CN102778529B

CN102778529B - Method for testing quality of tibetan medicine composition Liuwei Datuoyeyunshi preparation

Info

Publication number: CN102778529B
Application number: CN201210279814.4A
Authority: CN
Inventors: 张义智; 高英涵; 刘玉芹; 任松鹏; 李怀平
Original assignee: Shandong Arura Pharmaceutical Research & Development Co Ltd
Current assignee: JINHE TIBETAN MEDICINE CO., LTD.
Priority date: 2012-08-08
Filing date: 2012-08-08
Publication date: 2015-03-11
Anticipated expiration: 2032-08-08
Also published as: CN102778529A

Abstract

The invention discloses a method for testing the quality of tibetan medicine composition Liuwei Datuoyeyunshi preparation. The method comprises the following steps: carrying out the qualitative identification on the Datuoyeyunshi and Amomum kravanh in the Liuwei Datuoyeyunshi San by susing a thin layer chromatography (TLC method), and adopting a high performance liquid chromatography (HPLC method) to carry out the qualitative identification on the gallic acid contained in the Datuoyeyunshi and the pomegranate seed, the cinnamaldehyde contained in the cinnamon, the piperine contained in the fructus piperis longi and the hydroxyl carthamin yellow A contained in the safflower carthamus in the Liuwei Datuoyeyunshi San. The invention provides the method for identifying the Datuoyeyunshi and Amomum kravanh in the preparation, and a method for testing the contents of effective ingredients such as the gallic acid, the cinnamaldehyde, piperine, the hydroxyl carthamin yellow A and the like, the quality standard of a product is improved, the security, effectiveness and controllable quality of the medicine are ensured, and thus the preparation can be used for curing diseases more effectively.

Description

A kind of detection method of medicine composition Liuwei bonduc preparation

Technical field

The present invention relates to a kind of detection method of Tibetan medicinal composition preparation, particularly a kind of medicine composition Liuwei bonduc falls apart and the detection method of preparation.

Background technology

Six-element bonduc is loose to be recorded in the national drug standards, standard number: WS ₃-BC-0282-95, has the effect of warm kidney, enriching yin, is mainly used in women's leukorrhea.This compound preparation is made up of bonduc 90g, seed of pomegranate 90g, Chinese cassia tree 5g, cardamom 40g, Bi roots of grass 5g, safflower 50g.Bonduc is the dry mature seed of legume bonduc Caesalpinia crista L., has warm kidney, by cold effect; Seed of pomegranate is the dry seed of Punicaceae plant pomegranate Punica granatum L., cures mainly Baconic's sympotoms caused by cold factors, stomach cold disease and all stomach trouble; Chinese cassia tree is the dry bark of canella Chinese cassia tree Cinnamomum cassis Presl, has benefit fire supporing yang, guiding fire to origin, eliminating cold to stop pain, effect of warming meridian; Cardamom is the dry mature fruit of zingiberaceous plant Amomum cardamomum Amomum kravanh Pierre ex Gagnep. or amomum compactum Soland ex Maton Amomum compactum Soland ex Maton, has effect of dampness elimination promoting the circulation of qi, warming middle energizer to arrest vomiting, appetite-stimulating indigestion-relieving; The Bi roots of grass is the nearly ripe or mature fruit cluster of the drying of Piperaceae plant Bi roots of grass Piper longum L., has effect of warming spleen and stomach for dispelling cold, lower gas pain relieving; Safflower is the dried floral of feverfew safflower Carthamus tinctorius L., has effect of activating blood to promote menstruation, blood stasis removing analgesic.

It is normal before and after some women menstrual period that with the symptom such as irregular menstruation and abnormal leucorrhea, endocrine is not normal is arch-criminal, and the true cause of the abdominal pain cold and stomach cold that is kidney.If left for a long time, the development of this situation, probably damages uterus, causes infertile.Live in the Tibetan of Tibetan area, to take after mixing it with water bonduc often to the girl after the initial stage.Tibetan medicine's ancient books and records are recorded, effect that bonduc has warm kidney, dispels cold, and are applicable to that kidney is cold, stomach cold disease.Six-element bonduc is loose is main ingredient with bonduc, is aided with the number taste tonic herbs such as Chinese cassia tree, is treatment female irregular menstruation, the good medicine of abnormal leucorrhea.

Six-element bonduc falls apart in proper mass standard, only has every regulation relevant under should meeting powder item, has not both had TLC distinguish item, also without any assay index.Drug quality problems and human health are closely related, nowadays also more and more higher to the requirement of Drug's control, but said preparation quality standard is on the low side, and effectively can not ensure the quality of said preparation, said preparation quality standard is in urgent need to be improved.

Summary of the invention

The object of the invention is for overcoming above-mentioned the deficiencies in the prior art, a kind of quality determining method of medicine composition Liuwei bonduc preparation is provided.

Term illustrates:

Six-element bonduc is loose is the nomenclature of drug that Tibetan medicine (standard number WS3-BC-0282-95) promulgated by the ministries or commissions of the Central Government is recorded.

Six-element bonduc preparation (or being " Six-element bonduc loose and preparation ") comprises Six-element bonduc and falls apart and to fall apart other preparations prepared by Raw formula with Six-element bonduc, as micropill, dripping pill, tablet, capsule, particle, medicine materical crude slice or dispersing tablet etc., the proportioning of the bulk drug of each preparation can be fallen apart identical with Six-element bonduc, also can need to carry out suitable adjustment according to treatment.

For achieving the above object, the present invention adopts following technical proposals:

A kind of quality determining method of medicine composition Liuwei bonduc preparation, said preparation is the various preparations be made up by pharmacy conventional method of bonduc, seed of pomegranate, Chinese cassia tree, cardamom, the Bi roots of grass, safflower, quality determining method comprises the thin-layer chromatography qualitative identification method to bonduc, cardamom in preparation, one or more in the liquid chromatography content assaying method of the hydroxyl radical carthamin yellow carthamus A that the pipering that the cinnaldehydrum that the gallic acid contained bonduc and seed of pomegranate, Chinese cassia tree contain, the Bi roots of grass contain, safflower contain.

Preferably as the present invention, quality determining method is one or more in following methods:

Differentiate:

A. the discriminating of bonduc: it is for contrast with bonduc control medicinal material, with volume parts than 10:(0.1-0.5): the boiling range of 0.1 is 60-90 DEG C of petroleum ether-ethyl acetate-glacial acetic acid is that developping agent launches on silica gel g thin-layer plate, the thin-layered chromatography being developer with the ethanol solution of sulfuric acid of volume parts than 10%;

B. the discriminating of cardamom: it is for contrast with cardamom control medicinal material, with volume parts ratio (9-8): the boiling range of (1-2) is 60-90 DEG C of petroleum ether-ethyl acetate is that developping agent launches on silica gel g thin-layer plate, the thin-layered chromatography being developer with the vanillin-sulfuric acid solution of quality volume portion rate 1%;

C. the discriminating of Chinese cassia tree: it is for contrast with Chinese cassia tree control medicinal material, with volume parts ratio (6-10): the boiling range of (1-3) is 60-90 DEG C of petroleum ether-ethyl acetate is that developping agent launches on silica gel g thin-layer plate, the thin-layered chromatography being developer with the vanillin-sulfuric acid solution of quality volume portion rate 1-5%;

Assay:

A. the assay of gallic acid: it is with gallic acid reference substance for contrast, take octadecylsilane chemically bonded silica as filling agent; With volume parts ratio (1-3): the methyl alcohol-volume parts of (97-99) is than 1% glacial acetic acid aqueous solution for mobile phase, and determined wavelength is the high performance liquid chromatography of 275nm;

B. the assay of cinnaldehydrum: it is with cinnaldehydrum reference substance for contrast, take octadecylsilane chemically bonded silica as filling agent, with volume parts than the acetonitrile-water of 30-70:30-70 for mobile phase, determined wavelength is 290nm is high performance liquid chromatography;

C. the assay of pipering: it is with pipering reference substance for contrast, take octadecylsilane chemically bonded silica as filling agent, with volume parts than the methanol-water of 75-80:20-25 for mobile phase, determined wavelength is the high performance liquid chromatography of 343nm;

D. the assay of hydroxyl radical carthamin yellow carthamus A: it is for contrast with hydroxyl radical carthamin yellow carthamus A reference substance, take octadecylsilane chemically bonded silica as filling agent, with volume parts than the methyl alcohol-volume parts ratio of 25-35:65-75 be 1% glacial acetic acid aqueous solution for mobile phase, determined wavelength is the high performance liquid chromatography of 403nm.

