CN1600365A

CN1600365A - Method for controlling quality of tonic semifluid extract of ten ingredients

Info

Publication number: CN1600365A
Application number: CN 200410060978
Authority: CN
Inventors: 卢建中; 王伟兰; 李诒光; 徐昌瑞
Original assignee: Jiangzhong Pharmaceutical Co Ltd
Current assignee: Jiangzhong Pharmaceutical Co Ltd
Priority date: 2004-10-12
Filing date: 2004-10-12
Publication date: 2005-03-30
Anticipated expiration: 2024-10-12
Also published as: CN100543469C

Abstract

A quality control method for the Chinese medicine 'Cream of tea powerful tonics' which is prepared from 10 Chinese-medicinal materials including Chinese angelica root, Chuan-xiong rhizome, white peony root, astragalus root, etc features that the efficient liquid-phase chromatography is used to measure the content of paeoniflorin in said Chinese medicine.

Description

The method of quality control of SHIQUAN DABU GAO

Technical field

The present invention relates to a kind of method of quality control of the SHIQUAN DABU GAO of making by Chinese crude drug Radix Codonopsis 80g, the Rhizoma Atractylodis Macrocephalae (stir-fry) 80g, Poria 80g, Radix Glycyrrhizae (processed with honey) 40g, Radix Angelicae Sinensis 120g, Rhizoma Chuanxiong 40g, the Radix Paeoniae Alba (wine stir-fry) 80g, Radix Rehmanniae Preparata 120g, the Radix Astragali (processed with honey) 80g, Cortex Cinnamomi 20g, be specifically related to content of paeoniflorin in this medicine of high effective liquid chromatography for measuring, differentiate this medicine Chinese crude drug Radix Codonopsis, the Radix Astragali and Radix Glycyrrhizae with thin layer chromatography with the effect of temperature compensation QI and blood.

Background technology

The 3rd (WS of a kind of " Drug Standard of Ministry of Public Health of the Peoples Republic of China " (Chinese traditional patent formulation preparation) ₃-B-0471-91) in treatment deficiency of both QI and blood disease medicament SHIQUAN DABU GAO, this pharmaceutical formulation is made up of Chinese crude drug Radix Codonopsis 80g, the Rhizoma Atractylodis Macrocephalae (stir-fry) 80g, Poria 80g, Radix Glycyrrhizae (processed with honey) 40g, Radix Angelicae Sinensis 120g, Rhizoma Chuanxiong 40g, the Radix Paeoniae Alba (wine stir-fry) 80g, Radix Rehmanniae Preparata 120g, the Radix Astragali (processed with honey) 80g, Cortex Cinnamomi 20g; The 3rd (WS of " Drug Standard of Ministry of Public Health of the Peoples Republic of China " (Chinese traditional patent formulation preparation) ₃-B-0471-91) issued the SHIQUAN DABU GAO quality control standard, but in the existing SHIQUAN DABU GAO method of quality control that Ministry of Public Health is issued, has only the quality examination of relative density, insoluble matter inspection and minimum fill difference under the relevant unguentum item, the quality of restive SHIQUAN DABU GAO; Disclose the HPLC assay method of peoniflorin in " Chinese Journal of Pharmaceuticals " 2001,32 (12) documents " HPLC of peoniflorin measures in the medicinal piece of shiquan dabu capsule ", but its solvent extraction yield of selecting for use is low; " content of paeoniflorin in the high effective liquid chromatography for measuring bolus of ten powerful tonics " assay method compares in " Chinese medicine Leader " 2002,21 (11) documents, and the present invention omits the upper prop step, and it is fast and simple to handle sample, accuracy height, favorable reproducibility; The present invention overcomes the deficiencies in the prior art, improve the quality control standard of SHIQUAN DABU GAO, set up assay index and detection method thereof in the preparation, increase the wherein thin layer chromatography qualitative identification method of Radix Codonopsis, the Radix Astragali, licorice medicinal materials, guaranteed this compound preparation higher quality standard level.

Summary of the invention

The objective of the invention is the SHIQUAN DABU GAO that adopts extraction process by water is formulated new method of quality control, utilize content of paeoniflorin in this medicine of high effective liquid chromatography for measuring, and investigate test through methodology, confirmation method is easy, the result is accurate, favorable reproducibility; Differentiate this medicine Chinese crude drug Radix Codonopsis, differentiate this medicine Chinese crude drug Radix Astragali, differentiate this medicine Chinese crude drug Radix Glycyrrhizae that through the methodology test, confirmation method is exclusive, feasible, negative noiseless with thin layer chromatography with thin layer chromatography with thin layer chromatography; Guarantee the accuracy and the advance of quality inspection standard, can control the quality of liuwei Dihuang soft extract effectively.

Treatment deficiency of both QI and blood disease medicament SHIQUAN DABU GAO is recorded in the 3rd (WS of " Drug Standard of Ministry of Public Health of the Peoples Republic of China " (Chinese traditional patent formulation preparation) ₃-B-0471-91) in, this pharmaceutical formulation is by Chinese crude drug Radix Codonopsis 80g, the Rhizoma Atractylodis Macrocephalae (stir-fry) 80g, Poria 80g, Radix Glycyrrhizae (processed with honey) 40g, Radix Angelicae Sinensis 120g, Rhizoma Chuanxiong 40g, the Radix Paeoniae Alba (wine stir-fry) 80g, Radix Rehmanniae Preparata 120g, the Radix Astragali (processed with honey) 80g, Cortex Cinnamomi 20g forms, main flavour of a drug in the Radix Codonopsis side of being, be campanulaceae plant Radix Codonopsis Codonopsis pilosula (Franch.) Nannf., the dry root of plain flower Radix Codonopsis Codonopsis pilosula Nannf.Var.modesta (Nannf.) L.T.Shen or radix codonpsis tangshen Codonopsis tangshen Oliv, sweet in the mouth is flat, has invigorating the spleen and replenishing QI, the effect of promoting the production of body fluid and nourishing blood mainly contains the I of tangshenoside, the II of tangshenoside, the III of tangshenoside, the IV of tangshenoside, chemical constituents such as ligustrin; The Radix Astragali is the dry root of leguminous plant Radix Astagali Astragalus membranaceus (Fisch.) Bge.var.mongholicus (Bge.) Hsiao or Radix Astragali Astragalus membranaceus (Fisch.) Bge., sweet in the mouth, tepor, has tonifying Qi and lifting yang, benefit is defended consolidating superficial resistance, promoting pus discharge and tissue regeneration strengthening, the effect of diuretic detumescent mainly contains the effective ingredient of flavonoid, Saponin class, polysaccharide; Radix Glycyrrhizae is the dry root and rhizome of leguminous plant Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L. Glycyrrhizaglabra L., sweet in the mouth, flat, has invigorating the spleen and replenishing QI, nourishing the lung to arrest cough, relieving spasm to stop pain, the effect of the mitigation property of medicine, mainly contain glycyrrhizin, flavone compound.

