CN102778529A - Method for testing quality of tibetan medicine composition Liuwei Datuoyeyunshi preparation - Google Patents

Method for testing quality of tibetan medicine composition Liuwei Datuoyeyunshi preparation Download PDF

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CN102778529A
CN102778529A CN2012102798144A CN201210279814A CN102778529A CN 102778529 A CN102778529 A CN 102778529A CN 2012102798144 A CN2012102798144 A CN 2012102798144A CN 201210279814 A CN201210279814 A CN 201210279814A CN 102778529 A CN102778529 A CN 102778529A
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CN102778529B (en
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张义智
高英涵
刘玉芹
任松鹏
李怀平
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JINHE TIBETAN MEDICINE CO., LTD.
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Shandong Arura Pharmaceutical Research & Development Co Ltd
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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 quality determining method of Tibetan medicine composition Six-element bonduc preparation
Technical field
The present invention relates to a kind of quality determining method of Tibetan medicine composite preparation, particularly a kind of Tibetan medicine composition Six-element bonduc looses and the quality determining method of preparation.
Background technology
The Six-element bonduc looses and records in the national drug standards, standard numbering: WS 3-BC-0282-95 has the effect of warm kidney, enriching yin, and it is sick to be mainly used in the women 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, pursues 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, and it is fiery supporing yang to have benefit, guiding fire to origin, eliminating cold to stop pain, the 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 the 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 dry near maturation or the mature fruit cluster of Piperaceae plant Bi roots of grass Piper longum L., has the effect of warming spleen and stomach for dispelling cold, therapeutic method to keep the adverse qi flowing downward pain relieving; Safflower is the dried floral of feverfew safflower Carthamus tinctorius L., has the effect of activating blood to promote menstruation, blood stasis removing analgesic.
Often with symptoms such as irregular menstruation and abnormal leucorrheas, endocrine is not normal to be the arch-criminal before and after the part female menstrual period, and the true cause of abdominal pain to be kidney tremble with fear and stomach cold.This situation development probably damages the uterus if leave for a long time, causes infertile.Live in the Tibetan of Tibetan area, the bonduc of taking after mixing it with water of the girl after often giving the initial stage.The record of Tibetan medicine's ancient books and records, the effect that bonduc has warm kidney, dispels cold is applicable to that kidney is cold, stomach cold disease.It is main ingredient that the Six-element bonduc looses with the bonduc, is aided with number flavor tonic herbs such as Chinese cassia tree, is the treatment female irregular menstruation, the good medicine of abnormal leucorrhea.
In the diffusing proper mass standard of Six-element bonduc, have only to meet each item regulation relevant under the powder item, both do not had thin layer to differentiate item, also have no the assay index.Drug quality problem and human health are closely related, and be nowadays also increasingly high to the requirement of drug quality control, yet the said preparation quality standard is on the low side, can not effectively guarantee the quality of said preparation, and the said preparation quality standard is in urgent need to be improved.
Summary of the invention
The objective of the invention is for overcoming the deficiency of above-mentioned prior art, a kind of quality determining method of Tibetan medicine composition Six-element bonduc preparation is provided.
The term explanation:
It is the nomenclature of drug of Tibetan medicine (standard numbering WS3-BC-0282-95) promulgated by the ministries or commissions of the Central Government record that the Six-element bonduc looses.
Six-element bonduc preparation (or cry " Six-element bonduc loose and preparation ") comprise that the Six-element bonduc looses and loose with the Six-element bonduc in other preparations of preparing of composition of raw materials; Like 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 diffusing identical with the Six-element bonduc, also can carry out suitable adjustment according to the treatment needs.
For realizing above-mentioned purpose, the present invention adopts following technical proposals:
A kind of quality determining method of Tibetan medicine composition Six-element bonduc preparation; The various preparations of said preparation for processing by the pharmacy conventional method by 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 the preparation, one or more in the liquid chromatography content assaying method of the gallic acid that bonduc and seed of pomegranate are contained, the cinnaldehydrum that Chinese cassia tree contains, pipering that the Bi roots of grass contains, hydroxyl radical carthamin yellow carthamus A that safflower contains.
