CN102645508B - Detection method for six-component costustoot preparation - Google Patents

Abstract

The invention provides a detection method for a six-component costustoot preparation. The six-component costustoot preparation comprises six medicinal components, including costustoot, Veronica eriogyne H.Winkl, Phyllanthus emblica, cardamom, pomegranate seed and Fructus piperis longi. According to the method, costustoot, Veronica eriogyne H.Winkl, Phyllanthus emblica, cardamom, pomegranate seed and Fructus piperis longi in the six-component costustoot preparation are detected by using a thin layer chromatography method. The detection method has the advantages of good repeatability and stability, convenience in operation, high precision, high specificity, clear spot developing and high degree of separation. Through building of a reliable quality detection method with high specificity, the quality of the six-component costustoot preparation can be controlled effectively so that the quality of the six-component costustoot preparation become stable, safe and controllable.

Description

The detection method of six ingredients aucklandia root preparation

Technical field

The present invention relates to a kind of detection method of drug combination preparation, relate in particular to a kind of detection method of six ingredients aucklandia root preparation, belong to Chinese medicine detection technique field.

Background technology

Six ingredients aucklandia root ball is Tibetan medicine, records in " the Sanitation Ministry medicine standard " Tibetan medicine fascicle, and standard number is WS3-BC-0283-95.This standard is recorded content and is mainly comprised:

Prescription: banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g.

Method for making: above Six-element, be ground into fine powder, sieve, mix, with water pill, dry, to obtain final product.

Proterties: this product is the sepia water-bindered pill; The special fragrance of the tool banksia rose, taste is bitter.

Differentiate: get this product 2g, add ethyl acetate 20ml, reflux 30 minutes in 60～80 ℃ of water-baths, filter, filtrate is concentrated into about 2ml, as need testing solution; Separately get the banksia rose, the each 0.5g of seed of pomegranate control medicinal material, be made in the same way of control medicinal material solution; Get again pipering reference substance, accurately weighed, add methyl alcohol and make the solution of every ml containing pipering 1mg, product solution in contrast.Test according to thin-layered chromatography (57 pages of appendix), draw the each 10 μ l of above-mentioned four kinds of solution, point is on same silica gel g thin-layer plate, take cyclohexane-ethyl acetate (6: 2) as developping agent, launch, take out, dry, put under ultraviolet lamp (365nm) and inspect, in test sample chromatogram, with reference substance chromatogram relevant position on the fluorescence spot of aobvious same color.Spray is with 5% vanillic aldehyde concentrated sulfuric acid solution, in test sample chromatogram, with control medicinal material chromatogram relevant position on the spot of aobvious same color.

Check: should meet every regulation relevant under pill item.

Function with cure mainly: antiemetic, relieves the pain.The pain causing for " Baconic's wood cloth ", belch, abdominal distension, vomiting etc.

Usage and consumption: 5～6 balls, 3 times on the one.

Specification: the heavy 6.5g of every 10 balls.

Storage: airtight, put shady and cool dry place.

The banksia rose: record in one of " Chinese Pharmacopoeia " version in 2010, the 57th page, this product is the dry root of feverfew banksia rose Aucklandia lappa decene..Autumn, two seasons of winter excavate, and remove silt and fibrous root, and segment large is cutd open into lobe in again, hit tertia after dry.

BAXIAGA: be the dry aerial parts of bloodroot C. racemosa Corydalis racemosa (Thunb.) Pers, the full-bloom stage aerial part of gathering, dries or segment is dried for subsequent use.

Emblic: record in one of " Chinese Pharmacopoeia " version in 2010, the 167th page, this strain Tibetan conventional crude drugs, is the dry mature fruit of euphorbia plant emblic Phyllanthus emblica L..Winter to time spring gathers when fruit maturation, removes impurity, dry.

Cardamom: record in one of " Chinese Pharmacopoeia " version in 2010, the 156th page, this product is the dry mature fruit of zingiberaceous plant Amomum cardamomum Amomum kravanh Pierre ex Gagnep. or amomum compactum Soland ex Maton Amomumcompactun Soland ex Maton.Be divided into " former cardamom " and " Indonesia's cardamom " by place of production difference.

The Bi roots of grass: record in one of " Chinese Pharmacopoeia " version in 2010, the 219th page, this product is dry near maturation or the mature fruit cluster of Piperaceae plant Bi roots of grass Piper longum L..Fruit ear is gathered during by green blackening, removes impurity, dries.

Seed of pomegranate: record in first of Ministry of Health of the People's Republic of China's Tibetan medicine ministerial standard the 26th page (standard number: WS3-BC-0026-95).This product is the dry seed of Punicaceae plant pomegranate Punicagranatum L..Remove pericarp after fruit maturation autumn, dries.

From foregoing, in former ministerial standard, the thin layer of the banksia rose, seed of pomegranate, the Bi roots of grass is differentiated and all adopted unified extracting method and developping agent condition to carry out, in practice, find through thin layer discrimination test repeatedly, the banksia rose still has and negative disturbs, the Rf value of seed of pomegranate is higher, Bi roots of grass need testing solution is without any spot, repeatability and the specificity of standard are poor, cannot realize the controllability to six ingredients aucklandia root ball quality standard detecting method.

Summary of the invention

Therefore, the object of this invention is to provide a kind of detection method of six ingredients aucklandia root preparation, this detection method has been improved extracting method and the developping agent of the banksia rose in former ministerial standard, seed of pomegranate, the Bi roots of grass, detection method after improvement is compared with primary standard, repeatability and specificity are stronger, meet easy, principle fast; Set up the specificity thin-layer identification method of emblic in six ingredients aucklandia root preparation.

In the preferred embodiment of the present invention, further set up the content assaying method of the contained costunolide of the banksia rose and Decahydro-3,6,9-tris(methylene)azuleno[4,5-b in six ingredients aucklandia root preparation, to provide a kind of favorable reproducibility, specificity strong, meet accurate, easy, sensitive, principle fast, can effectively control the quality of product, make its steady quality, controlled safely.

For above-mentioned purpose, technical scheme of the present invention is as follows:

A detection method for six ingredients aucklandia root preparation, described six ingredients aucklandia root preparation comprises the banksia rose, BAXIAGA, emblic, cardamom, seed of pomegranate and six kinds of medicinal ingredients of the Bi roots of grass; It is characterized in that, the method is differentiated the banksia rose, seed of pomegranate, emblic and/or the Bi roots of grass in described six ingredients aucklandia root preparation by thin-layered chromatography; Wherein:

(A) discriminating of the described banksia rose and seed of pomegranate comprises the following steps:

(1) get described six ingredients aucklandia root preparation, add ethyl acetate, ultrasonic extraction, after gained extract is concentrated as the banksia rose, seed of pomegranate need testing solution;

(2) by the upper step gained banksia rose, seed of pomegranate need testing solution point sample in silica gel thin-layer plate, launch take cyclohexane-methylene chloride-ethyl acetate as developping agent to differentiate; And/or

(B) discriminating of described emblic and the Bi roots of grass comprises the following steps:

(1 ') gets described six ingredients aucklandia root preparation, adds ethanol, ultrasonic extraction, after gained extract is concentrated as emblic, Bi roots of grass need testing solution;

(2 ') in silica gel thin-layer plate, launches upper step gained emblic, Bi roots of grass need testing solution point sample to differentiate take petroleum ether-ethyl acetate-formic acid as developping agent.

Preferably, in described step (1), also comprise and get the banksia rose, seed of pomegranate control medicinal material, make respectively the banksia rose, seed of pomegranate control medicinal material solution according to the compound method of the described banksia rose, seed of pomegranate need testing solution; In step (2), also comprise the banksia rose, seed of pomegranate control medicinal material solution point sample in silica gel thin-layer plate, launch take cyclohexane-methylene chloride-ethyl acetate as developping agent; And/or

In described step (1 '), also comprise and get emblic control medicinal material, add ethanol, ultrasonic extraction, after gained extract is concentrated as emblic control medicinal material solution; Get pipering reference substance, add methyl alcohol and make the methanol solution of pipering, as pipering reference substance solution; In step (2 '), also comprise emblic control medicinal material solution, pipering reference substance solution point sample in silica gel thin-layer plate, launch take petroleum ether-ethyl acetate-formic acid as developping agent.

