer chromatography records the discrimination method of Herba Epimedii through reference, use silica gel g thin-layer plate, the butyl acetate-formic acid-water (1.3:1:1) that the carboxymethylcellulose sodium solution that contains the 0.1M sodium hydrogen phosphate is an adhesive instead and be developing solvent, speckle separates still undesirable, wouldn't be as the thin layer discrimination method of this product Herba Epimedii.

The thin layer Study on Identification of experimental example 7 granule Fructus Lycii of the present invention

Instrument and reagent: (usefulness feeds intake: be the dry mature fruit of plant of Solanaceae lycium barbarum LyciumbarbarumL., press check of Chinese Pharmacopoeia version in 2000, the result is up to specification for the Fructus Lycii control medicinal material.), (chemical pure, Qingdao Marine Chemical Co., Ltd. makes tlc silica gel G, lot number: 031215): ethyl acetate etc.: be analytical pure.

1, the preparation of need testing solution: get this product 20g, add water 30ml heating and make dissolving, put coldly, add ethyl acetate extraction 2 times, each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution

2, the preparation of control medicinal material solution: get Fructus Lycii control medicinal material 0.5g, add water 30ml, reflux 30 minutes filters, filtrate adds ethyl acetate extraction 2 times, and each 30ml merges ethyl acetate liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, in contrast medical material solution.

3, the preparation of negative control solution: make the negative sample that lacks inflexible Fructus Lycii in prescription ratio and method for making, get 20g, press the preparation method of test sample, make the negative control solution that lacks Fructus Lycii.

4, thin layer chromatography: according to thin chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be silica gel g thin-layer plate (the hands bed board of adhesive with the sodium carboxymethyl cellulose, thick 0.3mm) on, be developing solvent with ethyl acetate-chloroform-formic acid (3:2:0.2), room temperature is launched, exhibition is apart from 8cm, take out, dry, put under the purple lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical fluorescence speckle, the negative control chromatograph principal spot corresponding put interference arranged, wouldn't be as the thin layer discrimination method of this product Fructus Lycii

The thin layer Study on Identification of the experimental example 8 granule Radixs Astragali of the present invention

Instrument and reagent: 101-1-BS electric heating constant temperature air dry oven: astragaloside reference substance (calibrating of 0781-200210 Chinese biological goods provides): chromatography neutral alumina (Shanghai the May 4th chemical reagent company limited): tlc silica gel G (chemical pure, Haiyang Chemical Plant, Qingdao's company limited is made, lot number: 031215): n-butyl alcohol etc.: be analytical pure.

1, the preparation of need testing solution: get this product 20g, add water 30ml heating and make dissolving, put cold, with water saturated n-butanol extraction 2 times, each 30ml merges n-butyl alcohol liquid, adds ammonia solution and extracts 2 times, each 20ml, discard ammoniacal liquor, add water 20ml washing, evaporate to dryness, residue add methanol 1ml makes dissolving.Add neutral alumina 1g, mix thoroughly, water bath method, be contained in the neutral alumina pillar (100～200 orders, 3g,, internal diameter 10mm) on, with 40% methanol 50ml eluting, collect eluent, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.

2, the preparation of reference substance solution: get the astragaloside reference substance, add methanol and make the solution that every 1ml contains 0.5mg, in contrast product solution.

3, the preparation of negative control solution: make the negative sample that lacks the Radix Astragali in prescription ratio and method for making, get 20g, press the preparation method of test sample, make the negative control solution that lacks the Radix Astragali.

4, thin layer chromatography: according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000) test, draw each 10 μ l of need testing solution and negative control solution, the brilliant solution 5 μ l of contrast, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, placing the lower floor's solution that spends the night below 10 ℃ with chloroform-methanol-water (13:6:2) is developing solvent, and room temperature is launched, and exhibition is apart from 12cm, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to speckle colour developing at 105 ℃.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.The negative control chromatograph has interference on the relevant position, wouldn't be as the thin layer discrimination method of this product Radix Astragali.