As the present invention further preferably, described quality determining method is one or more in following methods:

Differentiate:

A. the discriminating of bonduc

Get powder formulation 2-4g to be detected, add methyl alcohol 10-30ml, ultrasonic process 20-40 minute, filter, filtrate is as need testing solution; Separately get bonduc control medicinal material 0.5-1g, with legal system for bonduc control medicinal material solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VIB), draw above-mentioned two kinds of solution each 2-5 μ l, put respectively on same silica gel g thin-layer plate, with volume parts than 10:(0.1-0.5): the boiling range of 0.1 is 60-90 DEG C of petroleum ether-ethyl acetate-glacial acetic acid is developping agent, launch, take out, dry, spray, with the ethanol solution of sulfuric acid of volume parts than 10%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

B. the discriminating of cardamom

Get powder formulation 1-3g to be detected, put in 100ml round-bottomed flask, add water 40-80ml, extract according to determination of volatile oil Division A League Matches of French Football method (Chinese Pharmacopoeia version in 2010 annex X D), adding water on the top of volatile oil extractor makes water overflow in flask, then adds ethyl acetate 1ml, refluxing extraction 1-3 hour, be cooled to room temperature, divide and get acetic acid ethyl fluid as need testing solution; Separately get cardamom control medicinal material 0.5-1g, with legal system for cardamom control medicinal material solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw above-mentioned two kinds of solution each 5-10 μ l, put respectively on same silica gel g thin-layer plate, with volume parts ratio (9 ~ 8): the boiling range of (1 ~ 2) is that 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent, launch, take out, dry, spray, with the vanillin-sulfuric acid solution of quality volume portion rate 1%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

C. Chinese cassia tree is differentiated

Get powder formulation 1-5g to be detected, put in 100ml round-bottomed flask, add 40-70ml water, extract according to determination of volatile oil Division A League Matches of French Football method (Chinese Pharmacopoeia version in 2010 annex X D), add water on the top of volatile oil extractor and water is overflowed in flask, then add ethyl acetate 1-2ml, refluxing extraction 1-3 hour, be cooled to room temperature, divide and get acetic acid ethyl fluid, as need testing solution.Get Chinese cassia tree control medicinal material 0.5-2g, with legal system for control medicinal material solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw above-mentioned two kinds of solution each 5-10 μ l, put respectively in same silica G chromatographic sheet, with volume parts ratio (6 ~ 10): the boiling range of (1 ~ 3) is that 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent, launch, take out, dry, spray is with quality volume portion rate 1-5% vanillin-sulfuric acid solution, and 105 DEG C to be heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

Assay:

A. the assay of gallic acid

Measure according to high performance liquid chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI D);

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; With volume parts ratio (1-3): the methyl alcohol-volume parts of (97-99) than 1% glacial acetic acid aqueous solution for mobile phase; Determined wavelength is 275nm; Number of theoretical plate calculates should be not less than 2000 by gallic acid peak;

The preparation of reference substance solution gets gallic acid reference substance in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing 0.1mg, to obtain final product;

Powder formulation 1-3g to be detected is got in the preparation of need testing solution, accurately weighed, and put in tool plug conical flask, precision adds methyl alcohol 20-30ml, close plug, weighed weight, ultrasonic process 30 minutes, lets cool, more weighed weight, supplies the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;

Determination method draws reference substance solution and need testing solution each 5-10 μ l respectively, injection liquid chromatography, measures, to obtain final product.

B. the assay of cinnaldehydrum

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; With volume parts than the acetonitrile-water of 30-70:30-70 for mobile phase; Determined wavelength is 290nm; Number of theoretical plate calculates should be not less than 3000 by cinnaldehydrum peak;

The preparation of reference substance solution gets cinnaldehydrum reference substance in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing 35 μ g, to obtain final product;

C. the assay of pipering

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; With volume parts than the methanol-water of 75-80:20-25 for mobile phase; Determined wavelength is 343nm; Number of theoretical plate calculates should be not less than 1500 by pipering peak;

The preparation of reference substance solution gets pipering reference substance in right amount, accurately weighed, puts in brown measuring bottle, adds absolute ethyl alcohol and makes the solution of every 1ml containing 20 μ g, to obtain final product;

Powder formulation 2-4g to be detected is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds absolute ethyl alcohol 20-30ml, close plug, weighed weight, ultrasonic process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with absolute ethyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;

D. the assay of hydroxyl radical carthamin yellow carthamus A

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that 1% glacial acetic acid aqueous solution is for mobile phase with volume parts than the methyl alcohol-volume parts ratio of 25-35:65-75; Determined wavelength is 403nm; Number of theoretical plate calculates should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak;

The preparation of reference substance solution gets hydroxyl radical carthamin yellow carthamus A reference substance in right amount, accurately weighed, puts in brown bottle, adds volume parts and makes the solution of every 1ml containing 0.13mg than 25% methanol aqueous solution, to obtain final product;

Powder formulation 1-3g to be detected is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds volume parts than 25% methanol aqueous solution 20-30ml, close plug, weighed weight, ultrasonic 40 minutes, let cool, more weighed weight, supply the weight of less loss than 25% methanol aqueous solution by volume parts, shake up, filter, get subsequent filtrate, to obtain final product;

Described preparation raw material medicine composition is preferably: bonduc 90 weight portion, seed of pomegranate 90 weight portion, Chinese cassia tree 5 weight portion, cardamom 40 weight portion, the Bi roots of grass 5 weight portion, safflower 50 weight portion.

Described preparation is that Six-element bonduc is loose, Six-element bonduc particle, Six-element bonduc ball, Six-element bonduc sheet or Six-element bonduc capsule.The bulk drug that Six-element bonduc particle, Six-element bonduc ball, Six-element bonduc sheet and Six-element bonduc capsule are loose with Six-element bonduc as can be seen here forms identical, therefore can adopt same or analogous quality determining method.

For above-mentioned preferred feedstock medicine composition or its preparation, described quality determining method specifically:

Differentiate:

A. the discriminating of bonduc

Get powder formulation 3g to be detected, add methyl alcohol 20ml, ultrasonic process 30 minutes, filter, filtrate is as need testing solution; Separately get bonduc control medicinal material 1g, with legal system for bonduc control medicinal material solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw each 3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume parts than the boiling range of 10:0.3:0.1 for 60-90 DEG C of petroleum ether-ethyl acetate-glacial acetic acid is for developping agent, launch, take out, dry, spray, with the ethanol solution of sulfuric acid of volume parts than 10%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

B. the discriminating of cardamom

Get powder formulation 2g to be detected, put in 100ml round-bottomed flask, add water 50ml, extract according to determination of volatile oil Division A League Matches of French Football method (Chinese Pharmacopoeia version in 2010 annex X D), adding water on the top of volatile oil extractor makes water overflow in flask, add ethyl acetate 1ml again, refluxing extraction 2 hours, divide and get acetic acid ethyl fluid as need testing solution; Separately get cardamom control medicinal material 1g, with legal system for cardamom control medicinal material solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume parts than the boiling range of 8:2 for 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent, launch, take out, dry, spray, with the vanillin-sulfuric acid solution of quality volume portion rate 1%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

C. the discriminating of Chinese cassia tree

Get powder formulation 2g to be detected, put in 100ml round-bottomed flask, add 60ml water, extract according to determination of volatile oil Division A League Matches of French Football method (Chinese Pharmacopoeia version in 2010 annex X D), add water on the top of volatile oil extractor and water is overflowed in flask, then add ethyl acetate 1ml, refluxing extraction 1 hour, be cooled to room temperature, divide and get acetic acid ethyl fluid, as need testing solution; Get Chinese cassia tree control medicinal material 1g, with legal system for control medicinal material solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same silica G chromatographic sheet, be that 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent with the boiling range of volume parts ratio (10:2), launch, take out, dry, spray is with quality volume portion rate 1% vanillin-sulfuric acid solution, and 105 DEG C to be heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

Assay:

A. the assay of gallic acid

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; With volume parts than the methyl alcohol-volume parts of 2:98 than 1% glacial acetic acid aqueous solution for mobile phase; Determined wavelength is 275nm; Number of theoretical plate calculates should be not less than 2000 by gallic acid peak;

Powder formulation 2g to be detected is got in the preparation of need testing solution, accurately weighed, and put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 30 minutes, lets cool, more weighed weight, supplies the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;

Determination method draws reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product;

This product gallic acid (C ₇h ₆o ₅) content must not be less than 2.32mg/g.