Preferably, in described step (1), also comprise in prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate respectively, and makes the banksia rose, seed of pomegranate negative sample solution according to the compound method of the described banksia rose, seed of pomegranate need testing solution; In step (2), also comprise the banksia rose, seed of pomegranate negative sample solution point sample in silica gel thin-layer plate, launch take cyclohexane-methylene chloride-ethyl acetate as developping agent; And/or

In described step (1 '), also comprise in prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass respectively, and makes emblic, Bi roots of grass negative sample solution according to the compound method of described emblic, Bi roots of grass need testing solution; In step (2 '), also comprise emblic, Bi roots of grass negative sample solution point sample in silica gel thin-layer plate, launch take petroleum ether-ethyl acetate-formic acid as developping agent.

Preferably, described step (1) is: get six ingredients aucklandia root preparation 0.5～5 weight portion, after porphyrize, add ethyl acetate 5～40 parts by volume, ultrasonic processing 10～60 minutes, filters, filtrate is concentrated into 0.5～5 parts by volume, as the banksia rose, seed of pomegranate need testing solution; Separately get the banksia rose, each 0.1～3 weight portion of seed of pomegranate control medicinal material, make respectively the banksia rose, seed of pomegranate control medicinal material solution according to the preparation method of the described banksia rose, seed of pomegranate need testing solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and makes the banksia rose, seed of pomegranate negative sample solution by the compound method of the described banksia rose, seed of pomegranate need testing solution; Wherein, when above-mentioned weight portion measures with g, parts by volume is in mL.

Preferably, described step (1) is: get six ingredients aucklandia root preparation 2 weight portions, after porphyrize, add ethyl acetate 20 parts by volume, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 2 parts by volume, as the banksia rose, seed of pomegranate need testing solution; Separately get the banksia rose, each 0.5 weight portion of seed of pomegranate control medicinal material, make respectively the banksia rose, seed of pomegranate control medicinal material solution according to the preparation method of the described banksia rose, seed of pomegranate need testing solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and makes the banksia rose, seed of pomegranate negative sample solution by the compound method of the described banksia rose, seed of pomegranate need testing solution; Wherein, when above-mentioned weight portion measures with g, parts by volume is in mL.

Preferably, described step (2) is: according to appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia " version in 2010, draw the sample solution making in step (1), it is put respectively on same silica gel g thin-layer plate, take cyclohexane-methylene chloride-ethyl acetate as developping agent, thin layer plate is put in expansion cylinder saturated, launch, take out, dry, spray is with vanillic aldehyde sulfuric acid solution, and wind is clear to spot colour developing.

Preferably, need testing solution in the sample solution of described absorption and negative sample solution are 5～20 μ l, control medicinal material solution is 1～10 μ l, and the need testing solution in the sample solution of preferred described absorption and negative sample solution are 10 μ l, control medicinal material solution is 5 μ l; The volume ratio of described developping agent cyclohexane-methylene chloride-ethyl acetate is 8～22: 3～8: 0.1～1, and preferred volume ratio is 15: 5: 0.5; Described thin layer plate is put in expansion cylinder saturated 0～40 minute, preferably saturated 20 minutes; Described vanillic aldehyde sulfuric acid solution percent weight in volume is 5% (to take 5 weight portion vanillic aldehydes, add 100 parts by volume sulfuric acid, stir and get final product.Weight portion/parts by volume=g/ml), it is clear to blow to spot colour developing with hot blast.

Preferably, described step (1 ') is: get six ingredients aucklandia root preparation 0.5～5 weight portion, after porphyrize, add ethanol 5～40 parts by volume, ultrasonic processing 10～60 minutes, filters, filtrate is concentrated into 0.5～5 parts by volume, as emblic, Bi roots of grass need testing solution; Separately get emblic control medicinal material 0.1～3 weight portion, make emblic control medicinal material solution according to the preparation method of described emblic, Bi roots of grass need testing solution; Get again pipering reference substance appropriate, add methyl alcohol and make the solution of every 1ml containing pipering 0.5～3mg, as pipering reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and makes emblic, Bi roots of grass negative sample solution by the compound method of described emblic, Bi roots of grass need testing solution; Wherein, when above-mentioned weight portion measures with g, parts by volume is in mL.

Preferably, described step (1 ') is: get six ingredients aucklandia root preparation 2 weight portions, after porphyrize, add ethanol 20 parts by volume, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 2 parts by volume, as emblic, Bi roots of grass need testing solution; Separately get emblic control medicinal material 0.5 weight portion, make emblic control medicinal material solution according to the preparation method of described emblic, Bi roots of grass need testing solution; Get again pipering reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing pipering 1mg, as pipering reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and makes emblic, Bi roots of grass negative sample solution by the compound method of described emblic, Bi roots of grass need testing solution; Wherein, when above-mentioned weight portion measures with g, parts by volume is in mL.

Preferably, described step (2 ') is: according to appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia " version in 2010, draw the sample solution making in step (1 '), it is put respectively in same silica G F ₂₅₄on thin layer plate, take petroleum ether-ethyl acetate-formic acid as developping agent, thin layer plate is put in expansion cylinder saturated, launches, and takes out, and dries, and under 254nm ultraviolet lamp, inspects.

Preferably, the sample solution of described absorption is 5～20 μ l, is preferably 10 μ l; The volume ratio of described petroleum ether-ethyl acetate-formic acid is 1～10: 2～6: 0.05～0.5, and preferred volume ratio is 6: 4: 0.25, the boiling range of described sherwood oil is 60～90 ℃; Described thin layer plate is put in expansion cylinder saturated 0～40 minute, preferably saturated 20 minutes.

Preferably, described detection method further comprises the content with the contained costunolide of the banksia rose and/or dehydro-α-curcumene in six ingredients aucklandia root preparation described in high effective liquid chromatography for measuring, wherein:

The step of described high effective liquid chromatography for measuring comprises:

(1 ") prepare banksia rose need testing solution, and costunolide and/or dehydro-α-curcumene reference substance solution;

(2 ") by upper step gained banksia rose need testing solution, and costunolide and/or dehydro-α-curcumene reference substance solution injection liquid chromatography are measured.

Preferably, in described step (1 "), also comprise and prepare banksia rose negative sample solution; In described step (2 "), also comprise banksia rose negative sample solution injection liquid chromatography is measured.

Preferably, the chromatographic condition of described high effective liquid chromatography for measuring is: with octadecylsilane chemically bonded silica be filling agent; Take volume ratio as 60～70: 30～40 acetonitrile-water is mobile phase; Detection wavelength is 225 ± 2nm; Column temperature: 20～40 ℃, number of theoretical plate calculates and is not less than 3000 by costunolide peak.

Preferably, the chromatographic condition of described high effective liquid chromatography for measuring is: with octadecylsilane chemically bonded silica be filling agent; Take volume ratio as the acetonitrile-water of 65: 35 is as mobile phase; Detection wavelength is 225nm; Column temperature: 25 ℃, number of theoretical plate calculates and is not less than 3000 by costunolide peak.

Preferably, described step (1 ") is: get six ingredients aucklandia root preparation 0.1～3 weight portion, after porphyrize; put in tool plug conical flask, precision adds methyl alcohol 10～50 parts by volume, weighed weight; at power 250W, under frequency 40kHz, ultrasonic processing 15～45 minutes, lets cool; weighed weight again; supply the weight of less loss with methyl alcohol, shake up, and filters; get subsequent filtrate, as banksia rose need testing solution; Precision takes costunolide reference substance in addition and dehydro-α-curcumene reference substance is appropriate, add methyl alcohol and make the mixed solution of every 1ml containing costunolide 20～140 μ g and dehydro-α-curcumene 40～200 μ g, mix reference substance solution as costunolide and dehydro-α-curcumene; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, and makes banksia rose negative sample solution by the compound method of described banksia rose need testing solution; Wherein, when above-mentioned weight portion measures with g, parts by volume is in mL.

Preferably, described step (1 ") is: get six ingredients aucklandia root preparation 1 weight portion, after porphyrize; put in tool plug conical flask, precision adds methyl alcohol 25 parts by volume, weighed weight; at power 250W, under frequency 40kHz, ultrasonic processing 30 minutes, lets cool; weighed weight again; supply the weight of less loss with methyl alcohol, shake up, and filters; get subsequent filtrate, as banksia rose need testing solution; Precision takes costunolide reference substance with appropriate with dehydro-α-curcumene reference substance in addition, accurately weighed, add methyl alcohol and make the mixed solution of every 1ml containing costunolide 40 μ g and dehydro-α-curcumene 60 μ g, mix reference substance solution as costunolide and dehydro-α-curcumene; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, and makes banksia rose negative sample solution by the compound method of described banksia rose need testing solution; Wherein, when above-mentioned weight portion measures with g, parts by volume is in mL.