The thin layer Study on Identification of experimental example 9 granule Radix Ginsengs of the present invention

Instrument and reagent: 101-1-BS electric heating constant temperature air dry oven; Ginsenoside Rb ₁Reference substance (calibrating of 0704-200313 Chinese biological goods provides); Ginsenoside Re's reference substance (calibrating of 0754-200313 Chinese biological goods provides); The ginsenoside Rg ₁Reference substance (calibrating of 110703-200323 Chinese biological goods provides): chromatography neutral alumina (Shanghai the May 4th chemical reagent company limited): tlc silica gel G (chemical pure, Qingdao Marine Chemical Co., Ltd. makes, lot number: 031215): n-butyl alcohol etc.: be analytical pure.

1, the preparation of need testing solution: get this product 20g, add water 30ml heating and make dissolving, put cold, with water saturated n-butanol extraction 2 times, each 30ml merges n-butyl alcohol liquid, adds water 20ml washing, evaporate to dryness, residue adds methanol 1ml makes dissolving, adds neutral alumina 1g, mixes thoroughly, water bath method is contained in neutral alumina pillar (100～200 orders, 3g, internal diameter 10mm) on,, collects eluent with 40% methanol 50ml eluting, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.

2, the preparation of reference substance solution: get ginsenoside Rb ₁, Re and Rg ₁Reference substance adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution.

3, the preparation of negative control solution: make the negative sample that the shortage of staff joins in prescription ratio and method for making, get 20g, press the preparation method of test sample, make the negative control solution of shortage of staff's ginseng.

4, thin layer chromatography: according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000) test, draw need testing solution and negative control solution each 10 μ l, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive, with the carboxymethylcellulose sodium solution with chloroform-ethyl acetate.Methanol-water (15:40:22:10) is placed the lower floor's solution that spends the night below 10 ℃ be developing solvent, launches, and takes out, and dries, and spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.The negative control chromatograph has interference on the relevant position, wouldn't be as the thin layer discrimination method of this product Radix Ginseng.

The thin layer Study on Identification of experimental example 10 granule Radix Angelicae Sinensis of the present invention, Rhizoma Chuanxiong

Instrument and reagent: Radix Angelicae Sinensis control medicinal material (calibrating of 0927-9806 Chinese biological goods provides); River medicine control medicinal material (0918-9603, the calibrating of Chinese biological goods provides); Tlc silica gel G (chemical pure, Qingdao Marine Chemical Co., Ltd. makes, lot number: 031215); Ethyl acetate etc.: be analytical pure.

1, the preparation of need testing solution: get this product 20g, add water 30ml heating and make dissolving, put coldly, add diethyl ether and extract 2 times, each 30ml merges ether solution, and evaporate to dryness, residue add dehydrated alcohol 1ml makes dissolving, as need testing solution.

2, the preparation of control medicinal material solution: get Radix Angelicae Sinensis and each 0.5g of Rhizoma Chuanxiong control medicinal material, the 30ml that respectively adds diethyl ether dipping 1 hour, the time add jolting, filter, filtrate volatilizes, residue adds dehydrated alcohol 1ml makes dissolving, in contrast medical material solution.

3, thin layer chromatography: according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same be silicon thigh G lamellae (the hands bed board of adhesive with the sodium carboxymethyl cellulose, thick 0.3mm) on, be developing solvent with n-hexane-ethyl acetate (9:1), room temperature is launched, exhibition is apart from 8cm, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with Radix Angelicae Sinensis control medicinal material and Rhizoma Chuanxiong control medicinal material chromatograph same position on, show the fluorescence speckle of identical blue and white.Owing to be non-exclusive, not as the thin layer discrimination method of this product Radix Angelicae Sinensis, Rhizoma Chuanxiong.