B. the assay of cinnaldehydrum

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; With volume parts than the acetonitrile-water of 36:64 for mobile phase; Determined wavelength is 290nm; Number of theoretical plate calculates should be not less than 3000 by cinnaldehydrum peak;

This product cinnaldehydrum (C ₉h ₈o) content must not be less than 0.21mg/g.

C. the assay of pipering

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; With volume parts than the methanol-water of 78:22 for mobile phase; Determined wavelength is 343nm; Number of theoretical plate calculates should be not less than 1500 by pipering peak;

Powder formulation 3g to be detected is got in the preparation of need testing solution, accurately weighed, and put in tool plug conical flask, precision adds absolute ethyl alcohol 25ml, close plug, weighed weight, ultrasonic process 30 minutes, lets cool, more weighed weight, supplies the weight of less loss with absolute ethyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;

This product pipering (C ₁₇h ₁₉nO ₃) content must not be less than 0.13mg/g.

D. the assay of hydroxyl radical carthamin yellow carthamus A

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent; Be that 1% glacial acetic acid aqueous solution is for mobile phase with volume parts than the methyl alcohol-volume parts ratio of 30:70; Determined wavelength is 403nm; Number of theoretical plate calculates should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak;

Powder formulation 2g to be detected is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds volume parts than 25% methanol aqueous solution 25ml, close plug, weighed weight, ultrasonic 40 minutes, let cool, more weighed weight, supply the weight of less loss than 25% methanol aqueous solution by volume parts, shake up, filter, get subsequent filtrate, to obtain final product;

This product hydroxyl radical carthamin yellow carthamus A (C ₂₇h ₃₀o ₁₅) content must not be less than 1.15mg/g.

Through Tibetan medicine hospital of Qinghai Province fame president, the qualification of Qinghai Province Tibetan medicine principal expert Nima, in the application, bonduc control medicinal material is the dry mature seed of legume bonduc Caesalpinia crista L.; Cardamom control medicinal material is the dry mature fruit of zingiberaceous plant Amomum cardamomum Amomum kravanh Pierre ex Gagnep..

The unit corresponding relation of the weight portion/mass parts described in this instructions and parts by volume is g/ml or kg/L.

The invention provides a kind of medicine composition Liuwei bonduc to fall apart and the thin-layer chromatography qualitative identification method of bonduc, cardamom in preparation, the liquid chromatography content assaying method of the hydroxyl radical carthamin yellow carthamus A that the pipering that the cinnaldehydrum that the gallic acid that bonduc and seed of pomegranate contain, Chinese cassia tree contain, the Bi roots of grass contain, safflower contain, the quality of comprehensive control said preparation, make said preparation while disease therapy, ensure that the safe and effective, quality controllable of medication.

The invention discloses a kind of medicine composition Liuwei bonduc to fall apart and the quality determining method of preparation.In using thin-layered chromatography (TLC method) loose to Six-element bonduc, bonduc, cardamom, Chinese cassia tree have carried out Qualitive test, adopt high performance liquid chromatography (HPLC method) to carry out quantitative detection to the Six-element bonduc hydroxyl radical carthamin yellow carthamus A that pipering that cinnaldehydrum that gallic acid that middle bonduc and seed of pomegranate contain, Chinese cassia tree contain, the Bi roots of grass contain, safflower contain that falls apart.The inventive method establishes the discrimination method of bonduc in said preparation, cardamom, Chinese cassia tree, the content assaying method of the effective constituents such as gallic acid, cinnaldehydrum, pipering, hydroxyl radical carthamin yellow carthamus A, improve target level of product quality, ensure that the safe and effective, quality controllable of medication, make said preparation can disease therapy more effectively.This law also can be used for other loose preparations of medicine composition Liuwei bonduc simultaneously, as Six-element bonduc particle, Six-element bonduc ball, Six-element bonduc sheet, Six-element bonduc capsule etc.

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

Experimental example 1: TLC distinguish is tested

Medicine composition Liuwei bonduc is loose scolds Tibetan medicine medicine company incorporated company by Qinghai gold provides.

A. the discriminating of bonduc

Get medicine composition Liuwei bonduc divided powder 3g, add methyl alcohol 20ml, ultrasonic process 30 minutes, filter, filtrate is as need testing solution; Get the negative control sample powder 3g lacking bonduc by prescription proportions, with the standby negative control solution lacking bonduc of legal system; Separately get bonduc control medicinal material 1g, with legal system for bonduc control medicinal material solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw each 3 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with volume parts than the boiling range of 10:0.3:0.1 for 60-90 DEG C of petroleum ether-ethyl acetate-glacial acetic acid is for developping agent, launch, take out, dry, spray, with the ethanol solution of sulfuric acid of volume parts than 10%, is heated to spot development at 105 DEG C clear.

Test findings: in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color, negative control does not disturb.The results are shown in Figure 1.

Investigate chloroform-methanol-glacial acetic acid respectively, boiling range has been 60-90 DEG C of sherwood oil-acetone-glacial acetic acid, boiling range has been chromatographic resolution effect under the different developping agent different volumes ratios such as 60-90 DEG C of petroleum ether-ethyl acetate-glacial acetic acid, result with volume parts than the boiling range of 10:0.1-0.5:0.1 for 60-90 DEG C of petroleum ether-ethyl acetate-glacial acetic acid is under developping agent condition, all have and launch effect preferably.Wherein, with volume parts than 10:0.3:0.1 boiling range for 60-90 DEG C of petroleum ether-ethyl acetate-glacial acetic acid for developping agent launch effect best.Result shows, this discrimination method specificity is strong, can be used as medicine composition Liuwei bonduc and to fall apart the thin-layer identification method of middle bonduc.

B. the discriminating of cardamom

Get medicine composition Liuwei bonduc divided powder 2g, put in 100ml round-bottomed flask, add water 50ml, extract according to determination of volatile oil Division A League Matches of French Football method (Chinese Pharmacopoeia version in 2010 annex X D), adding water on the top of volatile oil extractor makes water overflow in flask, add ethyl acetate 1ml again, refluxing extraction 2 hours, divide and get acetic acid ethyl fluid as need testing solution; Get the negative control sample powder 2g lacking cardamom by prescription proportions, with the standby negative control solution lacking cardamom of legal system; Separately get cardamom control medicinal material 1g, with legal system for cardamom control medicinal material solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with volume parts than the boiling range of 8:2 for 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent, launch, take out, dry, spray, with the vanillin-sulfuric acid solution of quality volume portion rate 1%, is heated to spot development at 105 DEG C clear.

Test findings: in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color, negative control does not disturb.The results are shown in Figure 2.

Under volume parts is 60-90 DEG C of petroleum ether-ethyl acetate developping agent condition than the boiling range of 9-8:1-2, all has and launch effect preferably.Wherein, with volume parts than 8:2 boiling range for 60-90 DEG C of petroleum ether-ethyl acetate for developping agent launch effect best.Result shows, this discrimination method specificity is strong, can be used as medicine composition Liuwei bonduc and to fall apart the thin-layer identification method of middle cardamom.

C. the discriminating of Chinese cassia tree

Get medicine composition Liuwei bonduc divided powder 2g, porphyrize, put in 100ml round-bottomed flask, add water 60ml, extracts according to determination of volatile oil Division A League Matches of French Football method (Chinese Pharmacopoeia version in 2010 annex X D), adding water on the top of volatile oil extractor makes water overflow in flask, add 1ml ethyl acetate again, refluxing extraction 1 hour, is cooled to room temperature, divide and get acetic acid ethyl fluid, as need testing solution.Get Chinese cassia tree control medicinal material 1g, with legal system for control medicinal material solution.Get the misrun osmanthus negative controls by prescription proportions, according to test sample preparation method, with legal system for negative sample solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 μ l of above-mentioned three kinds of solution, put respectively in same silica G chromatographic sheet, with volume parts than the boiling range of 10:2 for 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent, launch, take out, dry, spray the vanillin-sulfuric acid solution with 1%, 105 DEG C to be heated to spot development clear.