" method of quality control of 2000 editions SHIQUAN DABU GAO of recording of Chinese pharmacopoeia has the thin layer discrimination method of the Radix Paeoniae Alba, Radix Angelicae Sinensis, and a physics and chemistry and a microscopical identification are arranged, and physicochemical identification is because specificity is not strong, and the existing basic thin layer that adopts replaces physicochemical identification.Because of " technology of 2000 editions SHIQUAN DABU GAO of recording of Chinese pharmacopoeia is that water is carried, so microscopical identification is also inapplicable.The chemical constituent of Radix Angelicae Sinensis, Rhizoma Chuanxiong is basic identical, through experimental study, uses official method, and can only differentiate has Radix Angelicae Sinensis or Rhizoma Chuanxiong in the finished product, can't differentiate Radix Angelicae Sinensis separately; The medicinal piece of shiquan dabu mixture that tentative standard promulgated by the ministries or commissions of the Central Government is recorded has recorded the thin layer discrimination method of Radix Angelicae Sinensis and Rhizoma Chuanxiong, the Radix Paeoniae Alba, Radix Glycyrrhizae, the Radix Astragali.Through test, original discrimination method impurity is many, disturbs greatly the speckle inferior separating effect.Therefore the application provides the new thin layer discrimination method to wherein Radix Codonopsis, the Radix Astragali, Radix Glycyrrhizae, compares with original discrimination method, and specificity is strong, has avoided the interference between the speckle, and easy and simple to handle, sensitivity is strong; The application has further set up the content of paeoniflorin assay method." 2000 editions pills that record of Chinese pharmacopoeia, the medicinal piece of shiquan dabu dosage form that ministry standard is recorded does not all have the assay project except that the medicinal piece of shiquan dabu mixture is tentative standard.For improving the quality standard of SHIQUAN DABU GAO, be necessary to set the assay project, the quality of control and assurance SHIQUAN DABU GAO.Radix Paeoniae Alba cold nature, bitter in the mouth, acid.The pain relieving of tool suppressing the hyperactive liver, nourishing blood for regulating menstruation, the function of astringing YIN to stop sweating is main flavour of a drug in prescription.Because peoniflorin tool water solublity, in this preparation technology, more easily come out, so this standard application peoniflorin is quantitative target by water boiling and extraction, investigate.The medicinal piece of shiquan dabu mixture that tentative standard promulgated by the ministries or commissions of the Central Government is recorded selects for use ferulic acid as quantitative target, and owing to Radix Angelicae Sinensis, Rhizoma Chuanxiong two flavor medical materials all contain ferulic acid, specificity is not strong, so this standard application peoniflorin is a quantitative target, measures with high performance liquid chromatography.Content assaying method of the present invention compared with prior art, the solvent extraction yield height of selecting for use, extraction impurity is few, the flow phase system of selecting is to sample chromatogram peak good separating effect, the processing sample is fast and simple, accuracy height, favorable reproducibility, so select its index for use, can reflect and control the inherent quality of preparation better as control this product quality.

The present invention is by with content of paeoniflorin in this medicine of high effective liquid chromatography for measuring; Differentiate that with thin layer chromatography this medicine Chinese crude drug Radix Codonopsis, the Chinese crude drug Radix Astragali, Chinese crude drug Radix Glycyrrhizae effectively control the quality of medicinal piece of shiquan dabu cream drug.

The quality control detection method that SHIQUAN DABU GAO is new, this method can be undertaken by following determination step particularly: the main test instrument and equipment

ZF-type three is used ultraviolet analyzer: Shanghai Gu Cun electric light instrument plant

HH-4 digital display thermostat water bath: state China Electrical Appliances Co., Ltd

Hp1100 high performance liquid chromatograph: Agilent company

The Agilent100 chem workstation

SK-5200 ultrasonic cleaner: Shanghai High Kudos Science Instrument Co., Ltd.

The temperature digital display is regulated drying baker: Shanghai City instrument experiment head factory

Crude drug source

Radix Codonopsis, the Rhizoma Atractylodis Macrocephalae, Poria, Radix Glycyrrhizae Preparata, Radix Angelicae Sinensis, Rhizoma Chuanxiong, the Radix Paeoniae Alba, Radix Rehmanniae Preparata, Radix Astragali Preparata, Cortex Cinnamomi all derive from " 2000 editions one one of Chinese pharmacopoeia, all meet its standard code through check, medical material provides by letter Pharmaceutical Co., Ltd in the city of Haozhou, Anhui.

1, measures content of paeoniflorin with high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000)

The a chromatographic condition: with octadecylsilane chemically bonded silica is filler; Acetonitrile-0.1% phosphoric acid solution (10-30: 90-70) be mobile phase; The detection wavelength is 230 ± 2nm, flow velocity 0.5-1.0ml/min, and column temperature: 27-30 ℃, number of theoretical plate calculates by the peoniflorin peak should be not less than 2500.

B. the preparation of reference substance peoniflorin solution: it is an amount of to get the peoniflorin reference substance, accurate claims surely, adds in methanol, ethanol or the water of different solubility any and makes the solution that every 1ml contains 10-60 μ g, promptly.

C. the preparation of need testing solution: get this product 0.5-4g, the accurate title, decide, and puts in the tool plug conical flask, any 10-100ml in the methanol of the different solubility of accurate adding, ethanol, the water, claim to decide weight, supersound process 30 minutes is put cold, claim to decide weight again, methanol, ethanol or water with different solubility are supplied the weight that subtracts mistake, shake up, and filter, get subsequent filtrate with 0.45 μ m membrane filtration, promptly.

D. content of paeoniflorin assay method: precision is measured reference substance solution and each 5-20 μ l of need testing solution respectively, injects chromatograph of liquid, measures content of paeoniflorin; Every gram contains the Radix Paeoniae Alba in peoniflorin, must not be lower than 0.20mg.

2. differentiate this medicine Chinese crude drug Radix Codonopsis, Radix Glycyrrhizae, the Radix Astragali with thin layer chromatography

A. this medicine Chinese crude drug Radix Codonopsis is differentiated with thin layer chromatography

(1) preparation of control medicinal material solution: get Radix Codonopsis control medicinal material 0.25-2g, adding water 25-100ml decocted 30 minutes, Cotton Gossypii filters, filtrate adds hydrochloric acid 0.5-2ml and chloroform 10-100ml, and reflux 30 minutes is put cold, divide and get chloroform solution, evaporate to dryness, residue add ethyl acetate 0.5-1ml makes dissolving, in contrast medical material solution.

(2) preparation of need testing solution: get this product 5-60g, add water 10-100ml, hydrochloric acid 0.5-2ml and chloroform 10-60ml, reflux 15-60 minute, put coldly, divide and to get chloroform solution, evaporate to dryness, residue add ethyl acetate 0.5-1ml makes dissolving, as need testing solution.By this prescription, get and remove other outer each medical materials of Radix Codonopsis in addition, make the negative control solution that lacks Radix Codonopsis with method.

(3) according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000) test, draw above-mentioned two kinds of each 5-20ul of solution, put respectively in same be the silica gel G of adhesive with the sodium carboxymethyl cellulose _F254On the lamellae, (4: 8: 3: 0.2) be developing solvent, expansion was taken out, and dries with petroleum ether (60～90 ℃)-toluene-ethyl acetate-glacial acetic acid.Lamellae put under the ultra-violet lamp (254nm) inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the skin dark stain of same color.

B. this medicine Chinese crude drug Radix Astragali is differentiated with thin layer chromatography

(1) preparation of control medicinal material solution: get the astragaloside reference substance, add methanol and make the solution that every 1ml contains 0.1-1mg, in contrast product solution.