The research of experimental example 11 granule Fructus Cnidii assays of the present invention

This product prescription is made up of 22 flavor Chinese medicines, and wherein Fructus Cnidii is a principal agent, and osthole is its main effective ingredient, so be elected to be the assay index.With reference to " " Fructus Cnidii " content assaying method that records down of Chinese pharmacopoeia version in 2005 adopts the HPLC method to measure its content, and result of the test is as follows:

1, instrument and reagent

SP8810 high performance liquid chromatograph: Waters510 high performance liquid chromatograph: Spectra 100 UV-detector: SP4270 integrator; SEPU3000P work station: Hitachi's 3210 ultraviolet-uisible spectrophotometers; KQ-100 type ultrasonic cleaner.Osthole reference substance (0822-9801 for assay Nat'l Pharmaceutical ﹠ Biological Products Control Institute).Acetonitrile: chromatographically pure (Tianjin four friendly biomedical technology company limiteies): water is ultra-pure water: other reagent: be analytical pure;

2, high-efficient liquid phase chromatogram condition

The stainless steel column of Hypersil ODS2 (4.6 * 200mm, 5 μ m): mobile phase: acetonitrile-water (65:35): flow velocity: 1.0ml, 1.2ml/min: detect wavelength: 322nm: column temperature: room temperature.

3, standard curve

It is an amount of that precision takes by weighing the osthole reference substance, adds methanol and make the solution that every 1ml contains osthole 1.9539,3.8719,7.7438,15.4875,30.975 μ g.Draw 10 μ l respectively, inject high performance liquid chromatograph, press content assaying method and measure peak area.The results are shown in Table 9.

Table 9

As abscissa, the peak area A that records carries out linear regression as ordinate with reference substance concentration W, gets linear equation W=4.69018 * 10 ^-8+ 2.26068 * 10 ^-8A:r=1.00000: the range of linearity 1.9539-30.975 μ g.

4, the preparation of need testing solution

" extracting method under " Fructus Cnidii " assay of Chinese pharmacopoeia version in 2005 item is " the accurate dehydrated alcohol 25ml that adds placed ultrasonic 0.5 hour 2 hours ", through test, according to said method extracts granule for strengthening kidney, and content is on the low side.So reformulate the preparation method of need testing solution in this product content assaying method.

(1) selection of extracting method: for investigating extracting method, get 6 parts of each 1g of same test sample, accurate claim fixed: 1., with dehydrated alcohol as extracting solvent, supersound process was extracted 0.5 hour, moved to evaporating dish, evaporate to dryness adds dissolve with methanol and is settled to 5ml.2., with ethanol as extracting solvent, supersound process was extracted 0.5 hour, moved to evaporating dish, evaporate to dryness adds dissolve with methanol and is settled to 5ml.3., with ethanol as extracting solvent, heating and refluxing extraction 4 hours moves to evaporating dish, evaporate to dryness adds dissolve with methanol and is settled to 5ml.4., with dehydrated alcohol as extracting solvent, heating and refluxing extraction 4 hours moves to evaporating dish, evaporate to dryness adds dissolve with methanol and is settled to 5ml.5., with ethanol as extracting solvent, put in the apparatus,Soxhlet's, heating and refluxing extraction 4 hours moves to evaporating dish, evaporate to dryness adds dissolve with methanol and is settled to 5ml.6., with dehydrated alcohol as extracting solvent, put in the apparatus,Soxhlet's, heating and refluxing extraction 4 hours moves to evaporating dish, evaporate to dryness adds dissolve with methanol and is settled to 5ml.Content by the assay condition mensuration osthole of drafting the results are shown in Table 10.

Table 10

By The above results as can be known, the inside of 6 kinds of extracting method, preferable with " ethanol is solvent, puts in the apparatus,Soxhlet's reflux 4 hours ".

(2) selection of extraction time: get 4 parts of each 2g of same test sample, accurate claim fixed: 1., with ethanol as extracting solvent, put in the apparatus,Soxhlet's, heating and refluxing extraction 4 hours moves to evaporating dish, evaporate to dryness adds dissolve with methanol and is settled to 5ml.2., with ethanol as extracting solvent, put in the apparatus,Soxhlet's, heating and refluxing extraction 6 hours moves to evaporating dish, evaporate to dryness adds dissolve with methanol and is settled to 5ml.3., with ethanol as extracting solvent, put in the apparatus,Soxhlet's, heating and refluxing extraction 8 hours moves to evaporating dish, evaporate to dryness adds dissolve with methanol and is settled to 5ml.4., with ethanol as extracting solvent, put in the apparatus,Soxhlet's, heating and refluxing extraction 10 hours moves to evaporating dish, evaporate to dryness adds dissolve with methanol and opens and be settled to 5ml.Content by the assay condition mensuration machine tool element of drafting the results are shown in Table 11.