Test findings: in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color, negative control is noiseless.The results are shown in Figure 3.

Under the petroleum ether-ethyl acetate developping agent condition that volume parts is 60-90 DEG C than the boiling range of 6-10:1-3, all have and launch effect preferably.Wherein, best to launch effect under the boiling range of the volume ratio 10:2 petroleum ether-ethyl acetate developping agent condition that is 60-90 DEG C.Result shows, this discrimination method specificity is strong, can be used as medicine composition Liuwei bonduc and to fall apart the thin-layer identification method of middle Chinese cassia tree

Experimental example 2: the assay experiment of gallic acid

1. instrument, reagent and test sample

Instrument: 1220 type Agilent high performance liquid chromatographs; Shimadzu AuW220D electronic balance.

Reference substance: gallic acid reference substance (Nat'l Pharmaceutical & Biological Products Control Institute) lot number: 110831-200803.

Sample: Six-element bonduc falls apart (Qinghai gold is scolded Tibetan medicine medicine company incorporated company and provided), and lot number is respectively: 20110516,20110517,20110518.

2. the selection of determined wavelength

Get gallic acid reference substance solution, scan in 190 ~ 400nm wavelength coverage, record it and have absorption maximum at 215nm and 275nm wavelength place, the relatively chromatogram of sample under 215nm and 275nm wavelength under identical chromatographic conditions, found that determined wavelength is that the chromatogram gallic acid peak of 275nm and adjacent chromatographic peak reach good baseline separation, and impurity interference is less, therefore selected 275nm is determined wavelength.

3. mobile phase is selected

Research finds, with volume parts than the methyl alcohol-volume parts of 1-3:99-97 than 1% glacial acetic acid aqueous solution for mobile phase time, gallic acid all can reach good chromatographic resolution effect.Wherein with methyl alcohol-volume parts than the volume parts of 1% glacial acetic acid aqueous solution than 2:98 for mobile phase is optimum.

4. system suitability

Under above-mentioned chromatographic condition, accurate absorption reference substance solution, each 10 μ l of need testing solution respectively, injection liquid chromatography, record chromatogram.Result shows, the degree of separation that in reference substance, test sample chromatogram, gallic acid is adjacent chromatographic peak is all greater than 1.5.The results are shown in Figure 3, Fig. 4.

5. reference substance preparation

Get gallic acid reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 0.1mg, to obtain final product.

6. test sample preparation

The investigation of 6.1 extracting method

By method under assay item, need testing solution is detected.3 parts are prepared, respectively ultrasonic, backflow, Zhen Oscillating process 30 minutes by the method under the preparation of need testing solution.With the content of gallic acid in every gram of medicine for index determination extracting method.The results are shown in Table 1.

Table 1 extracting method investigates test findings

Result shows, ultrasonic basically identical with the content of gallic acid in refluxing extraction gained every gram of medicine, considers that ultrasonic extraction is easy, easy to operate, therefore selects extracting method to be ultrasonic extraction.

The investigation of 6.2 Extraction solvent

By method under assay item, need testing solution is detected.Prepare 3 parts by the method under the preparation of need testing solution, precision adds water, ethanol, each 25ml of methyl alcohol respectively.With the content of gallic acid in every gram of medicine for index determination Extraction solvent.The results are shown in Table 2.

Table 2 Extraction solvent investigates test findings

Result shows, the gallic acid content that methyl alcohol is surveyed is the highest, therefore selects Extraction solvent to be methyl alcohol.

The investigation of 6.3 extraction times

By method under assay item, need testing solution is detected.3 parts are prepared, respectively ultrasonic process 20 minutes, 30 minutes, 40 minutes by the method under the preparation of need testing solution.With the content of gallic acid in every gram of medicine for index determines extraction time.The results are shown in Table 3.

Table 3 extraction time investigates test findings

Result shows, in ultrasonic extraction 30 minutes and 40 minutes gained every gram medicines, the content of gallic acid is basically identical, therefore selects extraction time to be 30 minutes.

7. the preparation of typical curve and the investigation of linear relationship

Precision measures gallic acid reference substance stock solution solution (gallic acid content is 251.2 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml, put in 10ml volumetric flask respectively, with methanol dilution to scale, shake up, each accurate sample introduction 10 μ l, with peak area (A), linear regression is carried out to reference substance concentration (C), obtain gallic acid regression equation: A=14.005C+4.1658, related coefficient: R=0.9997.Result shows, gallic acid is within the scope of 25.12 μ g/ml ~ 251.2 μ g/ml, and the peak area (A) of gallic acid is good with reference substance concentration (C) linear relationship.The results are shown in Table 4.

Table 4 gallic acid linear relationship investigates result

8. precision test

Accurate absorption gallic acid reference substance solution 10 μ l, injection liquid chromatography, each METHOD FOR CONTINUOUS DETERMINATION 6 times, record peak area also calculates relative standard deviation.Result shows, instrument precision is good.The results are shown in Table 5.

Table 5 gallic acid Precision test result

9. stability test

After prepared by need testing solution, accurate absorption 10 μ l, injection liquid chromatography, record peak area, measured once every 2 hours later, investigates 8 hours, and record peak area also calculates relative standard deviation.Result shows, in need testing solution, gallic acid measurement result in 8 hours is stablized.The results are shown in Table 6.

Table 6 gallic acid stability test result

10. replica test

Get and to fall apart sample (product batch number: 20110516) 2g with a collection of medicine composition Liuwei bonduc, accurately weighed, totally 6 parts, need testing solution is prepared by the method under the preparation of need testing solution, accurate absorption 10 μ l respectively, injection liquid chromatography, the content of gallic acid in calculation sample.Result shows, this analytical approach repeatability is good.The results are shown in Table 7.

Table 7 gallic acid replica test result

11. recovery tests

Precision take with a collection of medicine composition Liuwei bonduc fall apart sample (product batch number: 20110516) 6 parts, each precision adds gallic acid reference substance, measures its content, calculate the recovery.Result shows, this assay method measurement result is accurate.The results are shown in Table 8.

Table 8 gallic acid recovery test result

12. sample determinations

Get loose three batches of medicine composition Liuwei bonduc, measure and calculate gallic acid content.The results are shown in Table 9.

Table 9 sample size measurement result

Experimental example 3: the assay experiment of cinnaldehydrum

1. instrument, reagent and test sample

Reference substance: cinnaldehydrum reference substance (Nat'l Pharmaceutical & Biological Products Control Institute) lot number: 110710-200714.

2. the selection of determined wavelength

Get cinnaldehydrum reference substance solution, scan in 190 ~ 400nm wavelength coverage, recording it has absorption maximum at 290nm wavelength place, and therefore selected 290nm is determined wavelength.

3. mobile phase is selected

Research finds, with volume parts than the acetonitrile-water of 30-70:70-40 for mobile phase time, cinnaldehydrum all can reach good chromatographic resolution effect.Wherein with the volume parts of acetonitrile-water than 36:64 for mobile phase is optimum.

4. the investigation of system suitability and negative interference

Under above-mentioned chromatographic condition, accurate absorption reference substance solution, need testing solution, each 10 μ l of negative control solution respectively, injection liquid chromatography, record chromatogram.Result shows, the degree of separation that in reference substance, test sample chromatogram, cinnaldehydrum is adjacent chromatographic peak is all greater than 1.5, and negative control does not disturb.The results are shown in Figure 5, Fig. 6, Fig. 7.

5. reference substance preparation

Get cinnaldehydrum reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 35 μ g, to obtain final product.

6. test sample preparation

The investigation of 6.1 extracting method

By method under assay item, need testing solution is detected.3 parts are prepared, respectively ultrasonic, backflow, Zhen Oscillating process 30 minutes by the method under the preparation of need testing solution.With the content of cinnaldehydrum in every gram of medicine for index determination extracting method.The results are shown in Table 10.

Table 10 extracting method investigates test findings

Result shows, in ultrasonic extraction gained every gram of medicine, the content of cinnaldehydrum extracts apparently higher than Zhen Oscillating, therefore selects extracting method to be ultrasonic extraction.

The investigation of 6.2 Extraction solvent

By method under assay item, need testing solution is detected.Prepare 3 parts by the method under the preparation of need testing solution, precision adds methyl alcohol, ethanol, each 25ml of ethyl acetate respectively.With the content of cinnaldehydrum in every gram of medicine for index determination Extraction solvent.The results are shown in Table 11.