Preferably, described step (2 ") be: accurate need testing solution, reference substance solution, each 5～20 μ l of negative sample solution of drawing respectively, preferably each 10 μ l, injection liquid chromatography, measures, and to obtain final product.

Preferably, the bulk drug of described six ingredients aucklandia root preparation composition comprises: the banksia rose 200 weight portions, BAXIAGA 360 weight portions, emblic 500 weight portions, cardamom 80 weight portions, seed of pomegranate 400 weight portions and the Bi roots of grass 100 weight portions.

Preferably, the preparation method of described six ingredients aucklandia root preparation is: described bulk drug is ground into fine powder, sieves, mix, the appropriate pill that adds water, obtains pill; Or described bulk drug is ground into fine powder, and sieve, mix, by pharmacy conventional method, add conventional auxiliary material, make the oral formulations of accepting clinically: granule, pill, capsule, tablet or powder.

Preferably, described six ingredients aucklandia root preparation is granule, pill, capsule, tablet or powder.

This Tibetan medicine six ingredients aucklandia root preparation has and stomach, pain relieving, antiemetic effect.Be used for the treatment of the upper abdomen severe pain that " Baconic's wood cloth " (being equivalent to gastritis, digestive tract ulcer) causes, n and V, belch etc.

Detection method of the present invention has been carried out thin layer to the banksia rose, seed of pomegranate, emblic and the Bi roots of grass in prescription and has been differentiated research.The present invention has further set up the contained costunolide of the banksia rose and dehydro-α-curcumene high performance liquid chromatography content assaying method.The advantages such as detection method of the present invention has reappearance, good stability, method of operating is easy, precision is high, specificity is strong, spot colour developing is clear, degree of separation is good; The quality determining method reliable by method for building up, specificity is strong, can effectively control the quality of six ingredients aucklandia root preparation, makes the quality of six ingredients aucklandia root preparation reach stable, safety is controlled.

Accompanying drawing explanation

Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:

Fig. 1 is the thin-layer chromatogram that ministerial standard discrimination method is differentiated the banksia rose, seed of pomegranate, the Bi roots of grass; In figure, 1-3 is test sample, and 4 is banksia rose control medicinal material, and 5 is banksia rose negative sample, 6 seed of pomegranate control medicinal materials, and 7 is seed of pomegranate negative sample, and 8 is pipering reference substance, and 9 is Bi roots of grass negative sample.

Fig. 2 is the thin-layer chromatogram of the banksia rose of the present invention, seed of pomegranate; In figure, 1-3 is test sample, and 4-5 is banksia rose control medicinal material, and 6 is banksia rose negative sample, and 7-9 is test sample, and 10-11 is seed of pomegranate control medicinal material, and 12 is seed of pomegranate negative sample.

Fig. 3 is the thin-layer chromatogram of emblic of the present invention, the Bi roots of grass; In figure, 1-4 is test sample, and 5 is emblic control medicinal material, and 6 is emblic negative sample, and 7-10 is test sample, and 11 is pipering reference substance, and 12 is Bi roots of grass negative sample.

Fig. 4 is the chromatogram of the contained costunolide of the banksia rose of the present invention and dehydro-α-curcumene; Wherein chromatogram A is costunolide and dehydro-α-curcumene reference substance; Chromatogram B is banksia rose test sample; Chromatogram C is banksia rose negative sample.

Fig. 5 is the peak area of costunolide of the present invention and the linear relationship chart of reference substance concentration.

Fig. 6 is the peak area of costunolide of the present invention and the linear relationship chart of reference substance concentration.

Embodiment

Following experimental example and embodiment are just for illustrating the present invention rather than restriction the present invention.

Unless otherwise indicated, in the embodiment of the present invention, developping agent used and the ratio of mobile phase are volume ratio.

experimental example 1: the thin layer of the banksia rose, seed of pomegranate, the Bi roots of grass is differentiated

1.1 differentiate the banksia rose, seed of pomegranate and the Bi roots of grass according to ministerial standard discrimination method

The preparation of need testing solution: get described six ingredients aucklandia root preparation 2g, add ethyl acetate 20ml, reflux 30 minutes in 60～80 ℃ of water-baths, filter, filtrate is concentrated into about 2ml, as need testing solution;

The preparation of control medicinal material solution: get the banksia rose, the each 0.5g of seed of pomegranate control medicinal material, be made in the same way of control medicinal material solution;

The preparation of reference substance solution: get pipering reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing pipering 1mg, product solution in contrast;

The preparation of banksia rose negative sample solution: in prescription ratio, preparation does not contain the negative sample of the banksia rose, and makes banksia rose negative sample solution according to the preparation method of above-mentioned need testing solution;

The preparation of seed of pomegranate negative sample solution: in prescription ratio, preparation does not contain the negative sample of seed of pomegranate, and makes seed of pomegranate negative sample solution according to the preparation method of above-mentioned need testing solution;

The preparation of Bi roots of grass negative sample solution: in prescription ratio, preparation does not contain the negative sample of the Bi roots of grass, and makes Bi roots of grass negative sample solution according to the preparation method of above-mentioned need testing solution.

According to appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia " version in 2010, draw the each 10 μ l of above-mentioned six kinds of solution, point is on same silica gel g thin-layer plate, take cyclohexane-ethyl acetate (6: 2) as developping agent, launch, take out, dry, put under ultraviolet lamp (365nm) and inspect, in test sample chromatogram, with reference substance chromatogram relevant position on the fluorescence spot of aobvious same color.Spray (takes 5 weight portion vanillic aldehydes, adds 100 parts by volume sulfuric acid, stir and get final product take percent weight in volume as 5%.Weight portion/parts by volume=g/ml) vanillic aldehyde concentrated sulfuric acid solution, in test sample chromatogram, with control medicinal material chromatogram relevant position on the spot of aobvious same color.The results are shown in shown in accompanying drawing 1: the feminine gender of the banksia rose has interference; The Rf value of seed of pomegranate is too large; In Bi roots of grass need testing solution with the corresponding position of reference substance chromatogram on immaculate, repeat that result is still undesirable several times, therefore only the banksia rose and seed of pomegranate have been adopted to unified approach in this detection method, and extracting method and developping agent have been carried out to Improvement and perfection, the Bi roots of grass has carried out thin layer discriminating in addition.

1.2 the present invention differentiate the thin-layer chromatography shaker test of the banksia rose, seed of pomegranate

(1) instrument

Mortar, graduated cylinder, round-bottomed flask, reflux, evaporating dish, sample applicator, chromatography cylinder, spray bottle, analytical balance (Mei Teletuo benefit, model: XS205), ultrasonic extractor (Shanghai High Kudos Science Instrument Co., Ltd., model: SK8200HP), silica gel g thin-layer plate (Haiyang Chemical Plant, Qingdao, specification: 200 × 200mm).

(2) control medicinal material

Banksia rose control medicinal material (lot number: 120921-200506), seed of pomegranate control medicinal material (lot number: 121431-200501), all purchased from Nat'l Pharmaceutical & Biological Products Control Institute.

(3) reagent

Ethyl acetate, methylene chloride, ethanol, methyl alcohol, the concentrated sulphuric acid, vanillic aldehyde, benzene, ethyl formate, formic acid, cyclohexane, distilled water.

(4) method of inspection:

Extract the selection of solvent: adopt respectively ethanol, ethyl acetate, methyl alcohol for extracting solvent;

The selection of extracting method: adopt respectively ultrasonic extraction and refluxing extraction;

The selection of developping agent: adopting respectively cyclohexane-ethyl acetate (6: 2), benzene-ethyl acetate (19: 1), cyclohexane-ethyl formate-formic acid (15: 5: 1), cyclohexane-methylene chloride-ethyl acetate (15: 5: 0.5) is developping agent.