Table 11

4 hours	6 hours	8 hours	10 hours
4 hours	6 hours	8 hours	10 hours	9.36ug/g	14.17ug/g	15.33ug/g	14.8ug/g

By The above results as can be known, heating and refluxing extraction 8 hours is extracted osthole fully substantially, so the extraction time of adopting is 8 hours.In conjunction with the recovery test result, make the preparation method of the described need testing solution of technical scheme.

5, the preparation of reference substance solution

It is an amount of that precision takes by weighing the osthole reference substance, and methanol is made the solution that contains 4 μ g among every 1ml, promptly.

6, condition determination

1. measure the selection of wavelength: get the osthole reference substance solution, in the week interscan of 230～430nm wavelength model, the result has absorption maximum at 322nm wavelength place.

2. mobile phase selection: select to determine that through experimental study mobile phase is acetonitrile-water (65:35).

As described in technical scheme, accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, press content assaying method and measure.

7, blank assay

Get scarce Fructus Cnidii negative sample, handle and measure by the preparation and the assay method of need testing solution in prescription ratio and method for making preparation.The result does not see that blank assay has interference.

8, precision test

Accurate osthole reference substance solution (3.8719 μ g/ml) the 10 μ l that draw press content assaying method and repeat sample introduction 5 times, measure the absworption peak area.The results are shown in Table 12.

The test of table 12 precision

9, replica test

Get same test sample, precision takes by weighing 5 parts, presses content assaying method and measures.The results are shown in Table 13.

Table 13 replica test

10, stability test

The same need testing solution of accurate absorption, high performance liquid chromatograph is injected in 0,1,2,3,4 and 24 hour after starting shooting 2 hours respectively, presses content assaying method and measures.The results are shown in Table 14.

Table 14 stability test

11, application of sample recovery test

Get test sample (lot number: 20040403; Contain osthole 14.70ug/g) evenly, get 5 parts of each 2g, the accurate title, decide, accurate Fructus Cnidii reference substance solution (55.11ug/ml) 1ml that adds, water bath method is measured by the content assaying method that technical scheme is drafted, and the results are shown in Table 15.

Table 15 application of sample recovery test

12, sample size is measured

Get 3 batches of this product, measure, with the content of osthole in the external standard method calculation sample by method under the assay item.The results are shown in Table 16.

Table 16 sample size is measured

According to above-mentioned result of the test, calculate by 75% of meansigma methods, determine that every bag of this product contains Fructus Cnidii by osthole (C ₁₅H ₁₆O ₃) meter, must not be less than 0.10mg.

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

Embodiment 1:Granule of the present invention

Fructus Cnidii 28g Rhizoma Chuanxiong 28.3g Semen Cuscutae 66g Fructus Psoraleae 28.5g

Poria 30g Radix Ginseng Rubra 20g Fructus Foeniculi 14.4g Fructus Schisandrae Chinensis 36g

Fructus Rosae Laevigatae 94.6g Rhizoma Atractylodis Macrocephalae 14.2g Radix Angelicae Sinensis 46.8g Fructus Rubi 32.9g

Radix Polygoni Multiflori Preparata 74.4g Semen Plantaginis 16.5g Radix Rehmanniae Preparata 94g Fructus Lycii 66g

Rhizoma Dioscoreae 46.3g Herba Epimedii 94.6g Semen Trigonellae 94g Radix Astragali 51.4g

Herba Cistanches 47.3g Radix Glycyrrhizae Preparata 14.2g

More than 22 the flavor, Fructus Cnidii, Herba Epimedii, Radix Angelicae Sinensis, Rhizoma Chuanxiong, fennel are ground into coarse powder, according to fluid extract and the percolation (an appendix I of Chinese Pharmacopoeia version in 2000 O) that soaks under the high agent item, make solvent with 70% ethanol, flood after 48 hours, slowly percolation, collect percolate, reclaim ethanol, filter filtrate for later use; Red ginseng powder is broken into coarse powder, makes solvent, flood after 8 hours with 20% ethanol, the backflow secondary each 2 hours, merges backflow, reclaim ethanol, filter filtrate for later use: get ten Six-elements such as all the other Fructus Rubies, with the Radix Ginseng Rubra medicinal residues, decoct with water secondary, each 2 hours, merge decoction liquor, filter, filtrate is concentrated into relative density about 1.10, put coldly, add ethanol, make that to contain the alcohol amount be 70%, stir evenly, cooling was left standstill 48 hours, got supernatant and reclaimed ethanol, merge above-mentioned filtrate, be evaporated to relative density and be 1.30～1.33 clear paste, add cane sugar powder, make granule, drying is distributed into 100 bags, promptly.