Table 11 Extraction solvent investigates test findings

Result shows, the Determination of Cinnamaldehyde that methyl alcohol is surveyed is the highest, therefore selects Extraction solvent to be methyl alcohol.

The investigation of 6.3 extraction times

By method under assay item, need testing solution is detected.3 parts are prepared, respectively ultrasonic process 20 minutes, 30 minutes, 40 minutes by the method under the preparation of need testing solution.With the content of cinnaldehydrum in every gram of medicine for index determines extraction time.The results are shown in Table 12.

Table 12 extraction time investigates test findings

Result shows, in ultrasonic extraction 30 minutes and 40 minutes gained every gram medicines, the content of cinnaldehydrum is basically identical, therefore selects extraction time to be 30 minutes.

Precision measures cinnaldehydrum reference substance stock solution solution (Determination of Cinnamaldehyde is 72.4 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml, put in 10ml volumetric flask respectively, with methanol dilution to scale, shake up, each accurate sample introduction 10 μ l, with peak area (A), linear regression is carried out to reference substance concentration (C), obtain cinnaldehydrum regression equation: A=20.391C+2.2135, related coefficient: R=0.9999.Result shows, cinnaldehydrum is within the scope of 7.24 μ g/ml ~ 72.4 μ g/ml, and the peak area (A) of cinnaldehydrum is good with reference substance concentration (C) linear relationship.The results are shown in Table 13.

Table 13 cinnaldehydrum linear relationship investigates result

8. precision test

Accurate absorption cinnaldehydrum reference substance solution 10 μ l, injection liquid chromatography, each METHOD FOR CONTINUOUS DETERMINATION 6 times, record peak area also calculates relative standard deviation.Result shows, instrument precision is good.The results are shown in Table 14.

Table 14 cinnaldehydrum Precision test result

9. stability test

After prepared by need testing solution, accurate absorption 10 μ l, injection liquid chromatography, record peak area, measured once every 2 hours later, investigates 8 hours, and record peak area also calculates relative standard deviation.Result shows, in need testing solution, cinnaldehydrum measurement result in 8 hours is stablized.The results are shown in Table 15.

Table 15 cinnaldehydrum stability test result

10. replica test

Get with a collection of medicine composition Liuwei bonduc fall apart sample (product batch number: 20110516) 2g, accurately weighed, totally 6 parts, need testing solution is prepared by the method under the preparation of need testing solution, accurate absorption 10 μ l respectively, injection liquid chromatography, the content of cinnaldehydrum in calculation sample.Result shows, this analytical approach repeatability is good.The results are shown in Table 16.

Table 16 cinnaldehydrum replica test result

11. recovery tests

Precision take with a collection of medicine composition Liuwei bonduc fall apart sample (product batch number: 20110516) 6 parts, each precision adds cinnaldehydrum reference substance, measures its content, calculate the recovery.Result shows, this assay method measurement result is accurate.The results are shown in Table 17.

Table 17 cinnaldehydrum recovery test result

12. sample determinations

Get loose three batches of medicine composition Liuwei bonduc, measure and calculate Determination of Cinnamaldehyde.The results are shown in Table 18.

Table 18 sample size measurement result

Experimental example 4: the assay experiment of pipering

1. instrument, reagent and test sample

Reference substance: pipering reference substance (Nat'l Pharmaceutical & Biological Products Control Institute) lot number: 0775-200203.

2. the selection of determined wavelength

Get pipering reference substance solution, scan in 190 ~ 400nm wavelength coverage, pipering has absorption maximum at 343nm wavelength place, therefore to select 343nm according to ultraviolet absorpting spectrum be determined wavelength.

3. mobile phase is selected

Research finds, with volume parts than the methanol-water of 75-80:20-25 for mobile phase time, pipering all can reach good chromatographic resolution effect.Wherein with the volume parts of methanol-water than 78:22 for mobile phase is optimum.

4. the investigation of system suitability and negative interference

Under above-mentioned chromatographic condition, accurate absorption reference substance solution, need testing solution, each 10 μ l of negative control solution respectively, injection liquid chromatography, record chromatogram.Result shows, the degree of separation that in reference substance, test sample chromatogram, pipering is adjacent chromatographic peak is all greater than 1.5, and negative control does not disturb.The results are shown in Figure 8, Fig. 9, Figure 10.

5. reference substance preparation

Get pipering reference substance appropriate, accurately weighed, put in brown measuring bottle, add absolute ethyl alcohol and make the solution of every 1ml containing 20 μ g, to obtain final product.

6. test sample preparation

The investigation of 6.1 extracting method

By method under assay item, need testing solution is detected.3 parts are prepared, respectively ultrasonic, backflow, Zhen Oscillating process 30 minutes by the method under the preparation of need testing solution.With the content of pipering in every gram of medicine for index determination extracting method.The results are shown in Table 19.

Table 19 extracting method investigates test findings

Result shows, ultrasonic basically identical with the content of pipering in refluxing extraction gained every gram of medicine, considers that ultrasonic extraction is easy, easy to operate, therefore selects extracting method to be ultrasonic extraction.

The investigation of 6.2 Extraction solvent

By method under assay item, need testing solution is detected.Prepare 3 parts by the method under the preparation of need testing solution, precision adds methyl alcohol, absolute ethyl alcohol, volume parts than each 25ml of 80% ethanol water respectively.With the content of pipering in every gram of medicine for index determination Extraction solvent.The results are shown in Table 20.

Table 20 Extraction solvent investigates test findings

Result shows, methyl alcohol, absolute ethyl alcohol, volume parts are substantially more suitable than 80% ethanol water, three kinds of content that solvent measures, but with the test sample solvent that absolute ethyl alcohol extracts, impurity peaks is minimum, therefore selects Extraction solvent to be absolute ethyl alcohol.

The investigation of 6.3 extraction times

By method under assay item, need testing solution is detected.3 parts are prepared, respectively ultrasonic process 20 minutes, 30 minutes, 40 minutes by the method under the preparation of need testing solution.With the content of pipering in every gram of medicine for index determines extraction time.The results are shown in Table 21.

Table 21 extraction time investigates test findings

Result shows, in ultrasonic extraction 30 minutes and 40 minutes gained every gram medicines, the content of pipering is basically identical, therefore selects extraction time to be 30 minutes.

Precision measures pipering reference substance stock solution solution (content of piperine is 42.8 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml, put in 10ml volumetric flask respectively, absolute ethyl alcohol is diluted to scale, shake up, each accurate sample introduction 10 μ l, with peak area (A), linear regression is carried out to reference substance concentration (C), obtain pipering regression equation: A=22.850C+0.6620, related coefficient: R=0.9998.Result shows, pipering is within the scope of 4.28 μ g/ml ~ 42.8 μ g/ml, and the peak area (A) of pipering is good with reference substance concentration (C) linear relationship.The results are shown in Table 22.

Table 22 pipering linear relationship investigates result

8. precision test

Accurate absorption pipering reference substance solution 10 μ l, injection liquid chromatography, each METHOD FOR CONTINUOUS DETERMINATION 6 times, record peak area also calculates relative standard deviation.Result shows, instrument precision is good.The results are shown in Table 23.

Table 23 pipering Precision test result

9. stability test

After prepared by need testing solution, accurate absorption 10 μ l, injection liquid chromatography, record peak area, measured once every 2 hours later, investigates 8 hours, and record peak area also calculates relative standard deviation.Result shows, in need testing solution, pipering measurement result in 8 hours is stablized.The results are shown in Table 24.

Table 24 pipering stability test result

10. replica test

Get with a collection of medicine composition Liuwei bonduc fall apart sample (product batch number: 20110516) 3g, accurately weighed, totally 6 parts, need testing solution is prepared by the method under the preparation of need testing solution, accurate absorption 10 μ l respectively, injection liquid chromatography, the content of pipering in calculation sample.Result shows, this analytical approach repeatability is good.The results are shown in Table 25.

Table 25 pipering replica test result

11. recovery tests

Precision take with a collection of medicine composition Liuwei bonduc fall apart sample (product batch number: 20110516) 6 parts, each precision adds pipering reference substance, measures its content, calculate the recovery.Result shows, this assay method measurement result is accurate.The results are shown in Table 26.

Table 26 pipering recovery test result

12. sample determinations

Get loose three batches of medicine composition Liuwei bonduc, measure and calculate content of piperine.The results are shown in Table 27.