(2) preparation of need testing solution: get this product 20-50 gram, add water 10-50ml, with water saturated n-butanol extraction 4 times (40,30,30,20ml), combining extraction liquid is with 0.5mol/LNaHCO ₃Wash 3 times (10,10,5ml), reuse water 10ml washing 1 time discards washing liquid, butanol solution is put evaporate to dryness in the water-bath, and residue 5ml water dissolution, aqueous solution add to D101 type macroporous resin column (in the 1cm * 10cm), use the 50ml water elution, discard water liquid, reuse 100ml30% ethanol elution, discard eluent, continue to collect eluent, put evaporate to dryness in the water-bath with 70% ethanol 30ml eluting, residue adds methanol 0.5-1ml dissolving, as need testing solution.

(3) according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000) test, draw reference substance 5-20ul, need testing solution 10-30ul, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-butanone-formic acid-water (5: 3: 1: 1) be developing solvent, launch, take out, dry.Spray was dried by the fire about 10 minutes in 100 ℃ with 10% ethanol solution of sulfuric acid solution.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on show the speckle of same color.

C. this medicine Chinese crude drug Radix Glycyrrhizae is differentiated with thin layer chromatography

(1) preparation of control medicinal material solution: extracting liquorice control medicinal material 0.5-2g, adding water 25-100ml decocted 30 minutes, filter, filtrate adds hydrochloric acid 0.5-2ml and chloroform 10-60ml, reflux 15-60 minute, puts cold, divide and get chloroform solution, evaporate to dryness, residue add ethyl acetate 0.5-1ml makes dissolving, in contrast medical material solution.

(2) preparation of need testing solution: get this product 6-30g, add water 10-100ml, hydrochloric acid 0.5-2ml and chloroform 10-60ml, reflux 15-60 minute, put coldly, divide and to get chloroform solution, evaporate to dryness, residue add ethyl acetate 0.5-1ml makes dissolving, as need testing solution.

(3) according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000) test, draw need testing solution 5-40 μ l, control medicinal material solution 0.5-4 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with toluene-ethyl acetate-formic acid (15: 5: 1) is developing solvent, launch, take out, dry.Spray is with 20% ethanol solution of sulfuric acid, in about 10 minutes of 100 ℃ of bakings.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show two identical yellow spottings.

Optimized technical scheme of the present invention can be:

1. measure content of paeoniflorin in this medicine with high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000):

The a chromatographic condition: with octadecylsilane chemically bonded silica is filler; Acetonitrile-0.1% phosphoric acid solution (15: 85) is a mobile phase; The detection wavelength is 230nm.

The preparation of b reference substance solution: it is an amount of to get the peoniflorin reference substance, and accurate the title decides, and adds 50% ethanol and makes the solution that every 1ml contains 40 μ g, promptly.

The preparation of c need testing solution: get this product 2g, the accurate title, decide, and puts in the tool plug conical flask, and the accurate 50% ethanol 25ml that adds claims to decide weight, supersound process 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with 50% ethanol, shake up, filter, get subsequent filtrate with 0.45 μ m membrane filtration, promptly

D. content of paeoniflorin assay method: precision is measured reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, measures content of paeoniflorin;

E. every gram contains the Radix Paeoniae Alba in peoniflorin, must not be lower than 0.20mg.

2, this medicine Chinese crude drug Radix Codonopsis is differentiated with thin layer chromatography

A. the preparation of control medicinal material solution: get Radix Codonopsis control medicinal material 1g, add water 50ml and decocted 30 minutes, Cotton Gossypii filters, and filtrate adds hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, in contrast medical material solution.

.b. the preparation of need testing solution: get this product 10g, add water 15ml, hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, as need testing solution.

By this prescription, get and remove other outer each medical materials of Radix Codonopsis in addition, make the negative control solution that lacks Radix Codonopsis with method.

C. according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw above-mentioned two kinds of each 10ul of solution, put respectively in same be the silica gel G of adhesive with the sodium carboxymethyl cellulose _F254On the lamellae, (4: 8: 3: 0.2) be developing solvent, expansion was taken out, and dries with petroleum ether (60～90 ℃)-toluene-ethyl acetate-glacial acetic acid.Lamellae put under the ultra-violet lamp (254nm) inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the skin dark stain of same color.

3, this medicine Chinese crude drug Radix Astragali is differentiated with thin layer chromatography

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

B. the preparation of need testing solution: get this product 30 gram, add water 20ml, with water saturated n-butanol extraction 4 times (40,30,30,20ml), combining extraction liquid is with 0.5mol/LNaHCO ₃Wash 3 times (10,10,5ml), reuse water 10ml washing 1 time discards washing liquid, butanol solution is put evaporate to dryness in the water-bath, and residue 5ml water dissolution, aqueous solution add to D101 type macroporous resin column (in the 1cm * 10cm), use the 50ml water elution, discard water liquid, reuse 100ml 30% ethanol elution, discard eluent, continue to collect eluent, put evaporate to dryness in the water-bath with 70% ethanol 30ml eluting, residue adds methanol 1ml dissolving, as need testing solution.

C. test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw reference substance 5ul, need testing solution 15ul, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-butanone-formic acid-water (5: 3: 1: 1) be developing solvent, launch, take out, dry.Spray was dried by the fire about 10 minutes in 100 ℃ with 10% ethanol solution of sulfuric acid solution.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on show the speckle of same color.

4, this medicine Chinese crude drug Radix Glycyrrhizae is differentiated with thin layer chromatography

A. the preparation of control medicinal material solution: extracting liquorice control medicinal material 0.5g, add water 50ml and decocted 30 minutes, filter, filtrate adds hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, in contrast medical material solution.

B. the preparation of need testing solution: get this product 8g, add water 15ml, hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, as need testing solution.

C. test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000), draw need testing solution 20 μ l, control medicinal material solution 2 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with toluene-ethyl acetate-formic acid (15: 5: 1) is developing solvent, launch, take out, dry.Spray is with 20% ethanol solution of sulfuric acid, in about 10 minutes of 100 ℃ of bakings.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show two identical yellow spottings.

Through repeated trials repeatedly, confirmation method is easy, the result is accurate, can be used as the SHIQUAN DABU GAO quality control and investigates the index of the stability of technology.

Treatment temperature compensation QI and blood of the present invention, being used for deficiency of both QI and blood causes: pale complexion, the cardiopalmus of breathing hard, fatigue and asthenia, the preparation method of the SHIQUAN DABU GAO that extremity are not warm is according to the 3rd (WS of " Drug Standard of Ministry of Public Health of the Peoples Republic of China " (Chinese traditional patent formulation preparation) ₃-preparation method in B-0471-91) is made; The Fang Zhongshi medical material of distinguishing the flavor of, decoct with water three times, first and second time each 2 hours, 1 hour for the third time, add six times of water gagings for the first time, second and third time adds quintuple water, collecting decoction, filter, filtrate was left standstill 24 hours, got supernatant and was evaporated to the clear paste that relative density is 1.28～1.32 (85～90 ℃).Every 100g clear paste adds refined honey 700g, and mixing is heated to and boils, and filters, promptly; Instructions of taking: a 10～15g, 2 times on the one.