Embodiment 2:The discrimination method of granule of the present invention

A, get granule 20g of the present invention, add water 30ml heating and make dissolving, put coldly, add ethyl acetate extraction 2 times, each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.Other gets the osthole reference substance, adds methanol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with benzene-ethyl acetate (30:1) 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 reference substance chromatograph on, show the fluorescence speckle of same color.

B, get psoralen, isopsoralen reference substance, add ethyl acetate and make the mixed solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin layer chromatography (appendix V1B of Chinese Pharmacopoeia version in 2000), draw need testing solution 10 μ l, reference substance solution 5 μ l under [discriminating] A item, put respectively in same be on the silica gel g thin-layer plate of adhesive with the Sodium Tvlose, with petroleum ether (60～90 ℃)-ethyl acetate (4:1) 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 reference substance chromatograph on, show two identical blue and white fluorescence speckles.

C, get Radix Polygoni Multiflori control medicinal material 1g, add water 30ml, reflux 30 minutes filters, and filtrate adds ethyl acetate extraction 2 times, and each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methanol 1ml makes dissolving, in contrast medical material solution.Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw need testing solution and each 10 μ l of control medicinal material solution under [discriminating] A item, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 1% sodium hydroxide, placing stratified upper strata liquid with toluene-ethyl acetate-formic acid (15:2:1) 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 identical fluorescent orange speckle; Put in the ammonia steam smoked after, speckle becomes redness.

D, get the aqueous solution behind the ethyl acetate extraction under [discriminating] A item, with water saturated n-butanol extraction 2 times, each 30ml, merge n-butyl alcohol liquid, add water 20ml washing, evaporate to dryness, residue adds methanol 1ml makes dissolving, adds neutral alumina 1g, mixes thoroughly, water bath method is contained in neutral alumina pillar (100～200 orders, 3g, internal diameter 10mm) on,, collects eluent with 40% methanol 50ml eluting, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.Extracting liquorice 0.2g adds water 30ml in addition, and reflux 30 minutes filters, and the n-butyl alcohol that filtrate water is saturated shines medical material solution in pairs with legal system.Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw need testing solution 10 μ l, control medicinal material solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 1% sodium hydroxide, with ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) is developing solvent, launch, take 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 ℃.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.

Embodiment 3:The content assaying method of granule of the present invention

Measure according to high performance liquid chromatography (appendix VI D)

Chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica: with acetonitrile-water (65:35) is mobile phase; The detection wavelength is 322nm, and number of theoretical plate calculates by the osthole peak and is not less than 3000.

It is an amount of that the preparation precision of reference substance solution takes by weighing the osthole reference substance, adds methanol and make the solution that every 1ml contains 4ug, promptly.

Content under the granule content uniformity item of the present invention is got in the preparation of need testing solution, and porphyrize is got 2g, the accurate title, decide, and puts in the apparatus,Soxhlet's, and it is an amount of to add ethanol, reflux was put cold in 8 hours, move in the evaporating dish, evaporate to dryness, residue add methanol make the dissolving and move in the 5ml measuring bottle, add methanol to scale, shake up, filter, promptly.

Accurate respectively reference substance solution and each 10ul of need testing solution of drawing of algoscopy injects chromatograph of liquid, measures, promptly.

Embodiment 4:The method of quality control of granule of the present invention

Differentiate A, get granule 20g of the present invention, add water 30ml heating and make dissolving, put coldly, add ethyl acetate extraction 2 times, each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.Other gets the osthole reference substance, adds methanol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with benzene-ethyl acetate (30:1) 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 reference substance chromatograph on, show the fluorescence speckle of same color.