Table 27 sample size measurement result

Experimental example 5: the assay experiment of hydroxyl radical carthamin yellow carthamus A

1. instrument, reagent and test sample

Reference substance: hydroxyl radical carthamin yellow carthamus A reference substance (Nat'l Pharmaceutical & Biological Products Control Institute), lot number: 111637-200905.

2. the selection of determined wavelength

Get hydroxyl radical carthamin yellow carthamus A reference substance solution, scan in 190 ~ 500nm wavelength coverage, hydroxyl radical carthamin yellow carthamus A has absorption maximum at 403nm wavelength place, therefore to select 403nm according to ultraviolet absorpting spectrum be determined wavelength.

3. mobile phase is selected

Research find, with volume parts than the methyl alcohol-volume parts ratio of 25-35:65-75 be 1% glacial acetic acid aqueous solution for mobile phase time, hydroxyl radical carthamin yellow carthamus A all can reach good chromatographic resolution effect.Wherein with methyl alcohol-volume parts than be the volume parts of 1% glacial acetic acid aqueous solution than 30:70 for mobile phase is optimum.

4. the investigation of system suitability and negative interference

Under above-mentioned chromatographic condition, accurate absorption reference substance solution, need testing solution, each 10 μ l of negative control solution respectively, injection liquid chromatography, record chromatogram.Result shows, the degree of separation that in reference substance, test sample chromatogram, hydroxyl radical carthamin yellow carthamus A is adjacent chromatographic peak is all greater than 1.5, and negative control does not disturb.The results are shown in Figure 11, Figure 12, Figure 13.

5. reference substance preparation

Get hydroxyl safflower anthocyanidin A reference substance appropriate, accurately weighed, put in brown bottle, add volume parts and make the solution of every 1ml containing 0.13mg than 25% methanol aqueous solution, to obtain final product.

6. test sample preparation

The investigation of 6.1 extracting method

By method under assay item, need testing solution is detected.3 parts are prepared, respectively ultrasonic, backflow, Zhen Oscillating process 40 minutes by the method under the preparation of need testing solution.With the content of hydroxyl radical carthamin yellow carthamus A in every gram of medicine for index determination extracting method.The results are shown in Table 28.

Table 28 extracting method investigates test findings

Result shows, in ultrasonic extraction gained every gram of medicine, the content of hydroxyl radical carthamin yellow carthamus A is the highest, therefore selects extracting method to be ultrasonic extraction.

The investigation of 6.2 Extraction solvent

By method under assay item, need testing solution is detected.Prepare 3 parts by the method under the preparation of need testing solution, precision adds water, volume parts 25ml more each than 25% methanol aqueous solution, methyl alcohol respectively.With the content of hydroxyl radical carthamin yellow carthamus A in every gram of medicine for index determination Extraction solvent.The results are shown in Table 29.

Table 29 Extraction solvent investigates test findings

Result shows, the hydroxyl radical carthamin yellow carthamus A content that water, volume parts are surveyed than 25% methanol aqueous solution and methyl alcohol is basically identical, and owing to using water as Extraction solvent filtration difficulty, pure methyl alcohol toxicity is large, therefore selects Extraction solvent to be that volume parts is than 25% methanol aqueous solution.

The investigation of 6.3 extraction times

By method under assay item, need testing solution is detected.3 parts are prepared, respectively ultrasonic process 30 minutes, 40 minutes, 50 minutes by the method under the preparation of need testing solution.With the content of hydroxyl radical carthamin yellow carthamus A in every gram of medicine for index determines extraction time.The results are shown in Table 30.

Table 30 extraction time investigates test findings

Result shows, in ultrasonic extraction 40 minutes and 50 minutes gained every gram medicines, the content of hydroxyl radical carthamin yellow carthamus A is basically identical, therefore selects extraction time to be 40 minutes.

Precision measures hydroxyl radical carthamin yellow carthamus A reference substance stock solution solution (hydroxyl radical carthamin yellow carthamus A content is 245.7 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml, put in 10ml volumetric flask respectively, volume parts is diluted to scale than 25% methanol aqueous solution, shake up, each accurate sample introduction 10 μ l, with peak area (A), linear regression is carried out to reference substance concentration (C), obtain hydroxyl radical carthamin yellow carthamus A regression equation: A=22.933C+7.4763, related coefficient: R=0.9998.Result shows, hydroxyl radical carthamin yellow carthamus A is within the scope of 24.57 μ g/ml ~ 245.7 μ g/ml, and the peak area (A) of hydroxyl radical carthamin yellow carthamus A is good with reference substance concentration (C) linear relationship.The results are shown in Table 31.

Table 31 hydroxyl radical carthamin yellow carthamus A linear relationship investigates result

8. precision test

Accurate absorption hydroxyl radical carthamin yellow carthamus A reference substance solution 10 μ l, injection liquid chromatography, each METHOD FOR CONTINUOUS DETERMINATION 6 times, record peak area also calculates relative standard deviation.Result shows, instrument precision is good.The results are shown in Table 32.

Table 32 hydroxyl radical carthamin yellow carthamus A Precision test result

9. stability test

After prepared by need testing solution, accurate absorption 10 μ l, injection liquid chromatography, record peak area, measured once every 2 hours later, investigates 8 hours, and record peak area also calculates relative standard deviation.Result shows, in need testing solution, hydroxyl radical carthamin yellow carthamus A measurement result in 8 hours is stablized.The results are shown in Table 33.

Table 33 hydroxyl radical carthamin yellow carthamus A stability test result

10. replica test

Get and to fall apart sample (product batch number: 20110516) 2g with a collection of medicine composition Liuwei bonduc, accurately weighed, totally 6 parts, need testing solution is prepared by the method under the preparation of need testing solution, accurate absorption 10 μ l respectively, injection liquid chromatography, the content of hydroxyl radical carthamin yellow carthamus A in calculation sample.Result shows, this analytical approach repeatability is good.The results are shown in Table 34.

Table 34 hydroxyl radical carthamin yellow carthamus A replica test result

11. recovery tests

Precision take with a collection of medicine composition Liuwei bonduc fall apart sample (product batch number: 20110516) 6 parts, each precision adds hydroxyl radical carthamin yellow carthamus A reference substance, measures its content, calculate the recovery.Result shows, this assay method measurement result is accurate.The results are shown in Table 35.

Table 35 hydroxyl radical carthamin yellow carthamus A recovery test result

12. sample determinations

Get loose three batches of medicine composition Liuwei bonduc, measure and calculate hydroxyl radical carthamin yellow carthamus A content.The results are shown in Table 36.

Table 36 sample size measurement result

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

Accompanying drawing explanation

Fig. 1 is the TLC distinguish chromatogram of bonduc;

Fig. 2 is the TLC distinguish chromatogram of cardamom;

Fig. 3 is the TLC distinguish chromatogram of Chinese cassia tree;

Fig. 4 is gallic acid contrast high-efficient liquid phase chromatogram;

Fig. 5 is that gallic acid content measures test sample high-efficient liquid phase chromatogram;

Fig. 6 is cinnaldehydrum contrast high-efficient liquid phase chromatogram;

Fig. 7 is that Determination of Cinnamaldehyde measures test sample high-efficient liquid phase chromatogram;

Fig. 8 is misrun osmanthus negative control high-efficient liquid phase chromatogram;

Fig. 9 is pipering contrast high-efficient liquid phase chromatogram;

Figure 10 is that content of piperine measures test sample high-efficient liquid phase chromatogram;

Figure 11 is scarce Bi roots of grass negative control high-efficient liquid phase chromatogram;

Figure 12 is hydroxyl radical carthamin yellow carthamus A contrast high-efficient liquid phase chromatogram;

Figure 13 is hydroxyl radical carthamin yellow carthamus A assay test sample high-efficient liquid phase chromatogram;

Figure 14 is scarce safflower negative control high-efficient liquid phase chromatogram;

Wherein, in Fig. 1, Fig. 2, Fig. 3, a is control medicinal material, and b is test sample, and c is negative control; The horizontal ordinate of Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Figure 10, Figure 11, Figure 12, Figure 13, Figure 14 is time (min), and ordinate is absorbance (mAU).

Embodiment

Below in conjunction with embodiment, detailed elaboration is done to the present invention, but be not limited to these embodiments specifically recorded.The medicine composition Liuwei bonduc detected fall apart for Qinghai gold scold Tibetan medicine medicine company incorporated company produce sell.