Select respectively above solvent, extracting method and the developping agent of extracting to test, result is as follows:

Pass through in the above conditions repetition test, finally determined that the specificity thin-layer identification method of the banksia rose, seed of pomegranate is as follows:

Get described six ingredients aucklandia root preparation 2g, porphyrize, adds ethyl acetate 20ml, and ultrasonic extraction 30 minutes filters, and filtrate is concentrated into about 2ml, as the banksia rose, seed of pomegranate need testing solution; Separately get the banksia rose, the each 0.5g of seed of pomegranate control medicinal material, be made in the same way of the banksia rose, seed of pomegranate control medicinal material solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate respectively, and makes the banksia rose, seed of pomegranate negative sample solution according to the preparation method of above-mentioned need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 10 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 5 μ l, put respectively on same silica G plate, take cyclohexane-methylene chloride-ethyl acetate (15: 5: 0.5) as developping agent, thin layer plate is put in expansion cylinder saturated 20 minutes, launch, take out, dry, the vanillic aldehyde sulfuric acid solution of spray take percent weight in volume as 5%, it is clear that hot blast blows to spot colour developing, the results are shown in shown in accompanying drawing 2.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, and negative noiseless.The method is through many people test of many times operation, favorable reproducibility, and specificity is strong, meets easy, sensitive, principle fast, can be used as the detection method of the banksia rose, seed of pomegranate in six ingredients aucklandia root preparation.

1.3 the present invention differentiate the thin-layer chromatography shaker test of emblic, the Bi roots of grass

(1) instrument

Mortar, graduated cylinder, round-bottomed flask, reflux, evaporating dish, sample applicator, chromatography cylinder, spray bottle, analytical balance (Mei Teletuo benefit, model: XS205), ultrasonic extractor (Shanghai High Kudos Science Instrument Co., Ltd., model: SK8200HP), silica gel g thin-layer plate (Haiyang Chemical Plant, Qingdao, specification: 200 × 200mm).

(2) control medicinal material and reference substance

Emblic control medicinal material (lot number: 121289-200301), pipering reference substance (lot number: 110775-200203), all purchased from Nat'l Pharmaceutical & Biological Products Control Institute.

(3) reagent

Sherwood oil (boiling range 60-90 ℃), ethyl acetate, acetone, methyl alcohol, ethanol, toluene, formic acid, normal hexane, cyclohexane, glacial acetic acid, distilled water.

(4) method of inspection:

Extract the selection of solvent: adopting respectively percent by volume is that 70% ethanol (is got 70ml ethanol, add 30ml distilled water, stir evenly and get final product), ethanol, percent by volume is 70% methyl alcohol (is got 70ml methyl alcohol, add 30ml distilled water, stir evenly and get final product), methyl alcohol for extract solvent;

The selection of extracting method: adopt respectively extraction, heating reflux method after ultrasonic extraction, alcohol extract;

The selection of developping agent: adopt respectively toluene-ethyl acetate-acetone (7: 2: 1), cyclohexane-ethyl acetate-acetone (7: 2: 1), normal hexane-ethyl acetate-glacial acetic acid-methyl alcohol (4: 5: 0.4: 0.6), sherwood oil (boiling range 60-90 ℃)-ethyl acetate-formic acid (6: 4: 0.25) is developping agent.

Select respectively above extraction solvent, extracting method, developping agent to test, result is as follows:

Finally determine that through repetition test the specificity thin-layer identification method of emblic, the Bi roots of grass is as follows in the above conditions:

Get described six ingredients aucklandia root preparation 2g, porphyrize, adds ethanol 20ml, and ultrasonic extraction 30 minutes filters, and filtrate is concentrated into about 2ml, as emblic, Bi roots of grass need testing solution; Separately get emblic control medicinal material 0.5g, be made in the same way of emblic control medicinal material solution; It is appropriate that essence is got pipering reference substance again, accurately weighed, adds methyl alcohol and make the solution of 1ml containing pipering 1mg, as pipering reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass respectively, and makes emblic, Bi roots of grass negative sample solution according to the preparation method of above-mentioned need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 10 μ l of above-mentioned four kinds of solution, put respectively in same silica G F ₂₅₄on plate, take sherwood oil (60～90 ℃)-ethyl acetate-formic acid (6: 4: 0.25) as developping agent, thin layer plate is put in expansion cylinder saturated 20 minutes, launch, take out, dry, put under ultraviolet lamp (254nm) and inspect, the results are shown in shown in accompanying drawing 3.In test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color, and negative noiseless.The method is through many people test of many times operation, favorable reproducibility, and specificity is strong, meets easy, sensitive, principle fast, can be used as the detection method of emblic in six ingredients aucklandia root preparation, the Bi roots of grass.

experimental example 2: the assay of the contained costunolide of the banksia rose and dehydro-α-curcumene

(1) instrument

Conical flask, volumetric flask, transfer pipet, syringe, beaker, funnel, filter paper, filter, electronic balance (Mei Teletuo benefit, model: XS205), high performance liquid chromatograph (Agilent company of the U.S., model: Aglient 1100).

(2) reference substance: costunolide (lot number: 111524-201006), dehydro-α-curcumene (lot number: 111525-200907), all identify institute purchased from Chinese pharmaceutical biological product.

(3) reagent: methyl alcohol, acetonitrile, ethanol, water.

(4) method of inspection:

Extract the selection of solvent: adopt respectively methyl alcohol, ethanol;

The selection of extraction time: adopt respectively test for ultrasonic 20 minutes, 30 minutes, 45 minutes;

The selection of mobile phase: adopt respectively acetonitrile-water (65: 35), acetonitrile-water (60: 40), methanol-water (65: 35).

Select respectively above solvent, extraction time and the mobile phase of extracting to test, result is as shown in the table:

Repetition test in the above conditions, finally determine that the method for the contained costunolide of the banksia rose, dehydro-α-curcumene assay is as follows:

Chromatographic condition and system suitability high performance liquid chromatograph (Agilent company of the U.S.): comprise G1314A UV-vis detector, G1314A binary pump, Agilent1100 chromatographic work station; With octadecylsilane chemically bonded silica be filling agent; Take acetonitrile-water (65: 35) as mobile phase; Detection wavelength is 225nm; Column temperature: 25 ℃.Number of theoretical plate calculates and should be not less than 3000 by costunolide peak.

Get described six ingredients aucklandia root preparation 1g, porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, and filters, get subsequent filtrate, as banksia rose need testing solution; Separately get costunolide reference substance with appropriate with dehydro-α-curcumene reference substance, accurately weighed, add methyl alcohol and make the mixed solution of every 1ml containing costunolide 40 μ g, dehydro-α-curcumene 60 μ g, mix reference substance solution as costunolide and dehydro-α-curcumene.In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, and makes banksia rose negative sample solution by the compound method of described banksia rose need testing solution.According to 2010 editions one appendix VI D test of high performance liquid chromatography " Chinese Pharmacopoeia ", the accurate each 10 μ l of above-mentioned three kinds of solution that draw respectively, injection liquid chromatography, measures, and the results are shown in as shown in Figure 4.The every 1g of preparation counts 2.43mg containing the banksia rose with costunolide and dehydro-α-curcumene total amount.

experimental example 3: the assay-methodology of the contained costunolide of the banksia rose and dehydro-α-curcumene investigate

(1) instrument and reagent

Aglient 1100 high performance liquid chromatographs (Agilent company of the U.S.): G1314A UV-vis detector, G1314A binary pump, Agilent1100 chromatographic work station;

SK8200HP ultrasonic cleaner (power 250W, frequency 40kHz) (Shanghai High Kudos Science Instrument Co., Ltd.);

Acetonitrile (chromatographically pure), methyl alcohol (analyzing pure);

Costunolide (lot number: 111524-201006), dehydro-α-curcumene (lot number: 111525-200907), all identify institute purchased from Chinese pharmaceutical biological product;

Six ingredients aucklandia root preparation: provided by XiZang QiZheng Tibetan pharmaceuticals Co., Ltd.

(2) chromatographic condition

Octadecyl silane is filling agent;

Chromatographic column: Kromasil E56526-C18 (Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S. for 4.6 × 250mm, 5.0 μ m);

Mobile phase: acetonitrile-water (V: V=65: 35);

Flow velocity: 1.0ml/min;

Detect wavelength: 225nm;

Column temperature: 25 ℃.

(3) chromatographic system employment and suitability test (E & ST)

Under above-mentioned chromatographic condition, costunolide and dehydro-α-curcumene and all the other impurity peaks separating effects are better, and theoretical cam curve is calculated and should be not less than 3000 by costunolide peak.