The TCL that the invention provides Chinese crude drug Radix Codonopsis, Radix Glycyrrhizae, the Radix Astragali differentiates, and is feasible, negative noiseless through methodology test confirmation method; Having selected peoniflorin is testing index, and has carried out following methodological study test, has set up the HPLC content assaying method of SHIQUAN DABU GAO; Through repeated trials repeatedly, confirmation method is easy, the result is accurate, and favorable reproducibility can be used as the SHIQUAN DABU GAO quality control and investigates the index of the stability of technology.

Methodological study

(1) investigation of extraction solvent: what extracting method was commonly used is reflux, extract, and supersound extraction, because this product white Peony Root is made soft extract through water boiling and extraction, only be the process of from soft extract, disperseing, dissolving peoniflorin when therefore preparing need testing solution, so adopt ultrasonic extraction.Peoniflorin is a water soluble ingredient, " that adopts in relevant paeoniflorin content of Chinese pharmacopoeia version in 2000 is dissolved as methanol, ethanol, for this reason, investigate the extraction situation of 50% ethanol, methanol, ethanol, 50% methanol; get this product 2g; accurately claim surely, added 50% ethanol, methanol and extract solvent and investigate, the results are shown in Table 1.50% ethanol extraction yield is higher as can be seen from the results, and it is less to extract impurity, so select 50% ethanol as extracting solvent, sees Table 1.

Table 1 extracts the investigation of solvent

Extract solvent peak area/g

Methanol 195.80

Ethanol 80.06

50% methanol 224.30

50% ethanol 237.70

(2) investigation of supersound extraction time: get this product 2g, the accurate title, decide, and adds 50% ethanol and carry out the investigation of supersound extraction time, the results are shown in Table 2.From the result, extraction time does not have obvious influence to the content of paeoniflorin result, determines can extract in ultrasonic 30 minutes fully, sees Table 2.

The investigation of table 2 supersound extraction time

Extraction time (min) peak area/g

15 224.54

30 238.19

45 235.09

60 232.96

(3) investigation of reference substance linear relationship: getting concentration is 0.0104mg/ml, 0.0208mg/ml, 0.0416mg/ml, 0.0624mg/ml, 0.0832mg/ml, 0.1040mg/ml reference substance solution, difference sample introduction 10 μ l, press the text chromatographic condition and measure peak area, the results are shown in Table 3.With the peak area is vertical coordinate, and the reference substance amount is an abscissa, and the drawing standard curve calculates regression equation: y=1436.76x-0.03631, R=0.9998

The investigation of table 3 linear relationship

The reference substance amount

0.104 0.208 0.416 0.624 0.832 1.040

(μg)

Area 1 155.71 293.61 591.09 911.65 1180.87 1503.14

Peak area 2 155.52 292.94 592.44 909.96 1178.66 1499.19

Average peak

155.62 293.28 591.76 910.80 1179.76 1501.16

Area

The result shows: the peoniflorin reference substance solution has good linear relationship in 0.104 μ g～1.04 μ g scopes, and passes through initial point.

(5) precision test: get the peoniflorin reference substance solution of concentration 0.0624mg/ml, sample introduction 10ul presses liquid phase chromatogram condition in the text, repeats sample introduction 5 times, and measurement result sees Table 4.

Table 4 Precision test result

Numbering peak area RSD

1 906.71

2 903.36

3 908.65 0.37％

4 908.28

5 91?2.73

The result shows: this method precision is better.

Table 5 reproducible test results

The paeoniflorin content average content

RSD

Sequence number

(mg/g) (mg/g)

1 0.362

2 0.367

3 0.364 0.364 0.57％

4 0.363

5 0.366

(6) stability test: (lot number: 021023), the accurate 10 μ l that draw by above-mentioned chromatographic condition, measured peoniflorin peak area value in the need testing solution in 6 hours, the results are shown in Table 6 to get need testing solution.Illustrate that test sample is good at 6 hours internal stabilities, sees Table 6.

Table 6 stability test result

Time (hour) peak area RSD (%)

0 519.36

2 531.03

1.40％

3 515.44

6 528.19

(7) recovery test: this product (lot number: 021023) 5 parts of known content decided in accurate title, every part of about 1g, the accurate respectively peoniflorin reference substance that adds, adopt the application of sample recovery test, press chromatographic condition mensuration in the text, calculate recovery rate, RSD are 1.76%, show that this method response rate is better, the results are shown in Table 7.

Table 7 recovery test result

Sample contains and adds Chinese herbaceous peony and measure Chinese herbaceous peony and on average return

On average

Response rate RSD

Sequence number amount medicine glycosides amount medicine glycosides amount yield

Peak area

(％)

％

(mg) (mg) (mg) (％)

1 0.4932 0.416 511.97 0.9014 98.12

2 0.4592 0.416 493.82 0.8695 98.63

1.76

3 0.4400 0.416 479.64 0.8445 97.23 99.21％

％

4 0.4379 0.416 488.01 0.8592 101.27

5 0.4362 0.416 485.885 0.8555 100.79

(8) mensuration of sample: get 8 batches of this product, measure, record and the results are shown in Table 8 according to chromatographic condition in the text

Table 8 sample determination result

White Peony Root contains

The finished product peoniflorin contains meansigma methods

Sequence number lot number white Peony Root in batches

Number (mg/g) amount (mg/g) (mg/g)

1 021105 020706 23.98 0.384 0.384

0.384

2 021110 23.98 0.383

020706 0.382

0.397

3 021115 020706 23.98 0.395

0.393

0.369

4 021023 020706 23.98 0.369

0.369

0.379

5 021028 020706 23.98 0.376

0.374

0.388

6 021101 020706 23.98 0.386

0.384

0.266

7 021024 020711 16.40 0.264

0.263

0.257

8 021029 020711 16.40 0.257

0.256

According to above-mentioned result of the test, consider source, the factors such as processing processs of preparing Chinese medicine, storage of white Peony Root, fix tentatively the every gram of this product and contain peoniflorin and must not be lower than 0.20mg.

The specific embodiment

Embodiment 1:

Get Chinese crude drug Radix Codonopsis 80g, the Rhizoma Atractylodis Macrocephalae (stir-fry) 80g, Poria 80g, Radix Glycyrrhizae (processed with honey) 40g, Radix Angelicae Sinensis 120g, Rhizoma Chuanxiong 40g, the Radix Paeoniae Alba (wine stir-fry) 80g, Radix Rehmanniae Preparata 120g, the Radix Astragali (processed with honey) 80g, Cortex Cinnamomi 20g, above-mentioned raw materials decocts with water three times, about six times of water gagings for the first time, second and third time quintuple water, first and second time each 2 hours, 1 hour for the third time, collecting decoction, filter, filtrate was left standstill 24 hours, got supernatant and was evaporated to the clear paste that relative density is 1.28～1.32 (85-90 ℃).Every 100g clear paste adds refined honey 700g, and mixing is heated to and boils, and filters, promptly.Every bag of cream heavily is 10g.

1, measure content of paeoniflorin in this medicine with high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D):

The a chromatographic condition: with octadecylsilane chemically bonded silica is filler; Acetonitrile-0.1% phosphoric acid solution (15: 85) is a mobile phase; The detection wavelength is 230nm, flow velocity 1.0ml/min, column temperature: 27 ℃.