Embodiment 1: the quality determining method that Six-element bonduc is loose

Differentiate:

A. the discriminating of bonduc

Get medicine composition Liuwei bonduc divided powder 3g, add methyl alcohol 20ml, ultrasonic process 30 minutes, filter, filtrate is as need testing solution; Separately get bonduc control medicinal material 1g, with legal system for bonduc control medicinal material solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw each 3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume parts than the boiling range of 10:0.3:0.1 for 60-90 DEG C of petroleum ether-ethyl acetate-glacial acetic acid is for developping agent, launch, take out, dry, spray, with the ethanol solution of sulfuric acid of volume parts than 10%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

B. the discriminating of cardamom

Get medicine composition Liuwei bonduc divided powder 2g, put in 100ml round-bottomed flask, add water 50ml, extract according to determination of volatile oil Division A League Matches of French Football method (Chinese Pharmacopoeia version in 2010 annex X D), adding water on the top of volatile oil extractor makes water overflow in flask, add ethyl acetate 1ml again, refluxing extraction 2 hours, divide and get acetic acid ethyl fluid as need testing solution; Separately get cardamom control medicinal material 1g, with legal system for cardamom control medicinal material solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VIB), draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume parts than the boiling range of 8:2 for 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent, launch, take out, dry, spray, with the vanillin-sulfuric acid solution of quality volume portion rate 1%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

C. the discriminating of Chinese cassia tree

Get medicine composition Liuwei bonduc divided powder 2g, put in 100ml round-bottomed flask, add 60ml water, extract according to determination of volatile oil Division A League Matches of French Football method (Chinese Pharmacopoeia version in 2010 annex X D), add water on the top of volatile oil extractor and water is overflowed in flask, then add ethyl acetate 1ml, refluxing extraction 1 hour, be cooled to room temperature, divide and get acetic acid ethyl fluid, as need testing solution; Get Chinese cassia tree control medicinal material 1g, with legal system for control medicinal material solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same silica G chromatographic sheet, be that 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent with the boiling range of volume parts ratio (10:2), launch, take out, dry, spray is with quality volume portion rate 1% vanillin-sulfuric acid solution, and 105 DEG C to be heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

Assay:

A. the assay of gallic acid

Medicine composition Liuwei bonduc divided powder 2g is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;

B. the assay of cinnaldehydrum

C. the assay of pipering

Medicine composition Liuwei bonduc divided powder 3g is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds absolute ethyl alcohol 25ml, close plug, weighed weight, ultrasonic process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with absolute ethyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;

D. the assay of hydroxyl radical carthamin yellow carthamus A

Medicine composition Liuwei bonduc divided powder 2g is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds volume parts than 25% methanol aqueous solution 25ml, close plug, weighed weight, ultrasonic 40 minutes, let cool, more weighed weight, supply the weight of less loss than 25% methanol aqueous solution by volume parts, shake up, filter, get subsequent filtrate, to obtain final product;

Embodiment 2: the quality determining method of Six-element bonduc particle

Differentiate:

A. the discriminating of bonduc

Get medicine composition Liuwei bonduc particle powder 3g, add methyl alcohol 20ml, ultrasonic process 30 minutes, filter, filtrate is as need testing solution; Separately get bonduc control medicinal material 1g, with legal system for bonduc control medicinal material solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw each 3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume parts than the boiling range of 10:0.3:0.1 for 60-90 DEG C of petroleum ether-ethyl acetate-glacial acetic acid is for developping agent, launch, take out, dry, spray, with the ethanol solution of sulfuric acid of volume parts than 10%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

B. the discriminating of cardamom

Get medicine composition Liuwei bonduc particle powder 2g, put in 100ml round-bottomed flask, add water 50ml, extract according to determination of volatile oil Division A League Matches of French Football method (Chinese Pharmacopoeia version in 2010 annex X D), adding water on the top of volatile oil extractor makes water overflow in flask, add ethyl acetate 1ml again, refluxing extraction 2 hours, divide and get acetic acid ethyl fluid as need testing solution; Separately get cardamom control medicinal material 1g, with legal system for cardamom control medicinal material solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume parts than the boiling range of 8:2 for 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent, launch, take out, dry, spray, with the vanillin-sulfuric acid solution of quality volume portion rate 1%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

C. the discriminating of Chinese cassia tree

Get medicine composition Liuwei bonduc particle powder 2g, put in 100ml round-bottomed flask, add 60ml water, extract according to determination of volatile oil Division A League Matches of French Football method (Chinese Pharmacopoeia version in 2010 annex X D), add water on the top of volatile oil extractor and water is overflowed in flask, then add ethyl acetate 1ml, refluxing extraction 1 hour, be cooled to room temperature, divide and get acetic acid ethyl fluid, as need testing solution; Get Chinese cassia tree control medicinal material 1g, with legal system for control medicinal material solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same silica G chromatographic sheet, be that 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent with the boiling range of volume parts ratio (10:2), launch, take out, dry, spray is with quality volume portion rate 1% vanillin-sulfuric acid solution, and 105 DEG C to be heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

Assay:

A. the assay of gallic acid

Medicine composition Liuwei bonduc particle powder 2g is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;

B. the assay of cinnaldehydrum

C. the assay of pipering

Medicine composition Liuwei bonduc particle powder 3g is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds absolute ethyl alcohol 25ml, close plug, weighed weight, ultrasonic process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with absolute ethyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;

D. the assay of hydroxyl radical carthamin yellow carthamus A

Medicine composition Liuwei bonduc particle powder 2g is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds volume parts than 25% methanol aqueous solution 25ml, close plug, weighed weight, ultrasonic 40 minutes, let cool, more weighed weight, supply the weight of less loss than 25% methanol aqueous solution by volume parts, shake up, filter, get subsequent filtrate, to obtain final product;

Described medicine composition Liuwei bonduc particle refers to and to fall apart bulk drug formula bonduc 90g, seed of pomegranate 90g, Chinese cassia tree 5g, cardamom 40g, Bi roots of grass 5g, safflower 50g with Six-element bonduc, add customary adjuvant, conveniently the granule prepared of technique.

Embodiment 3: the quality determining method of Six-element bonduc ball

Differentiate:

A. the discriminating of bonduc

Get medicine composition Liuwei bonduc ball powder 3g, add methyl alcohol 20ml, ultrasonic process 30 minutes, filter, filtrate is as need testing solution; Separately get bonduc control medicinal material 1g, with legal system for bonduc control medicinal material solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw each 3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume parts than the boiling range of 10:0.3:0.1 for 60-90 DEG C of petroleum ether-ethyl acetate-glacial acetic acid is for developping agent, launch, take out, dry, spray, with the ethanol solution of sulfuric acid of volume parts than 10%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

B. the discriminating of cardamom

Get medicine composition Liuwei bonduc ball powder 2g, put in 100ml round-bottomed flask, add water 50ml, extract according to determination of volatile oil Division A League Matches of French Football method (Chinese Pharmacopoeia version in 2010 annex X D), adding water on the top of volatile oil extractor makes water overflow in flask, add ethyl acetate 1ml again, refluxing extraction 2 hours, divide and get acetic acid ethyl fluid as need testing solution; Separately get cardamom control medicinal material 1g, with legal system for cardamom control medicinal material solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VIB), draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume parts than the boiling range of 8:2 for 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent, launch, take out, dry, spray, with the vanillin-sulfuric acid solution of quality volume portion rate 1%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

C. the discriminating of Chinese cassia tree

Get medicine composition Liuwei bonduc ball powder 2g, put in 100ml round-bottomed flask, add 60ml water, extract according to determination of volatile oil Division A League Matches of French Football method (Chinese Pharmacopoeia version in 2010 annex X D), add water on the top of volatile oil extractor and water is overflowed in flask, then add ethyl acetate 1ml, refluxing extraction 1 hour, be cooled to room temperature, divide and get acetic acid ethyl fluid, as need testing solution; Get Chinese cassia tree control medicinal material 1g, with legal system for control medicinal material solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same silica G chromatographic sheet, be that 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent with the boiling range of volume parts ratio (10:2), launch, take out, dry, spray is with quality volume portion rate 1% vanillin-sulfuric acid solution, and 105 DEG C to be heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

Assay:

A. the assay of gallic acid

Medicine composition Liuwei bonduc ball powder 2g is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;

B. the assay of cinnaldehydrum

C. the assay of pipering

Medicine composition Liuwei bonduc ball powder 3g is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds absolute ethyl alcohol 25ml, close plug, weighed weight, ultrasonic process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with absolute ethyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;

D. the assay of hydroxyl radical carthamin yellow carthamus A

Medicine composition Liuwei bonduc ball powder 2g is got in the preparation of need testing solution, accurately weighed, puts in tool plug conical flask, precision adds volume parts than 25% methanol aqueous solution 25ml, close plug, weighed weight, ultrasonic 40 minutes, let cool, more weighed weight, supply the weight of less loss than 25% methanol aqueous solution by volume parts, shake up, filter, get subsequent filtrate, to obtain final product;

Described medicine composition Liuwei bonduc ball refers to and to fall apart bulk drug formula bonduc 90g, seed of pomegranate 90g, Chinese cassia tree 5g, cardamom 40g, Bi roots of grass 5g, safflower 50g with Six-element bonduc, add customary adjuvant, conveniently the pill prepared of technique.