(4) preparation of reference substance solution, need testing solution, negative sample solution

The preparation of reference substance solution: get costunolide, dehydro-α-curcumene reference substance is appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing costunolide 40 μ g, dehydro-α-curcumene 60 μ g, shake up, obtain reference substance solution;

The preparation of need testing solution: get the about 1g of described six ingredients aucklandia root preparation, porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, let cool, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate as need testing solution;

The preparation of negative sample solution: get the negative sample 1g that lacks banksia rose medicinal material, be made in the same way of negative sample solution, for subsequent use.

(5) negative interference test

Accurate reference substance solution, need testing solution and the each 10 μ l of negative sample solution of drawing, in injection liquid chromatography, from chromatogram, with the corresponding position of reference substance chromatographic peak retention time on, the chromatographic peak that need testing solution has identical retention time occurs, and negative sample solution at this wavelength without absorption, noiseless to the assay of costunolide in preparation and dehydro-α-curcumene.The results are shown in Figure chromatogram A, B, the C shown in 4.

(6) investigation of linear relationship

Get costunolide, dehydro-α-curcumene reference substance is appropriate, accurately weighed, adds methyl alcohol and makes the mixed solution of every 1ml containing costunolide 120 μ g and dehydro-α-curcumene 170 μ g, shake up, as storing solution.Accurate absorption in above-mentioned stock solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 10ml to 10ml volumetric flask, be diluted to scale with methyl alcohol, shake up, precision measures 10 μ l injection liquid chromatography respectively, measure peak area, costunolide between 12～120 μ g/ml in the concentration of peak area and reference substance be good linear relationship, its regression equation is y=23.793x+10.253 (r=0.9997); Dehydro-α-curcumene between 17～170 μ g/ml in the concentration of peak area and reference substance be good linear relationship, its regression equation is y=14.982x+0.0062 (r=0.9996).The results are shown in Table 1 and Fig. 5, table 2 and Fig. 6.

Table 1 costunolide contrast concentration and peak area result

Table 2 dehydro-α-curcumene contrast concentration and peak area result

(7) precision test

The same reference substance solution 10 μ l of accurate absorption, continuous sample introduction 5 times, the peak area of mensuration chromatographic peak, the results are shown in Table 3.

Table 3 Precision test result

Test shows, precision is good, and RSD is respectively 0.61% and 1.34%.

(8) stability test

Accurate draw same need testing solution 10 μ l, respectively at 0,2,4,6,12h sample introduction, measure the peak area of chromatographic peak, the results are shown in Table 4.

Table 4 stability test result

Test shows, result is good at 0～12h internal stability, and RSD is respectively 0.79% and 1.55%.

(9) reappearance test

(lot number: 20100821) 5 parts, preparation method processes according to test sample, distinguishes sample introduction, calculates respectively content, total amount and the RSD of costunolide and dehydro-α-curcumene, the results are shown in Table 5 to get described six ingredients aucklandia root preparation.

Table 5 reproducible test results

Test shows, 5 parts of mensuration of same batch sample sampling, and result reappearance is better, and RSD is respectively 0.44%, 1.31% and 0.94%.

(10) recovery test

Get the six ingredients aucklandia root preparation (lot number: 20100821 of known content, content is 2.656mg/g) the about 0.5g of powder, accurately weighed, add a certain amount of costunolide and dehydro-α-curcumene, measure in accordance with the law, according to chromatographic peak peak area value, try to achieve respectively the content of costunolide and dehydro-α-curcumene, calculate recovery rate, measurement result is in table 6, table 7.

The average recovery test of table 6 costunolide

Result shows, this law recovery is good, and RSD is 1.26%.

The average recovery test of table 7 dehydro-α-curcumene

Result shows, this law recovery is good, RSD1.74%.

(11) mensuration of test sample

Get the about 1g of described six ingredients aucklandia root preparation, porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, with 0.45 μ m filter membrane filtration, get subsequent filtrate, obtain need testing solution.The accurate need testing solution 10 μ l that draw measure in accordance with the law, calculate the content of costunolide and dehydro-α-curcumene, the results are shown in Table 8.

The assay result of costunolide and dehydro-α-curcumene in table 8 test sample

Known according to the measurement result of above-mentioned three multiple batches of test samples of producer, the every 1g of described six ingredients aucklandia root preparation containing the banksia rose in costunolide and dehydro-α-curcumene total amount between 1.352～2.656mg, therefore, should be not less than 1.30mg containing the banksia rose with costunolide and dehydro-α-curcumene total amount in the tentative every 1g of minimum limitation.

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

test example 1: the detection of six ingredients aucklandia root ball

Banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g;

Above Six-element, is ground into fine powder, sieves, and the appropriate pill that adds water, to obtain final product.

A. the thin layer of the banksia rose, seed of pomegranate is differentiated

Get above-mentioned six ingredients aucklandia root ball 2g, porphyrize, adds ethyl acetate 20ml, and ultrasonic extraction 30 minutes filters, and filtrate is concentrated into about 2ml, as need testing solution; Separately get the banksia rose, the each 0.5g of seed of pomegranate control medicinal material, be made in the same way of control medicinal material solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and makes the banksia rose, seed of pomegranate negative sample solution by the compound method of the above-mentioned banksia rose, seed of pomegranate need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 10 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 5 μ l, put respectively on same silica G plate, take cyclohexane-methylene chloride-ethyl acetate (15: 5: 0.5) as developping agent, thin layer plate is put in expansion cylinder saturated 20 minutes, launches, take out, dry, the vanillic aldehyde sulfuric acid solution of spray take percent weight in volume as 5%, it is clear that hot blast blows to spot colour developing.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, and negative noiseless.

B. the thin layer of emblic, the Bi roots of grass is differentiated

Get above-mentioned six ingredients aucklandia root ball 2g, porphyrize, adds ethanol 20ml, and ultrasonic extraction 30 minutes filters, and filtrate is concentrated into about 2ml, as need testing solution; Separately get emblic control medicinal material 0.5g, be made in the same way of control medicinal material solution; Get again pipering reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing pipering 1mg, product solution in contrast; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and makes emblic, Bi roots of grass negative sample solution by the compound method of above-mentioned emblic, Bi roots of grass need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 10 μ l of above-mentioned four kinds of solution, put respectively in same silica G F ₂₅₄on plate, take sherwood oil (60～90 ℃)-ethyl acetate-formic acid (6: 4: 0.25) as developping agent, thin layer plate is put in expansion cylinder saturated 20 minutes, launches, and takes out, and dries, and puts under ultraviolet lamp (254nm) and inspects.In test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color, and negative noiseless.

C. the assay of the contained costunolide of the banksia rose and dehydro-α-curcumene

According to 2010 editions one appendix VI D of high performance liquid chromatography " Chinese Pharmacopoeia "

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile-water (65: 35) as mobile phase; Detection wavelength is 225nm, and column temperature is 25 ℃, and number of theoretical plate calculates and should be not less than 3000 by costunolide peak.

The preparation of reference substance solution: get costunolide reference substance and dehydro-α-curcumene reference substance is appropriate, accurately weighed, add methyl alcohol and make the mixed solution of every 1ml containing costunolide 40 μ g, dehydro-α-curcumene 60 μ g, to obtain final product.

The preparation of need testing solution: get six ingredients aucklandia root ball 1g, porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, and filters, get subsequent filtrate, to obtain final product.

Determination method: accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.

The every 1g of six ingredients aucklandia root ball contains the banksia rose with costunolide (C ₁₅h ₂₀o ₂) and dehydro-α-curcumene (C ₁₅h ₁₈o ₂) total amount count 2.43mg.

test example 2: the detection of six ingredients aucklandia root hard shell capsules

Banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g;

Above Six-element, is ground into fine powder, sieves, and mixes by pharmacy conventional method, adds conventional auxiliary material, makes acceptable six ingredients aucklandia root hard shell capsules clinically.

A. the thin layer of the banksia rose, seed of pomegranate is differentiated

Get above-mentioned six ingredients aucklandia root hard shell capsules 0.8g, porphyrize, adds ethyl acetate 8ml, and ultrasonic extraction 15 minutes filters, and filtrate is concentrated into about 0.5ml, as need testing solution; Separately get the banksia rose, the each 0.2g of seed of pomegranate control medicinal material, be made in the same way of control medicinal material solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and makes the banksia rose, seed of pomegranate negative sample solution by the compound method of the above-mentioned banksia rose, seed of pomegranate need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 5 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 10 μ l, put respectively on same silica G plate, take cyclohexane-methylene chloride-ethyl acetate (12: 4: 0.3) as developping agent, thin layer plate is put in expansion cylinder saturated 30 minutes, launches, take out, dry, the vanillic aldehyde sulfuric acid solution of spray take percent weight in volume as 5%, it is clear that hot blast blows to spot colour developing.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, and negative noiseless.