D content of paeoniflorin assay method: precision is measured reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, measures content of paeoniflorin;

The every gram of e contains the Radix Paeoniae Alba in peoniflorin, must not be lower than 0.20mg.

B. the preparation of need testing solution: get this product 10g, add water 15ml, hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, as need testing solution.By this prescription, get and remove other outer each medical materials of Radix Codonopsis in addition, make the negative control solution that lacks Radix Codonopsis with method.

C. according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000) test, draw above-mentioned two kinds of each 10ul of solution, put respectively in same be the silica gel G of adhesive with the sodium carboxymethyl cellulose _F254On the lamellae, (4: 8: 3: 0.2) be developing solvent, expansion was taken out, and dries with petroleum ether (60～90 ℃)-toluene-ethyl acetate-glacial acetic acid.Lamellae put under the ultra-violet lamp (254nm) inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the skin dark stain of same color.

C. test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw reference substance 5ul, need testing solution 15ul, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-butanone-formic acid-water (5: 3: 1: 1) be developing solvent, launch, take out, dry.Spray was dried by the fire about 10 minutes in 100 ℃ with 10% ethanol solution of sulfuric acid solution.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on show the speckle of same color.

Embodiment 2:

The unguentum preparation method is with embodiment 1, and the method for quality control of this unguentum is as follows:

1 usefulness high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D) is measured content of paeoniflorin in this medicine:

The a chromatographic condition: with octadecylsilane chemically bonded silica is filler; Acetonitrile-0.1% phosphoric acid solution (30: 70) is a mobile phase; The detection wavelength is 230nm, flow velocity 0.5ml/min, 30 ℃ of column temperatures.

The preparation of b reference substance solution: it is an amount of to get the peoniflorin reference substance, and accurate the title decides, and adds 50% ethanol and makes the solution that every 1ml contains 10 μ g, promptly.

The preparation of c need testing solution: get this product 0.5g, the accurate title, decide, and puts in the tool plug conical flask, the accurate 50% ethanol 25ml that adds, claim to decide weight, supersound process 30 minutes is put cold, claim to decide weight again, supply the weight that subtracts mistake with 50% ethanol, shake up, filter, get subsequent filtrate with 0.45 μ m membrane filtration, promptly

D. content of paeoniflorin assay method: precision is measured reference substance solution and each 20 μ l of need testing solution respectively, injects chromatograph of liquid, measures content of paeoniflorin;

A. the preparation of control medicinal material solution: get Radix Codonopsis control medicinal material 0.25g, add water 50ml and decocted 30 minutes, Cotton Gossypii filters, and filtrate adds hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, in contrast medical material solution.

B. the preparation of need testing solution: get this product 5g, add water 15ml, hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, as need testing solution.By this prescription, get and remove other outer each medical materials of Radix Codonopsis in addition, make the negative control solution that lacks Radix Codonopsis with method.

A. the preparation of control medicinal material solution: get the astragaloside reference substance, add methanol and make the solution that every 1ml contains 0.2mg, in contrast product solution.

B. the preparation of need testing solution: get this product 20 gram, add water 20ml, with water saturated n-butanol extraction 4 times (40,30,30,20ml), combining extraction liquid is with 0.5mol/LNaHCO ₃Wash 3 times (10,10,5ml), reuse water 10ml washing 1 time discards washing liquid, butanol solution is put evaporate to dryness in the water-bath, and residue 5ml water dissolution, aqueous solution add to D101 type macroporous resin column (in the 1cm * 10cm), use the 50ml water elution, discard water liquid, reuse 100ml 30% ethanol elution, discard eluent, continue to collect eluent, put evaporate to dryness in the water-bath with 70% ethanol 30ml eluting, residue adds methanol 1ml dissolving, as need testing solution.

With the index of said method as the stability of this drug quality control and investigation technology.

Embodiment 3:

Get Chinese crude drug Radix Codonopsis 80g, the Rhizoma Atractylodis Macrocephalae (stir-fry) 80g, Poria 80g, Radix Glycyrrhizae (processed with honey) 40g, Radix Angelicae Sinensis 120g, Rhizoma Chuanxiong 40g, the Radix Paeoniae Alba (wine stir-fry) 80g, Radix Rehmanniae Preparata 120g, the Radix Astragali (processed with honey) 80g, Cortex Cinnamomi 20g, above-mentioned raw materials decocts with water three times, about six times of water gagings for the first time, second and third time quintuple water, first and second time each 2 hours, 1 hour for the third time, collecting decoction, filter, filtrate was left standstill 24 hours, got supernatant and was evaporated to the clear paste that relative density is 1.28～1.32 (85-90 ℃).

A. chromatographic condition: with octadecylsilane chemically bonded silica is filler; Acetonitrile-0.1% phosphoric acid solution (10: 90) is a mobile phase; The detection wavelength is 230nm, flow velocity 0.5ml/min, and column temperature: 25 ℃, number of theoretical plate calculates by the peoniflorin peak should be not less than 2500.

B. the preparation of reference substance peoniflorin solution: it is an amount of to get the peoniflorin reference substance, and accurate the title decides, and adds 60% ethanol and makes the solution that every 1ml contains 60 μ g, promptly.

C. the preparation of need testing solution: get this product 4g, the accurate title, decide, and puts in the tool plug conical flask, and the accurate 60% ethanol 10ml that adds claims to decide weight, supersound process 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with 60% ethanol, shake up, filter, get subsequent filtrate with 0.45 μ m membrane filtration, promptly

D. content of paeoniflorin assay method: precision is measured reference substance solution and each 5 μ l of need testing solution respectively, injects chromatograph of liquid, measures content of paeoniflorin; Every gram contains the Radix Paeoniae Alba in peoniflorin, must not be lower than 0.20mg.

(1) preparation of control medicinal material solution: get Radix Codonopsis control medicinal material 2g, add water 100ml and decocted 30 minutes, Cotton Gossypii filters, and filtrate adds hydrochloric acid 2ml and chloroform 100ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, in contrast medical material solution.

(2) preparation of need testing solution: get this product 60g, add water 100ml, hydrochloric acid 02ml and chloroform 60ml, reflux 60 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, as need testing solution.By this prescription, get and remove other outer each medical materials of Radix Codonopsis in addition, make the negative control solution that lacks Radix Codonopsis with method.

(3) according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000) test, draw above-mentioned two kinds of each 5ul of solution, put respectively in same be the silica gel G of adhesive with the sodium carboxymethyl cellulose _F254On the lamellae, (4: 8: 3: 0.2) be developing solvent, expansion was taken out, and dries with petroleum ether (60～90 ℃)-toluene-ethyl acetate-glacial acetic acid.Lamellae put under the ultra-violet lamp (254nm) inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the skin dark stain of same color.

2. this medicine Chinese crude drug Radix Astragali is differentiated with thin layer chromatography

(1) preparation of control medicinal material solution: get the astragaloside reference substance, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution.

(2) preparation of need testing solution: get this product 50 gram, add water 50ml, with water saturated n-butanol extraction 4 times (40,30,30,20ml), combining extraction liquid is with 0.5mol/LNaHCO ₃Wash 3 times (10,10,5ml), reuse water 10ml washing 1 time discards washing liquid, butanol solution is put evaporate to dryness in the water-bath, and residue 5ml water dissolution, aqueous solution add to D101 type macroporous resin column (in the 1cm * 10cm), use the 50ml water elution, discard water liquid, reuse 100ml 30% ethanol elution, discard eluent, continue to collect eluent, put evaporate to dryness in the water-bath with 70% ethanol 30ml eluting, residue adds methanol 1ml dissolving, as need testing solution.