Claims

1. the detection method of a Six-element bonduc preparation, said preparation is the various preparations be made up by pharmacy conventional method of bonduc, seed of pomegranate, Chinese cassia tree, cardamom, the Bi roots of grass, safflower, it is characterized in that, detection method comprises the thin-layer chromatography qualitative identification method to bonduc, cardamom, Chinese cassia tree in preparation, the liquid chromatography content assaying method of the hydroxyl radical carthamin yellow carthamus A that the pipering that the cinnaldehydrum that the gallic acid contained bonduc and seed of pomegranate, Chinese cassia tree contain, the Bi roots of grass contain, safflower contain, specific as follows:

Differentiate:

B. the discriminating of cardamom: it is for contrast with cardamom control medicinal material, with volume parts ratio (8-9): the boiling range of (1-2) is 60-90 DEG C of petroleum ether-ethyl acetate is that developping agent launches on silica gel g thin-layer plate, the thin-layered chromatography being developer with the vanillin-sulfuric acid solution of quality volume portion rate 1%;

Assay:

2. detection method as claimed in claim 1, it is characterized in that, detection method is following methods:

Differentiate:

A. the discriminating of bonduc

Get powder formulation 2-4g to be detected, add methyl alcohol 10-30ml, ultrasonic process 20-40 minute, filter, filtrate is as need testing solution; Separately get bonduc control medicinal material 0.5-1g, with legal system for bonduc control medicinal material solution; Test according to thin-layered chromatography, draw above-mentioned two kinds of solution each 2-5 μ l, put respectively on same silica gel g thin-layer plate, with volume parts than 10:(0.1-0.5): the boiling range of 0.1 is 60-90 DEG C of petroleum ether-ethyl acetate-glacial acetic acid is developping agent, launch, take out, dry, spray, with the ethanol solution of sulfuric acid of volume parts than 10%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;

B. the discriminating of cardamom

Get powder formulation 1-3g to be detected, put in 100ml round-bottomed flask, add water 40-80ml, extract according to the determination of volatile oil Division A League Matches of French Football method on Chinese Pharmacopoeia version in 2010 annex XD, adding water on the top of volatile oil extractor makes water overflow in flask, add ethyl acetate 1ml again, refluxing extraction 1-3 hour, divide and get acetic acid ethyl fluid as need testing solution; Separately get cardamom control medicinal material 0.5-1g, with legal system for cardamom control medicinal material solution; Test according to thin-layered chromatography, draw above-mentioned two kinds of solution each 5-10 μ l, put respectively on same silica gel g thin-layer plate, with volume parts ratio (8-9): the boiling range of (1-2) is that 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent, launch, take out, dry, spray, with the vanillin-sulfuric acid solution of quality volume portion rate 1%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;

C. Chinese cassia tree is differentiated

Get powder formulation 1-5g to be detected, put in 100ml round-bottomed flask, add 40-70ml water, extract according to the determination of volatile oil Division A League Matches of French Football method on Chinese Pharmacopoeia version in 2010 annex XD, add water on the top of volatile oil extractor and water is overflowed in flask, then add ethyl acetate 1-2ml, refluxing extraction 1-3 hour, be cooled to room temperature, divide and get acetic acid ethyl fluid, as need testing solution;

Get Chinese cassia tree control medicinal material 0.5-2g, with legal system for control medicinal material solution; Test according to thin-layered chromatography, draw above-mentioned two kinds of solution 5-10 μ l, put respectively in same silica G chromatographic sheet, with volume parts ratio (6 ~ 10): the boiling range of (1 ~ 3) is that 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent, launch, take out, dry, spray is with quality volume portion rate 1-5% vanillin-sulfuric acid solution, and 105 DEG C to be heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;

Assay:

A. the assay of gallic acid

According to high effective liquid chromatography for measuring;

Determination method draws reference substance solution and need testing solution each 5-10 μ l respectively, injection liquid chromatography, measures, to obtain final product;

B. the assay of cinnaldehydrum

According to high effective liquid chromatography for measuring;

C. the assay of pipering

According to high effective liquid chromatography for measuring;

D. the assay of hydroxyl radical carthamin yellow carthamus A

According to high effective liquid chromatography for measuring;

3. detection method as claimed in claim 1, it is characterized in that, described preparation raw material medicine consists of: bonduc 90 weight portion, seed of pomegranate 90 weight portion, Chinese cassia tree 5 weight portion, cardamom 40 weight portion, the Bi roots of grass 5 weight portion, safflower 50 weight portion.

4. detection method as claimed in claim 3, is characterized in that, described preparation is that Six-element bonduc is loose, Six-element bonduc particle, Six-element bonduc ball, Six-element bonduc sheet or Six-element bonduc capsule.

5. the detection method as described in claim 3 or 4, is characterized in that, described detection method specifically:

Differentiate:

A. the discriminating of bonduc

Get powder formulation 3g to be detected, add methyl alcohol 20ml, ultrasonic process 30 minutes, filter, filtrate is as need testing solution; Separately get bonduc control medicinal material 1g, with legal system for bonduc control medicinal material solution; Test according to thin-layered chromatography, draw each 3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume parts than the boiling range of 10:0.3:0.1 for 60-90 DEG C of petroleum ether-ethyl acetate-glacial acetic acid is for developping agent, launch, take out, dry, spray, with the ethanol solution of sulfuric acid of volume parts than 10%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;

B. the discriminating of cardamom

Get powder formulation 2g to be detected, put in 100ml round-bottomed flask, add water 50ml, extract according to according to the determination of volatile oil Division A League Matches of French Football method on Chinese Pharmacopoeia version in 2010 annex XD, adding water on the top of volatile oil extractor makes water overflow in flask, add ethyl acetate 1ml again, refluxing extraction 2 hours, divide and get acetic acid ethyl fluid as need testing solution; Separately get cardamom control medicinal material 1g, with legal system for cardamom control medicinal material solution; Test according to thin-layered chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume parts than the boiling range of 8:2 for 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent, launch, take out, dry, spray, with the vanillin-sulfuric acid solution of quality volume portion rate 1%, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;

C. the discriminating of Chinese cassia tree

Get powder formulation 2g to be detected, put in 100ml round-bottomed flask, add 60ml water, extract according to the determination of volatile oil Division A League Matches of French Football method on Chinese Pharmacopoeia version in 2010 annex XD, add water on the top of volatile oil extractor and water is overflowed in flask, then add ethyl acetate 1ml, refluxing extraction 1 hour, be cooled to room temperature, divide and get acetic acid ethyl fluid, as need testing solution; Get Chinese cassia tree control medicinal material 1g, with legal system for control medicinal material solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same silica G chromatographic sheet, be that 60-90 DEG C of petroleum ether-ethyl acetate is for developping agent with the boiling range of volume parts ratio (10:2), launch, take out, dry, spray is with quality volume portion rate 1% vanillin-sulfuric acid solution, and 105 DEG C to be heated to spot development clear; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color;

Assay:

A. the assay of gallic acid

According to high effective liquid chromatography for measuring;

B. the assay of cinnaldehydrum

According to high effective liquid chromatography for measuring;

C. the assay of pipering

According to high effective liquid chromatography for measuring;

D. the assay of hydroxyl radical carthamin yellow carthamus A

According to high effective liquid chromatography for measuring;

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