B. the thin layer of emblic, the Bi roots of grass is differentiated

Get above-mentioned six ingredients aucklandia root hard shell capsules 2g, porphyrize, adds ethanol 20ml, and ultrasonic extraction 30 minutes filters, and filtrate is concentrated into about 2ml, as need testing solution; Separately get emblic control medicinal material 0.5g, be made in the same way of control medicinal material solution; Get again pipering reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing pipering 1mg, product solution in contrast; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and makes emblic, Bi roots of grass negative sample solution by the compound method of above-mentioned emblic, Bi roots of grass need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 10 μ l of above-mentioned four kinds of solution, put respectively in same silica G F ₂₅₄on plate, take sherwood oil (60～90 ℃)-ethyl acetate-formic acid (6: 4: 0.25) as developping agent, thin layer plate is put in expansion cylinder saturated 20 minutes, launches, and takes out, and dries, and puts under ultraviolet lamp (254nm) and inspects.In test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color, and negative noiseless.

C. the assay of the contained costunolide of the banksia rose and dehydro-α-curcumene

According to 2010 editions one appendix VI D of high performance liquid chromatography " Chinese Pharmacopoeia "

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile-water (64: 36) as mobile phase; Detection wavelength is 225nm, and column temperature is 20 ℃, and number of theoretical plate calculates and should be not less than 3000 by costunolide peak.

The preparation of reference substance solution: get costunolide reference substance and dehydro-α-curcumene reference substance is appropriate, accurately weighed, add methyl alcohol and make the mixed solution of every 1ml containing costunolide 30 μ g, dehydro-α-curcumene 50 μ g, to obtain final product.

The preparation of need testing solution: get six ingredients aucklandia root hard shell capsules 0.2g, porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 15ml, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 20 minutes, lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, and filters, get subsequent filtrate, to obtain final product.

Determination method: accurate reference substance solution and the each 5 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.

The every 1g of six ingredients aucklandia root hard shell capsules contains the banksia rose with costunolide (C ₁₅h ₂₀o ₂) and dehydro-α-curcumene (C ₁₅h ₁₈o ₂) total amount count 1.41mg.

test example 3: the detection of Liuwei aucklandia root tablet

Banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g;

Above Six-element, is ground into fine powder, sieves, and mixes by pharmacy conventional method, adds conventional auxiliary material, makes acceptable Liuwei aucklandia root tablet clinically.

A. the thin layer of the banksia rose, seed of pomegranate is differentiated

Get above-mentioned Liuwei aucklandia root tablet 1.5g, porphyrize, adds ethyl acetate 15ml, and ultrasonic extraction 20 minutes filters, and filtrate is concentrated into about 1ml, as need testing solution; Separately get the banksia rose, the each 0.8g of seed of pomegranate control medicinal material, be made in the same way of control medicinal material solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and makes the banksia rose, seed of pomegranate negative sample solution by the compound method of the above-mentioned banksia rose, seed of pomegranate need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 15 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 4 μ l, put respectively on same silica G plate, take cyclohexane-methylene chloride-ethyl acetate (18: 6: 0.8) as developping agent, launch, take out, dry, the vanillic aldehyde sulfuric acid solution of spray take percent weight in volume as 5%, it is clear that hot blast blows to spot colour developing.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, and negative noiseless.

B. the thin layer of emblic, the Bi roots of grass is differentiated

Get above-mentioned Liuwei aucklandia root tablet 0.8g, porphyrize, adds ethanol 8ml, and ultrasonic extraction 15 minutes filters, and filtrate is concentrated into about 0.5ml, as need testing solution; Separately get emblic control medicinal material 0.2g, be made in the same way of control medicinal material solution; Get again pipering reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing pipering 0.5mg, product solution in contrast; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and makes emblic, Bi roots of grass negative sample solution by the compound method of above-mentioned emblic, Bi roots of grass need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 5 μ l of above-mentioned four kinds of solution, put respectively in same silica G F ₂₅₄on plate, take sherwood oil (60～90 ℃)-ethyl acetate-formic acid (2: 3: 0.07) as developping agent, thin layer plate is put in expansion cylinder saturated 30 minutes, launches, and takes out, and dries, and puts under ultraviolet lamp (254nm) and inspects.In test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color, and negative noiseless.

C. the assay of the contained costunolide of the banksia rose and dehydro-α-curcumene

According to 2010 editions one appendix VI D of high performance liquid chromatography " Chinese Pharmacopoeia "

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile-water (65: 35) as mobile phase; Detection wavelength is 225nm, and column temperature is 20 ℃, and number of theoretical plate calculates and should be not less than 3000 by costunolide peak.

The preparation of reference substance solution: get costunolide reference substance and dehydro-α-curcumene reference substance is appropriate, accurately weighed, add methyl alcohol and make the mixed solution of every 1ml containing costunolide 40 μ g, dehydro-α-curcumene 60 μ g, to obtain final product.

The preparation of need testing solution: get Liuwei aucklandia root tablet 1g, porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, and filters, get subsequent filtrate, to obtain final product.

Determination method: accurate reference substance solution and the each 15 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.

The every 1g of Liuwei aucklandia root tablet contains the banksia rose with costunolide (C ₁₅h ₂₀o ₂) and dehydro-α-curcumene (C ₁₅h ₁₈o ₂) total amount count 2.15mg.

test example 4: the detection of six ingredients aucklandia root particle

Banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g;

Above Six-element, is ground into fine powder, sieves, and mixes by pharmacy conventional method, adds conventional auxiliary material, makes acceptable six ingredients aucklandia root particle clinically.

A. the thin layer of the banksia rose, seed of pomegranate is differentiated

Get above-mentioned six ingredients aucklandia root particle 3g, porphyrize, adds ethyl acetate 30ml, and ultrasonic extraction 45 minutes filters, and filtrate is concentrated into about 3ml, as need testing solution; Separately get the banksia rose, the each 1.5g of seed of pomegranate control medicinal material, be made in the same way of control medicinal material solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and makes the banksia rose, seed of pomegranate negative sample solution by the compound method of the above-mentioned banksia rose, seed of pomegranate need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 10 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 5 μ l, put respectively on same silica G plate, take cyclohexane-methylene chloride-ethyl acetate (10: 3.5: 0.2) as developping agent, thin layer plate is put in expansion cylinder saturated 40 minutes, launches, take out, dry, the vanillic aldehyde sulfuric acid solution of spray take percent weight in volume as 5%, it is clear that hot blast blows to spot colour developing.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, and negative noiseless.

B. the thin layer of emblic, the Bi roots of grass is differentiated

Get above-mentioned six ingredients aucklandia root particle 1.5g, porphyrize, adds ethanol 15ml, and ultrasonic extraction 20 minutes filters, and filtrate is concentrated into about 1ml, as need testing solution; Separately get emblic control medicinal material 0.8g, be made in the same way of control medicinal material solution; Get again pipering reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing pipering 1.5mg, product solution in contrast; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and makes emblic, Bi roots of grass negative sample solution by the compound method of above-mentioned emblic, Bi roots of grass need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 15 μ l of above-mentioned four kinds of solution, put respectively in same silica G F ₂₅₄on plate, take sherwood oil (60～90 ℃)-ethyl acetate-formic acid (8: 5: 0.3) as developping agent, launch, take out, dry, put under ultraviolet lamp (254nm) and inspect.In test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color, and negative noiseless.

C. the assay of the contained costunolide of the banksia rose and dehydro-α-curcumene

According to 2010 editions one appendix VI D of high performance liquid chromatography " Chinese Pharmacopoeia "

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile-water (66: 34) as mobile phase; Detection wavelength is 225nm, and column temperature is 30 ℃, and number of theoretical plate calculates and should be not less than 3000 by costunolide peak.

The preparation of reference substance solution: get costunolide reference substance and dehydro-α-curcumene reference substance is appropriate, accurately weighed, add methyl alcohol and make the mixed solution of every 1ml containing costunolide 60 μ g, dehydro-α-curcumene 90 μ g, to obtain final product.

The preparation of need testing solution: get six ingredients aucklandia root particle 0.8g, porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 20ml, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 15 minutes, lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, and filters, get subsequent filtrate, to obtain final product.