(3) according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw reference substance 5ul, need testing solution 10ul, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-butanone-formic acid-water (5: 3: 1: 1) be developing solvent, launch, take out, dry.Spray was dried by the fire about 10 minutes in 100 ℃ with 10% ethanol solution of sulfuric acid solution.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on show the speckle of same color.，

3. this medicine Chinese crude drug Radix Glycyrrhizae is differentiated with thin layer chromatography

(1) preparation of control medicinal material solution: extracting liquorice control medicinal material 2g, add water 100ml and decocted 30 minutes, filter, filtrate adds hydrochloric acid 2ml and chloroform 60ml, reflux 60 minutes is put coldly, divides and to get chloroform solution, evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, in contrast medical material solution.

(2) preparation of need testing solution: get this product 30g, add water 100ml, hydrochloric acid 2ml and chloroform 60ml, reflux 60 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, as need testing solution.

(3) according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw need testing solution 5 μ l, control medicinal material solution 0.5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with toluene-ethyl acetate-formic acid (15: 5: 1) is developing solvent, launch, take out, dry.Spray is with 20% ethanol solution of sulfuric acid, in about 10 minutes of 100 ℃ of bakings.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show two identical yellow spottings.

Embodiment 4: get Chinese crude drug Radix Codonopsis 80g, the Rhizoma Atractylodis Macrocephalae (stir-fry) 80g, Poria 80g, Radix Glycyrrhizae (processed with honey) 40g, Radix Angelicae Sinensis 120g, Rhizoma Chuanxiong 40g, the Radix Paeoniae Alba (wine stir-fry) 80g, Radix Rehmanniae Preparata 120g, the Radix Astragali (processed with honey) 80g, Cortex Cinnamomi 20g, above-mentioned raw materials decocts with water three times, about six times of water gagings for the first time, second and third time quintuple water, first and second time each 2 hours, 1 hour for the third time, collecting decoction, filter, filtrate was left standstill 24 hours, got supernatant and was evaporated to the clear paste that relative density is 1.28～1.32 (85-90 ℃).Every 100g clear paste adds refined honey 700g, and mixing is heated to and boils, and filters, promptly.Every bag of cream heavily is 10g.

A. chromatographic condition: with octadecylsilane chemically bonded silica is filler; Acetonitrile-0.1% phosphoric acid solution (12: 88) is a mobile phase; The detection wavelength is 230nm.Flow velocity 1.0ml/min, 28 ℃ of column temperatures, number of theoretical plate calculate by the peoniflorin peak should be not less than 2500.

The preparation of b reference substance solution: it is an amount of to get the peoniflorin reference substance, and accurate the title decides, and adds 50% ethanol and makes the solution that every 1ml contains 30 μ g, promptly.

C. the preparation of need testing solution: get this product 1g, the accurate title, decide, and puts in the tool plug conical flask, and the accurate 50% ethanol 25ml that adds claims to decide weight, supersound process 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with 50% ethanol, shake up, filter, get subsequent filtrate with 0.45 μ m membrane filtration, promptly

A. the preparation of control medicinal material solution: get Radix Codonopsis control medicinal material 2g, add water 50ml and decocted 30 minutes, Cotton Gossypii filters, and filtrate adds hydrochloric acid 1ml and chloroform 40ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, evaporate to dryness, residue add ethyl acetate 2ml makes dissolving, in contrast medical material solution.

B. the preparation of need testing solution: get this product 20g, add water 30ml, hydrochloric acid 2ml and chloroform 40ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 2ml makes dissolving, as need testing solution.By this prescription, get and remove other outer each medical materials of Radix Codonopsis in addition, make the negative control solution that lacks Radix Codonopsis with method.

B. the preparation of need testing solution: get this product 40 gram, add water 20ml, with water saturated n-butanol extraction 4 times (40,30,30,20ml), combining extraction liquid is with 0.5mol/LNaHCO ₃Wash 3 times (10,10,5ml), reuse water 10ml washing 1 time discards washing liquid, butanol solution is put evaporate to dryness in the water-bath, and residue 5ml water dissolution, aqueous solution add to D101 type macroporous resin column (in the 1cm * 10cm), use the 50ml water elution, discard water liquid, reuse 100ml 30% ethanol elution, discard eluent, continue to collect eluent, put evaporate to dryness in the water-bath with 70% ethanol 30ml eluting, residue adds methanol 1ml dissolving, as need testing solution.

B. the preparation of need testing solution: get this product 6g, add water 15ml, hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, as need testing solution.

C. test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw need testing solution 20 μ l, control medicinal material solution 2 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with toluene-ethyl acetate-formic acid (15: 5: 1) is developing solvent, launch, take out, dry.Spray is with 20% ethanol solution of sulfuric acid, in about 10 minutes of 100 ℃ of bakings.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show two identical yellow spottings.

Embodiment 5:

A. chromatographic condition: with octadecylsilane chemically bonded silica is filler; Acetonitrile-0.1% phosphoric acid solution (20: 80) is a mobile phase; The detection wavelength is 230nm.Flow velocity 1.0ml/min, 29 ℃ of column temperatures, number of theoretical plate calculate by the peoniflorin peak should be not less than 2500.

The preparation of b reference substance solution: it is an amount of to get the peoniflorin reference substance, and accurate the title decides, and adds 50% methanol and makes the solution that every 1ml contains 40 μ g, promptly.

C. the preparation of need testing solution: get this product 2g, the accurate title, decide, and puts in the tool plug conical flask, the accurate 50% methanol 25ml that adds claims to decide weight, supersound process 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 50% methanol, shake up, filter, get subsequent filtrate with 0.45 μ m membrane filtration, promptly get d. content of paeoniflorin assay method: precision is measured respectively 10 μ l of reference substance solution and need testing solution respectively, inject chromatograph of liquid, measure content of paeoniflorin;

Embodiment 6: get Chinese crude drug Radix Codonopsis 80g, the Rhizoma Atractylodis Macrocephalae (stir-fry) 80g, Poria 80g, Radix Glycyrrhizae (processed with honey) 40g, Radix Angelicae Sinensis 120g, Rhizoma Chuanxiong 40g, the Radix Paeoniae Alba (wine stir-fry) 80g, Radix Rehmanniae Preparata 120g, the Radix Astragali (processed with honey) 80g, Cortex Cinnamomi 20g, above-mentioned raw materials decocts with water three times, about six times of water gagings for the first time, second and third time quintuple water, first and second time each 2 hours, 1 hour for the third time, collecting decoction, filter, filtrate was left standstill 24 hours, got supernatant and was evaporated to the clear paste that relative density is 1.28～1.32 (85-90 ℃).Every 100g clear paste adds refined honey 700g, and mixing is heated to and boils, and filters, promptly.Every bag of cream heavily is 10g.

A. chromatographic condition: with octadecylsilane chemically bonded silica is filler; Acetonitrile-0.1% phosphoric acid solution (14: 86) is a mobile phase; The detection wavelength is 230nm.Flow velocity 1.0ml/min, 27 ℃ of column temperatures, number of theoretical plate calculate by the peoniflorin peak should be not less than 2500.