(assay) measured according to high performance liquid chromatography (appendix VID)

Content under this product content uniformity item is got in the preparation of need testing solution, and porphyrize is got 2g, the accurate title, decide, and puts in the apparatus,Soxhlet's, and it is an amount of to add ethanol, reflux was put cold in 8 hours, move in the evaporating dish, evaporate to dryness, residue add methanol make the dissolving and move in the 5ml measuring bottle, add methanol to scale, shake up, filter, promptly.

Claims

1, a kind of method of quality control for the treatment of the medicament composition granule agent of syndrome of deficiency of kidney-QI is characterized in that this method comprises the steps:

Differentiate:

A, get this product 20g, add water 30ml heating and make dissolving, put coldly, add ethyl acetate extraction 2 times, each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the osthole reference substance, adds methanol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; According to thin layer chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, benzene-ethyl acetate with 25-35:1 is developing solvent, launches, and takes out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

B, get psoralen, isopsoralen reference substance, add ethyl acetate and make the mixed solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw the need testing solution 10 μ l that differentiate under the A item, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the Sodium Tvlose, be developing solvent with petroleum ether-ethyl acetate of 3-5:1, wherein the petroleum ether temperature is 60～90 ℃, launch, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show two identical blue and white fluorescence speckles;

C, get Radix Polygoni Multiflori control medicinal material 1g, add water 30ml, reflux 30 minutes filters, and filtrate adds ethyl acetate extraction 2 times, and each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methanol 1ml makes dissolving, in contrast medical material solution; Test according to thin layer chromatography, draw need testing solution and each 10 μ l of control medicinal material solution of differentiating under the A item, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 1% sodium hydroxide, placing stratified upper strata liquid with 10-20:1-3:1 toluene-ethyl acetate-formic acid is developing solvent, launch, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical fluorescent orange speckle; Put in the ammonia steam smoked after, speckle becomes redness;

D, get and differentiate the aqueous solution behind the ethyl acetate extraction under the A item, with water saturated n-butanol extraction 2 times, 30ml at every turn, merge n-butyl alcohol liquid, add water 20ml washing, evaporate to dryness, residue adds methanol 1ml makes dissolving, adds neutral alumina 1g, mixes thoroughly, water bath method is contained on the neutral alumina pillar of 100～200 orders, 3g, internal diameter 10mm, with 40% methanol 50ml eluting, collect eluent, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Extracting liquorice 0.2g adds water 30ml in addition, and reflux 30 minutes filters, and the n-butyl alcohol that filtrate water is saturated shines medical material solution in pairs with legal system; Test according to thin layer chromatography, draw need testing solution 10 μ l, control medicinal material solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 1% sodium hydroxide, with 10-20:1:1:1-3 ethyl acetate-formic acid-glacial acetic acid-water is developing solvent, launch, take 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 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.

Assay:

According to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With the 65:35 acetonitrile-water is mobile phase; The detection wavelength is 322nm, and number of theoretical plate calculates by the osthole peak and is not less than 3000; It is an amount of that precision takes by weighing the osthole reference substance, adds methanol and make the solution that every 1ml contains 4ug, promptly gets reference substance solution; Get content under this product content uniformity item, porphyrize is got 2g, and accurate the title decides, and puts in the apparatus,Soxhlet's, it is an amount of to add ethanol, and reflux was put cold in 8 hours, move in the evaporating dish, evaporate to dryness, residue add methanol make the dissolving and move in the 5ml measuring bottle, add methanol to scale, shake up, filter, promptly get need testing solution; Accurate respectively reference substance solution and each 10ul of need testing solution of drawing injects chromatograph of liquid, measures, promptly; This product contains Fructus Cnidii by osthole for every bag, must not be less than 0.10mg;

The method of the medicament composition granule agent of described treatment syndrome of deficiency of kidney-QI is:

Choose crude drug:

Radix Glycyrrhizae Preparata 10-20 weight portion;