Determination method: accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.

The every 1g of six ingredients aucklandia root particle contains the banksia rose with costunolide (C ₁₅h ₂₀o ₂) and dehydro-α-curcumene (C ₁₅h ₁₈o ₂) total amount count 1.94mg.

test example 5: the detection of six ingredients aucklandia root soft capsule

Banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g;

Above Six-element, is ground into fine powder, sieves, and mixes by pharmacy conventional method, adds conventional auxiliary material, makes acceptable six ingredients aucklandia root soft capsule clinically.

A. the thin layer of the banksia rose, seed of pomegranate is differentiated

Get above-mentioned six ingredients aucklandia root soft capsule 4.5g, porphyrize, adds ethyl acetate 40ml, and ultrasonic extraction 60 minutes filters, and filtrate is concentrated into about 4ml, as need testing solution; Separately get the banksia rose, the each 2.5g of seed of pomegranate control medicinal material, be made in the same way of control medicinal material solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and makes the banksia rose, seed of pomegranate negative sample solution by the compound method of the above-mentioned banksia rose, seed of pomegranate need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 20 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 2 μ l, put respectively on same silica G plate, take cyclohexane-methylene chloride-ethyl acetate (20: 7.8: 0.6) as developping agent, thin layer plate is put in expansion cylinder saturated 20 minutes, launches, take out, dry, the vanillic aldehyde sulfuric acid solution of spray take percent weight in volume as 5%, it is clear that hot blast blows to spot colour developing.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, and negative noiseless.

B. the thin layer of emblic, the Bi roots of grass is differentiated

Get above-mentioned six ingredients aucklandia root soft capsule 3g, porphyrize, adds ethanol 30ml, and ultrasonic extraction 45 minutes filters, and filtrate is concentrated into about 3ml, as need testing solution; Separately get emblic control medicinal material 1.5g, be made in the same way of control medicinal material solution; Get again pipering reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing pipering 2mg, product solution in contrast; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and makes emblic, Bi roots of grass negative sample solution by the compound method of above-mentioned emblic, Bi roots of grass need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 10 μ l of above-mentioned four kinds of solution, put respectively in same silica G F ₂₅₄on plate, take sherwood oil (60～90 ℃)-ethyl acetate-formic acid (3: 3: 0.1) as developping agent, thin layer plate is put in expansion cylinder saturated 40 minutes, launches, and takes out, and dries, and puts under ultraviolet lamp (254nm) and inspects.In test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color, and negative noiseless.

C. the assay of the contained costunolide of the banksia rose and dehydro-α-curcumene

According to 2010 editions one appendix VI D of high performance liquid chromatography " Chinese Pharmacopoeia "

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile-water (63: 37) as mobile phase; Detection wavelength is 225nm, and column temperature is 25 ℃, and number of theoretical plate calculates and should be not less than 3000 by costunolide peak.

The preparation of reference substance solution: get costunolide reference substance and dehydro-α-curcumene reference substance is appropriate, accurately weighed, add methyl alcohol and make the mixed solution of every 1ml containing costunolide 120 μ g, dehydro-α-curcumene 140 μ g, to obtain final product.

The preparation of need testing solution: get six ingredients aucklandia root soft capsule 1.5g, porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 35ml, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 30 minutes, lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, and filters, get subsequent filtrate, to obtain final product.

Determination method: accurate reference substance solution and the each 20 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.

The every 1g of six ingredients aucklandia root soft capsule contains the banksia rose with costunolide (C ₁₅h ₂₀o ₂) and dehydro-α-curcumene (C ₁₅h ₁₈o ₂) total amount count 2.23mg.

test example 6: the detection of Liuwei Muxiang San

Banksia rose 200g, BAXIAGA 360g, emblic 500g, cardamom 80g, seed of pomegranate 400g, Bi roots of grass 100g;

Above Six-element, is ground into fine powder, sieves, and mixes by pharmacy conventional method, adds conventional auxiliary material, makes acceptable Liuwei Muxiang San clinically.

A. the thin layer of the banksia rose, seed of pomegranate is differentiated

Get above-mentioned Liuwei Muxiang San 2g, porphyrize, adds ethyl acetate 20ml, and ultrasonic extraction 30 minutes filters, and filtrate is concentrated into about 2ml, as need testing solution; Separately get the banksia rose, the each 0.5g of seed of pomegranate control medicinal material, be made in the same way of control medicinal material solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and makes the banksia rose, seed of pomegranate negative sample solution by the compound method of the above-mentioned banksia rose, seed of pomegranate need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 10 μ l of above-mentioned need testing solution and negative sample solution, control medicinal material solution 5 μ l, put respectively on same silica G plate, take cyclohexane-methylene chloride-ethyl acetate (15: 5: 0.5) as developping agent, launch, take out, dry, the vanillic aldehyde sulfuric acid solution of spray take percent weight in volume as 5%, it is clear that hot blast blows to spot colour developing.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, and negative noiseless.

B. the thin layer of emblic, the Bi roots of grass is differentiated

Get above-mentioned Liuwei Muxiang San 4.5g, porphyrize, adds ethanol 40ml, and ultrasonic extraction 60 minutes filters, and filtrate is concentrated into about 4ml, as need testing solution; Separately get emblic control medicinal material 2.5g, be made in the same way of control medicinal material solution; Get again pipering reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing pipering 2.5mg, product solution in contrast; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and makes emblic, Bi roots of grass negative sample solution by the compound method of above-mentioned emblic, Bi roots of grass need testing solution.According to 2010 editions one appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia ", draw the each 20 μ l of above-mentioned four kinds of solution, put respectively in same silica G F ₂₅₄on plate, take sherwood oil (60～90 ℃)-ethyl acetate-formic acid (9: 2: 0.4) as developping agent, thin layer plate is put in expansion cylinder saturated 20 minutes, launches, and takes out, and dries, and puts under ultraviolet lamp (254nm) and inspects.In test sample chromatogram, with control medicinal material, the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color, and negative noiseless.

C. the assay of the contained costunolide of the banksia rose and dehydro-α-curcumene

According to 2010 editions one appendix VI D of high performance liquid chromatography " Chinese Pharmacopoeia "

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile-water (67: 33) as mobile phase; Detection wavelength is 225nm, and column temperature is 40 ℃, and number of theoretical plate calculates and should be not less than 3000 by costunolide peak.

The preparation of reference substance solution: get costunolide reference substance and dehydro-α-curcumene reference substance is appropriate, accurately weighed, add methyl alcohol and make the mixed solution of every 1ml containing costunolide 80 μ g, dehydro-α-curcumene 170 μ g, to obtain final product.

The preparation of need testing solution: get Liuwei Muxiang San 2.5g, porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 45ml, weighed weight, ultrasonic processing (power 250W, frequency 40kHz) 45 minutes, lets cool, weighed weight again, the weight of supplying less loss with methyl alcohol, shakes up, and filters, get subsequent filtrate, to obtain final product.

Determination method: accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.

The every 1g of Liuwei Muxiang San contains the banksia rose with costunolide (C ₁₅h ₂₀o ₂) and dehydro-α-curcumene (C ₁₅h ₁₈o ₂) total amount count 2.56mg.

Claims (12)

1. a detection method for six ingredients aucklandia root preparation, described six ingredients aucklandia root preparation comprises the banksia rose, BAXIAGA, emblic, cardamom, seed of pomegranate and six kinds of medicinal ingredients of the Bi roots of grass; It is characterized in that, the method is differentiated the banksia rose, seed of pomegranate, emblic and/or the Bi roots of grass in described six ingredients aucklandia root preparation by thin-layered chromatography; Wherein:

(A) discriminating of the described banksia rose and seed of pomegranate comprises the following steps:

(1) get six ingredients aucklandia root preparation 0.5～5 weight portion, after porphyrize, add ethyl acetate 5～40 parts by volume, ultrasonic processing 10～60 minutes, filters, and filtrate is concentrated into 0.5～5 parts by volume, as the banksia rose, seed of pomegranate need testing solution; Separately get the banksia rose, each 0.1～3 weight portion of seed of pomegranate control medicinal material, make respectively the banksia rose, seed of pomegranate control medicinal material solution according to the preparation method of the described banksia rose, seed of pomegranate need testing solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and makes the banksia rose, seed of pomegranate negative sample solution by the compound method of the described banksia rose, seed of pomegranate need testing solution; Wherein, when above-mentioned weight portion measures with g, parts by volume is in mL;