The preparation of b reference substance solution: it is an amount of to get the peoniflorin reference substance, and accurate the title decides, and adds methanol and makes the solution that every 1ml contains 20 μ g, promptly.

C. the preparation of need testing solution: get this product 2g, the accurate title, decide, and puts in the tool plug conical flask, and the accurate methanol 25ml that adds claims to decide weight, supersound process 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate with 0.45 μ m membrane filtration, promptly

C. test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) formula, draw reference substance 5ul, need testing solution 15ul, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-butanone-formic acid-water (5: 3: 1: 1) be developing solvent, launch, take out, dry.Spray was dried by the fire about 10 minutes in 100 ℃ with 10% ethanol solution of sulfuric acid solution.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on show the speckle of same color.

Embodiment 7: get Chinese crude drug Radix Codonopsis 80g, the Rhizoma Atractylodis Macrocephalae (stir-fry) 80g, Poria 80g, Radix Glycyrrhizae (processed with honey) 40g, Radix Angelicae Sinensis 120g, Rhizoma Chuanxiong 40g, the Radix Paeoniae Alba (wine stir-fry) 80g, Radix Rehmanniae Preparata 120g, the Radix Astragali (processed with honey) 80g, Cortex Cinnamomi 20g, above-mentioned raw materials decocts with water three times, about six times of water gagings for the first time, second and third time quintuple water, first and second time each 2 hours, 1 hour for the third time, collecting decoction, filter, filtrate was left standstill 24 hours, got supernatant and was evaporated to the clear paste that relative density is 1.28～1.32 (85-90 ℃).Every 100g clear paste adds refined honey 700g, and mixing is heated to and boils, and filters, promptly.Every bag of cream heavily is 10g.

1 usefulness high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D) is measured content of paeoniflorin in this medicine: a. chromatographic condition: with octadecylsilane chemically bonded silica is filler; Acetonitrile-0.1% phosphoric acid solution (13: 87) is a mobile phase; The detection wavelength is 230nm.Number of theoretical plate calculates by the peoniflorin peak should be not less than 2500.

C. the preparation of need testing solution: get this product 2g, the accurate title, decide, and puts in the tool plug conical flask, the accurate 50% ethanol 25ml that adds claims to decide weight, supersound process 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 50% ethanol, shake up, filter, get subsequent filtrate with 0.45 μ m membrane filtration, promptly get d. content of paeoniflorin assay method: precision is measured respectively 10 μ l of reference substance solution and need testing solution respectively, inject chromatograph of liquid, measure content of paeoniflorin;

B. the preparation of need testing solution: get this product 10g, add water 15ml, hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, as need testing solution.

Embodiment 8: get Chinese crude drug Radix Codonopsis 80g, the Rhizoma Atractylodis Macrocephalae (stir-fry) 80g, Poria 80g, Radix Glycyrrhizae (processed with honey) 40g, Radix Angelicae Sinensis 120g, Rhizoma Chuanxiong 40g, the Radix Paeoniae Alba (wine stir-fry) 80g, Radix Rehmanniae Preparata 120g, the Radix Astragali (processed with honey) 80g, Cortex Cinnamomi 20g, above-mentioned raw materials decocts with water three times, about six times of water gagings for the first time, second and third time quintuple water, first and second time each 2 hours, 1 hour for the third time, collecting decoction, filter, filtrate was left standstill 24 hours, got supernatant and was evaporated to the clear paste that relative density is 1.28～1.32 (85-90 ℃).Every 100g clear paste adds refined honey 700g, and mixing is heated to and boils, and filters, promptly.Every bag of cream heavily is 10g, and every bottle of cream heavily is 200g.

A. chromatographic condition: with octadecylsilane chemically bonded silica is filler; Acetonitrile-0.1% phosphoric acid solution (16: 84) is a mobile phase; The detection wavelength is 230nm.Number of theoretical plate calculates by the peoniflorin peak should be not less than 2500.

C. the preparation of need testing solution: get this product 2g, the accurate title, decide, and puts in the tool plug conical flask, and the accurate 50% ethanol 25ml that adds claims to decide weight, supersound process 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with 50% ethanol, shake up, filter, get subsequent filtrate with 0.45 μ m membrane filtration, promptly

B. the preparation of need testing solution: get this product 20g, add water 15ml, hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, as need testing solution.By this prescription, get and remove other outer each medical materials of Radix Codonopsis in addition, make the negative control solution that lacks Radix Codonopsis with method.

C. according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw above-mentioned two kinds of each 5ul of solution, put respectively in same be the silica gel G of adhesive with the sodium carboxymethyl cellulose _F254On the lamellae, (4: 8: 3: 0.2) be developing solvent, expansion was taken out, and dries with petroleum ether (60～90 ℃)-toluene-ethyl acetate-glacial acetic acid.Lamellae put under the ultra-violet lamp (254nm) inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the skin dark stain of same color.

B. the preparation of need testing solution: get this product 20g, add water 15ml, hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving, as need testing solution.

C. test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw need testing solution 10 μ l, control medicinal material solution 2 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with toluene-ethyl acetate-formic acid (15: 5: 1) is developing solvent, launch, take out, dry.Spray is with 20% ethanol solution of sulfuric acid, in about 10 minutes of 100 ℃ of bakings.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show two identical yellow spottings.

Claims

1. the method for quality control of a SHIQUAN DABU GAO of being made by Chinese crude drug Radix Codonopsis 80g, the Rhizoma Atractylodis Macrocephalae (stir-fry) 80g, Poria 80g, Radix Glycyrrhizae (processed with honey) 40g, Radix Angelicae Sinensis 120g, Rhizoma Chuanxiong 40g, the Radix Paeoniae Alba (wine stir-fry) 80g, Radix Rehmanniae Preparata 120g, the Radix Astragali (processed with honey) 80g, Cortex Cinnamomi 20g is characterized in that this method comprises:

(1) with content of paeoniflorin in this medicine of high effective liquid chromatography for measuring;

(2) differentiate this medicine Chinese crude drug Radix Codonopsis with thin layer chromatography;

(3) differentiate this medicine Chinese crude drug Radix Astragali with thin layer chromatography;

(4) differentiate this medicine Chinese crude drug Radix Glycyrrhizae with thin layer chromatography.