More than 22 the flavor, Fructus Cnidii, Herba Epimedii, Radix Angelicae Sinensis, Rhizoma Chuanxiong, Fructus Foeniculi are ground into coarse powder, according to the percolation under Chinese Pharmacopoeia version appendix IO fluid extract in 2000 and the extractum item, make solvent with 60-80% ethanol, flood after 48 hours, slowly percolation, collect percolate, reclaim ethanol, filter filtrate for later use; Red ginseng powder is broken into coarse powder, makes solvent, flood after 8 hours with 10-30% ethanol, backflow 1-2 time, each 1-2 hour, merge backflow, reclaim ethanol, filter filtrate for later use; Get ten Six-elements such as all the other Fructus Rubies,, decoct with water 1-2 time, each 1-2 hour with the Radix Ginseng Rubra medicinal residues, merge decoction liquor, filter, it is about 1.10 that filtrate is concentrated into relative density, puts cold, add ethanol, make that to contain the alcohol amount be 70%, stir evenly, cooling is left standstill, and gets supernatant and reclaims ethanol, merges above-mentioned filtrate, be evaporated to relative density and be 1.30～1.33 clear paste, add cane sugar powder, make granule, drying is distributed into bag, promptly gets medicament composition granule agent of the present invention.

2, method of quality control as claimed in claim 1 is characterized in that this method comprises the steps:

Differentiate:

A, get this product 20g, add water 30ml heating and make dissolving, put coldly, add ethyl acetate extraction 2 times, each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the osthole reference substance, adds methanol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; According to thin layer chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, benzene-ethyl acetate with 30:1 is developing solvent, launches, and takes out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

B, get psoralen, isopsoralen reference substance, add ethyl acetate and make the mixed solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw the need testing solution 10 μ l that differentiate under the A item, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the Sodium Tvlose, be developing solvent with petroleum ether-ethyl acetate of 4:1, wherein the petroleum ether temperature is 60～90 ℃, launch, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show two identical blue and white fluorescence speckles;

C, get Radix Polygoni Multiflori control medicinal material 1g, add water 30ml, reflux 30 minutes filters, and filtrate adds ethyl acetate extraction 2 times, and each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methanol 1ml makes dissolving, in contrast medical material solution; Test according to thin layer chromatography, draw need testing solution and each 10 μ l of control medicinal material solution of differentiating under the A item, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 1% sodium hydroxide, placing stratified upper strata liquid with 15:2:1 toluene-ethyl acetate-formic acid is developing solvent, launch, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical fluorescent orange speckle; Put in the ammonia steam smoked after, speckle becomes redness;

D, get and differentiate the aqueous solution behind the ethyl acetate extraction under the A item, with water saturated n-butanol extraction 2 times, 30ml at every turn, merge n-butyl alcohol liquid, add water 20ml washing, evaporate to dryness, residue adds methanol 1ml makes dissolving, adds neutral alumina 1g, mixes thoroughly, water bath method is contained on the neutral alumina pillar of 100～200 orders, 3g, internal diameter 10mm, with 40% methanol 50ml eluting, collect eluent, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Extracting liquorice 0.2g adds water 30ml in addition, and reflux 30 minutes filters, and the n-butyl alcohol that filtrate water is saturated shines medical material solution in pairs with legal system; Test according to thin layer chromatography, draw need testing solution 10 μ l, control medicinal material solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 1% sodium hydroxide, with 15:1:1:2 ethyl acetate-formic acid-glacial acetic acid-water is developing solvent, launch, take 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 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;

Assay: according to high effective liquid chromatography for measuring

Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With the 65:35 acetonitrile-water is mobile phase; The detection wavelength is 322nm, and number of theoretical plate calculates by the osthole peak and is not less than 3000; It is an amount of that precision takes by weighing the osthole reference substance, adds methanol and make the solution that every 1ml contains 4ug, promptly gets reference substance solution; Get content under this product content uniformity item, porphyrize is got 2g, and accurate the title decides, and puts in the apparatus,Soxhlet's, it is an amount of to add ethanol, and reflux was put cold in 8 hours, move in the evaporating dish, evaporate to dryness, residue add methanol make the dissolving and move in the 5ml measuring bottle, add methanol to scale, shake up, filter, promptly get need testing solution; Accurate respectively reference substance solution and each 10ul of need testing solution of drawing injects chromatograph of liquid, measures, promptly; This product contains Fructus Cnidii by osthole for every bag, must not be less than 0.10mg.