(2) according to appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia " version in 2010, draw the need testing solution and negative sample solution 5～20 μ l, control medicinal material solution 1～10 μ l that in step (1), make, it is put respectively on same silica gel g thin-layer plate, cyclohexane-methylene chloride-ethyl acetate take volume ratio as 8～22:3～8:0.1～1 is developping agent, thin layer plate is put in expansion cylinder saturated 0～40 minute, launch, take out, dry, the vanillic aldehyde sulfuric acid solution of spray take percent weight in volume as 5%, it is clear that hot blast blows to spot colour developing; And/or

(B) discriminating of described emblic and the Bi roots of grass comprises the following steps:

(1 ') gets six ingredients aucklandia root preparation 0.5～5 weight portion, after porphyrize, adds ethanol 5～40 parts by volume, and ultrasonic processing 10～60 minutes filters, and filtrate is concentrated into 0.5～5 parts by volume, as emblic, Bi roots of grass need testing solution; Separately get emblic control medicinal material 0.1～3 weight portion, make emblic control medicinal material solution according to the preparation method of described emblic, Bi roots of grass need testing solution; Get again pipering reference substance appropriate, add methyl alcohol and make the solution of every 1ml containing pipering 0.5～3mg, as pipering reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and makes emblic, Bi roots of grass negative sample solution by the compound method of described emblic, Bi roots of grass need testing solution; Wherein, when above-mentioned weight portion measures with g, parts by volume is in mL;

(2 ') is according to appendix VI B test of thin-layered chromatography " Chinese Pharmacopoeia " version in 2010, draw each 5～20 μ l of need testing solution, control medicinal material solution, reference substance solution and negative sample solution that make in step (1 '), it is put respectively in same silica G F ₂₅₄on thin layer plate, the petroleum ether-ethyl acetate-formic acid take volume ratio as 1～10:2～6:0.05～0.5 is developping agent, and thin layer plate is put in expansion cylinder saturated 0～40 minute, launch, take out, dry, under 254nm ultraviolet lamp, inspect, the boiling range of described sherwood oil is 60～90 ℃.

2. detection method according to claim 1, wherein:

Described step (1) is: get six ingredients aucklandia root preparation 2 weight portions, after porphyrize, add ethyl acetate 20 parts by volume, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 2 parts by volume, as the banksia rose, seed of pomegranate need testing solution; Separately get the banksia rose, each 0.5 weight portion of seed of pomegranate control medicinal material, make respectively the banksia rose, seed of pomegranate control medicinal material solution according to the preparation method of the described banksia rose, seed of pomegranate need testing solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, seed of pomegranate, and makes the banksia rose, seed of pomegranate negative sample solution by the compound method of the described banksia rose, seed of pomegranate need testing solution; Wherein, when above-mentioned weight portion measures with g, parts by volume is in mL.

3. detection method according to claim 1, wherein:

Need testing solution and negative sample solution is 10 μ l, control medicinal material solution is 5 μ l in described step (2); The volume ratio of described developping agent cyclohexane-methylene chloride-ethyl acetate is 15:5:0.5; Described thin layer plate is put in expansion cylinder saturated 20 minutes.

4. detection method according to claim 1, wherein:

Described step (1 ') is: get six ingredients aucklandia root preparation 2 weight portions, after porphyrize, add ethanol 20 parts by volume, ultrasonic processing 30 minutes, filters, and filtrate is concentrated into 2 parts by volume, as emblic, Bi roots of grass need testing solution; Separately get emblic control medicinal material 0.5 weight portion, make emblic control medicinal material solution according to the preparation method of described emblic, Bi roots of grass need testing solution; Get again pipering reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing pipering 1mg, as pipering reference substance solution; In prescription ratio and preparation technology, preparation does not contain the negative sample of emblic, the Bi roots of grass, and makes emblic, Bi roots of grass negative sample solution by the compound method of described emblic, Bi roots of grass need testing solution; Wherein, when above-mentioned weight portion measures with g, parts by volume is in mL.

5. detection method according to claim 1, wherein:

Need testing solution, control medicinal material solution, reference substance solution and the negative sample solution in described step (2 '), drawn are 10 μ l; The volume ratio of described petroleum ether-ethyl acetate-formic acid is 6:4:0.25; Described thin layer plate is put in expansion cylinder saturated 20 minutes.

6. detection method according to claim 1, is characterized in that, described detection method further comprises the content with the contained costunolide of the banksia rose and/or dehydro-α-curcumene in six ingredients aucklandia root preparation described in high effective liquid chromatography for measuring, wherein:

The step of described high effective liquid chromatography for measuring comprises:

(1 ") got six ingredients aucklandia root preparation 0.1～3 weight portion, after porphyrize, puts in tool plug conical flask; precision adds methyl alcohol 10～50 parts by volume, and weighed weight, at power 250W; ultrasonic processing 15～45 minutes under frequency 40kHz; let cool, more weighed weight, supplies the weight of less loss; shake up with methyl alcohol; filter, get subsequent filtrate, as banksia rose need testing solution; Precision takes costunolide reference substance in addition and dehydro-α-curcumene reference substance is appropriate, add methyl alcohol and make the mixed solution of every 1ml containing costunolide 20～140 μ g and dehydro-α-curcumene 40～200 μ g, mix reference substance solution as costunolide and dehydro-α-curcumene; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, and makes banksia rose negative sample solution by the compound method of described banksia rose need testing solution; Wherein, when above-mentioned weight portion measures with g, parts by volume is in mL;

(2 ") be accurate upper step gained need testing solution, reference substance solution, each 5～20 μ l of negative sample solution of drawing respectively, and injection liquid chromatography, measures, and to obtain final product;

Wherein, the chromatographic condition of described high effective liquid chromatography for measuring is: with octadecylsilane chemically bonded silica be filling agent; Acetonitrile-water take volume ratio as 60～70:30～40 is mobile phase; Detection wavelength is 225 ± 2nm; Column temperature: 20～40 ℃, number of theoretical plate calculates and is not less than 3000 by costunolide peak.

7. detection method according to claim 6, the chromatographic condition of wherein said high effective liquid chromatography for measuring is: with octadecylsilane chemically bonded silica be filling agent; Acetonitrile-water take volume ratio as 65:35 is mobile phase; Detection wavelength is 225nm; Column temperature: 25 ℃, number of theoretical plate calculates and is not less than 3000 by costunolide peak.

8. detection method according to claim 6, (1 ") is wherein said step: get six ingredients aucklandia root preparation 1 weight portion, after porphyrize; put in tool plug conical flask, precision adds methyl alcohol 25 parts by volume, weighed weight; at power 250W, and under frequency 40kHz, ultrasonic processing 30 minutes, lets cool; weighed weight again; supply the weight of less loss with methyl alcohol, shake up, and filters; get subsequent filtrate, as banksia rose need testing solution; Precision takes costunolide reference substance with appropriate with dehydro-α-curcumene reference substance in addition, accurately weighed, add methyl alcohol and make the mixed solution of every 1ml containing costunolide 40 μ g and dehydro-α-curcumene 60 μ g, mix reference substance solution as costunolide and dehydro-α-curcumene; In prescription ratio and preparation technology, preparation does not contain the negative sample of the banksia rose, and makes banksia rose negative sample solution by the compound method of described banksia rose need testing solution; Wherein, when above-mentioned weight portion measures with g, parts by volume is in mL.

9. detection method according to claim 6, (2 ") are wherein said step: accurate need testing solution, reference substance solution, the each 10 μ l of negative sample solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.

10. according to the detection method described in any one in claim 1 to 9, wherein:

The bulk drug composition of described six ingredients aucklandia root preparation comprises: the banksia rose 200 weight portions, BAXIAGA 360 weight portions, emblic 500 weight portions, cardamom 80 weight portions, seed of pomegranate 400 weight portions and the Bi roots of grass 100 weight portions.

11. detection methods according to claim 10, wherein:

The preparation method of described six ingredients aucklandia root preparation is: described bulk drug is ground into fine powder, sieves, mix, the appropriate pill that adds water, obtains pill; Or described bulk drug is ground into fine powder, and sieve, mix, by pharmacy conventional method, add conventional auxiliary material, make the oral formulations of accepting clinically: granule, pill, capsule, tablet or powder.

12. according to the detection method described in any one in claim 1 to 9, and wherein, described six ingredients aucklandia root preparation is granule, pill, capsule, tablet or powder.

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