2. according to the method for quality control of the SHIQUAN DABU GAO of claim 1, it is characterized in that this method comprises the following steps:

(1) uses the high effective liquid chromatography for measuring content of paeoniflorin

The a chromatographic condition: with octadecylsilane chemically bonded silica is filler, and (10-30: 90-70) be mobile phase, the detection wavelength is 230 ± 2nm to acetonitrile-0.1% phosphoric acid solution, flow velocity 0.5-1.0ml/min, column temperature: 27-30 ℃;

B. the preparation of reference substance peoniflorin solution: it is an amount of to get the peoniflorin reference substance, adds in methanol, ethanol or the water of different solubility any and makes the solution that every 1ml contains 10-60 μ g;

C. the preparation of need testing solution: get this product 0.5-4g, any 10-100ml in methanol, ethanol or the water of the different solubility of accurate adding, claim to decide weight, supersound process 30 minutes is put coldly, claims to decide weight again, methanol, ethanol or water with different solubility are supplied the weight that subtracts mistake, shake up, filter, get subsequent filtrate with 0.45 μ m membrane filtration;

D. content of paeoniflorin assay method: precision is measured reference substance solution and each 5-20 μ l of need testing solution respectively, injects chromatograph of liquid;

(2) differentiate this medicine Chinese crude drug Radix Codonopsis with thin layer chromatography

A. the preparation of control medicinal material solution: get Radix Codonopsis control medicinal material 0.25-2g, add water 25-100ml and decocted 30 minutes, filter, filtrate adds hydrochloric acid 0.5-2ml and chloroform 10-60ml, reflux 15-60 minute, puts cold, divide and get chloroform solution, evaporate to dryness, residue add ethyl acetate 0.5-1ml makes dissolving;

B. the preparation of need testing solution: get this product 5-60g, add water 10-100ml, hydrochloric acid 0.5-2ml and chloroform 10-60ml, reflux 15-60 minute, put coldly, divide and to get chloroform solution, evaporate to dryness, residue add ethyl acetate 0.5-1ml makes dissolving;

C. according to thin layer chromatography test, draw above-mentioned two kinds of each 5-20ul of solution, put respectively in same be on the silica GF254 lamellae of adhesive with the sodium carboxymethyl cellulose, with petroleum ether-toluene-ethyl acetate-glacial acetic acid is developing solvent, launches, and takes out, dry, lamellae is put under the ultra-violet lamp inspected;

(2), differentiate this medicine Chinese crude drug Radix Astragali with thin layer chromatography

A. the preparation of control medicinal material solution: get the astragaloside reference substance, add methanol and make the solution that every 1ml contains 0.1-1mg;

B. the preparation of need testing solution: get this product 10-100 gram, add water 10-50ml, use water saturated n-butanol extraction, combining extraction liquid is with NaHCO ₃Washing washes with water again, discards washing liquid, butanol solution is put evaporate to dryness in the water-bath, and residue water dissolution, aqueous solution add in the macroporous resin column, the water eluting, discard water liquid, the reuse ethanol elution discards eluent, continue to use ethanol elution, collect eluent, put evaporate to dryness in the water-bath, residue adds methanol 0.5-1ml dissolving;

C. according to the thin layer chromatography test, draw reference substance 5-20ul, need testing solution 10-30ul, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with ethyl acetate-butanone-formic acid-water, launch, take out, dry, spray is with ethanol solution of sulfuric acid;

(3), differentiate this medicine Chinese crude drug Radix Glycyrrhizae with thin layer chromatography

A. the preparation of control medicinal material solution: extracting liquorice control medicinal material 0.5-2g, add water 25-100ml and decoct, filter, filtrate adds hydrochloric acid 0.5-2ml and chloroform 10-60ml, reflux 15-60 minute, puts cold, divide and get chloroform solution, evaporate to dryness, residue add ethyl acetate 0.5-1ml makes dissolving;

B. the preparation of need testing solution: get this product 6-30g, add water 10-100ml, hydrochloric acid 0.5-2ml and chloroform 10-60ml, reflux 15-60 minute, put coldly, divide and to get chloroform solution, evaporate to dryness, residue add ethyl acetate 0.5-1ml makes dissolving;

D. according to the thin layer chromatography test, draw need testing solution 5-40 μ l, control medicinal material solution 0.5-4 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with toluene-ethyl acetate-formic acid, launch, take out, dry, spray is with ethanol solution of sulfuric acid;

3. method according to claim 2 is characterized in that:

(1), with content of paeoniflorin in this medicine of high effective liquid chromatography for measuring:

A. chromatographic condition: with octadecylsilane chemically bonded silica is filler; Acetonitrile-0.1% phosphoric acid solution (15: 85) is a mobile phase; The detection wavelength is 230nm.

B. the preparation of reference substance solution: it is an amount of to get the peoniflorin reference substance, and accurate the title decides, and adds 50% ethanol and makes the solution that every 1ml contains 40 μ g;

C. the preparation of need testing solution: get this product 2g, the accurate title, decide, and puts in the tool plug conical flask, the accurate 50% ethanol 25ml that adds, claim decide weight, supersound process 30 minutes is put coldly, and weight decided in title again, supply the weight that subtracts mistake with 50% ethanol, shake up, filter, get subsequent filtrate with 0.45 μ m membrane filtration;

D. content of paeoniflorin assay method: precision is measured reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid;

(2), this medicine Chinese crude drug Radix Codonopsis is differentiated with thin layer chromatography

A. the preparation of control medicinal material solution: get Radix Codonopsis control medicinal material 1g, add water 50ml and decocted 30 minutes, Cotton Gossypii filters, and filtrate adds hydrochloric acid 1ml and chloroform 20ml, and reflux 30 minutes is put coldly, divides and gets chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving;

B. the preparation of need testing solution: get this product 10g, add water 15ml, hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving;

C. according to thin layer chromatography test, draw above-mentioned two kinds of each 10ul of solution, put respectively in same be the silica gel G of adhesive with the sodium carboxymethyl cellulose _F254On the lamellae, (4: 8: 3: 0.2) be developing solvent, expansion was taken out, and dries with petroleum ether (60～90 ℃)-toluene-ethyl acetate-glacial acetic acid.Lamellae put under the ultra-violet lamp (254nm) inspect;

(3), this medicine Chinese crude drug Radix Astragali is differentiated with thin layer chromatography

A. the preparation of control medicinal material solution: get the astragaloside reference substance, add methanol and make the solution that every 1ml contains 1mg;

B. the preparation of need testing solution: get this product 30 gram, add water 20ml, with water saturated n-butanol extraction 4 times (40,30,30,20ml), combining extraction liquid is with 0.5mol/LNaHCO ₃Wash 3 times (10,10,5ml), reuse water 10ml washing 1 time discards washing liquid, butanol solution is put evaporate to dryness in the water-bath, and residue 5ml water dissolution, aqueous solution add to D101 type macroporous resin column (in the 1cm * 10cm), use the 50ml water elution, discard water liquid, reuse 100ml30% ethanol elution discards eluent, continue with 70% ethanol 30ml eluting, collect eluent, put evaporate to dryness in the water-bath, residue adds methanol 1ml dissolving;

C. according to the thin layer chromatography test, draw reference substance 5ul, need testing solution 15ul, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-butanone-formic acid-water (5: 3: 1: 1) be developing solvent, launch, take out, dry; Spray was dried by the fire about 10 minutes in 100 ℃ with 10% ethanol solution of sulfuric acid;

(4), this medicine Chinese crude drug Radix Glycyrrhizae is differentiated with thin layer chromatography

B. the preparation of need testing solution: get this product 8g, add water 15ml, hydrochloric acid 1ml and chloroform 20ml, reflux 30 minutes is put coldly, divides and to get chloroform solution, and evaporate to dryness, residue add ethyl acetate 1ml makes dissolving;

E. according to the thin layer chromatography test, draw need testing solution 20 μ l, control medicinal material solution 2 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with toluene-ethyl acetate-formic acid (15: 5: 1), launch, take out, dry; Spray is with 20% ethanol solution of sulfuric acid, in about 10 minutes of 100 ℃ of bakings.

4. according to the arbitrary described method of claim 2-3, it is characterized in that every gram contains the Radix Paeoniae Alba in peoniflorin, must not be lower than 0.20mg.