Preferred as the present invention, quality determining method is one or more in the following method:
Differentiate:
A. the discriminating of bonduc: it is to be contrast with the bonduc control medicinal material; With volume parts than 10: (0.1-0.5): 0.1 boiling range is that 60-90 ℃ of petroleum ether-ethyl acetate-glacial acetic acid is that developping agent launches on silica gel g thin-layer plate, is the thin-layered chromatography of developer with volume parts than 10% ethanol solution of sulfuric acid;
B. the discriminating of cardamom: it is to be contrast with the cardamom control medicinal material; With volume parts than (9-8): boiling range (1-2) is that 60-90 ℃ of petroleum ether-ethyl acetate is that developping agent launches on silica gel g thin-layer plate, is the thin-layered chromatography of developer with the vanillic aldehyde sulfuric acid solution of mass and size portion rate 1%;
C. the discriminating of Chinese cassia tree: it is to be contrast with the Chinese cassia tree control medicinal material; With volume parts than (6-10): boiling range (1-3) is that 60-90 ℃ of petroleum ether-ethyl acetate is that developping agent launches on silica gel g thin-layer plate, is the thin-layered chromatography of developer with the vanillic aldehyde sulfuric acid solution of mass and size portion rate 1-5%;
Assay:
A. the assay of gallic acid: it is to be contrast with the gallic acid reference substance, is filling agent with the octadecylsilane chemically bonded silica; With volume parts than (1-3): methyl alcohol-volume parts (97-99) is a moving phase than 1% glacial acetic acid aqueous solution, and detecting wavelength is the high performance liquid chromatography of 275nm;
B. the assay of cinnaldehydrum: it is to be contrast with the cinnaldehydrum reference substance, is filling agent with the octadecylsilane chemically bonded silica, is moving phase with volume parts than the acetonitrile-water of 30-70:30-70, and detecting wavelength is that 290nm is a high performance liquid chromatography;
C. the assay of pipering: it is to be contrast with the pipering reference substance, is filling agent with the octadecylsilane chemically bonded silica, is moving phase with volume parts than the methanol-water of 75-80:20-25, and detecting wavelength is the high performance liquid chromatography of 343nm;
D. the assay of hydroxyl radical carthamin yellow carthamus A: it is to be contrast with the hydroxyl radical carthamin yellow carthamus A reference substance; With the octadecylsilane chemically bonded silica is filling agent; Is that 1% glacial acetic acid aqueous solution is a moving phase with volume parts than methyl alcohol-volume parts ratio of 25-35:65-75, and detecting wavelength is the high performance liquid chromatography of 403nm.
Further preferred as the present invention, said quality determining method is one or more in the following method:
Differentiate:
A. the discriminating of bonduc
Get preparation powder 2-4g to be detected, add methyl alcohol 10-30ml, sonicated 20-40 minute, filter, filtrating is as need testing solution; Other gets bonduc control medicinal material 0.5-1g, is equipped with bonduc control medicinal material solution with legal system; According to thin-layered chromatography (" appendix VIB of Chinese pharmacopoeia version in 2010) test, draw each 2-5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With volume parts than 10: (0.1-0.5): 0.1 boiling range is that 60-90 ℃ of petroleum ether-ethyl acetate-glacial acetic acid is developping agent; Launch, take out, dry; Than 10% ethanol solution of sulfuric acid, it is clear to be heated to the spot colour developing at 105 ℃ with volume parts in spray; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
B. the discriminating of cardamom
Get preparation powder 1-3g to be detected, put in the 100ml round-bottomed flask, add water 40-80ml; Extract according to determination of volatile oil Division A League Matches of French Football method (an appendix X of Chinese Pharmacopoeia version in 2010 D); Jia Shui overflows in the flask water on the top of volatile oil extractor, adds ethyl acetate 1ml again, refluxing extraction 1-3 hour; Be cooled to room temperature, obtain acetic acid ethyl fluid as need testing solution; Other gets cardamom control medicinal material 0.5-1g, is equipped with cardamom control medicinal material solution with legal system; According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw each 5-10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With volume parts than (9～8): the boiling range of (1～2) is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent; Launch, take out, dry; Spray is with the vanillic aldehyde sulfuric acid solution of mass and size portion rate 1%, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
C. Chinese cassia tree is differentiated
Get preparation powder 1-5g to be detected, put in the 100ml round-bottomed flask, add 40-70ml water; Extract according to determination of volatile oil Division A League Matches of French Football method (an appendix X of Chinese Pharmacopoeia version in 2010 D), Jia Shui overflows in the flask water on the top of volatile oil extractor, adds ethyl acetate 1-2ml again; Refluxing extraction 1-3 hour; Be cooled to room temperature, obtain acetic acid ethyl fluid, as need testing solution.Get Chinese cassia tree control medicinal material 0.5-2g, be equipped with control medicinal material solution with legal system; According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 5-10 μ l of above-mentioned two kinds of solution, put respectively on same silica G TLCP; With volume parts than (6～10): the boiling range of (1～3) is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent; Launch, take out, dry; Spray is with mass and size portion rate 1-5% vanillic aldehyde sulfuric acid solution, and 105 ℃ to be heated to the spot colour developing clear; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Assay:
A. the assay of gallic acid
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; With volume parts than (1-3): methyl alcohol-volume parts (97-99) is a moving phase than 1% glacial acetic acid aqueous solution; The detection wavelength is 275nm; Number of theoretical plate calculates by the gallic acid peak should be not less than 2000;
It is an amount of that the gallic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 0.1mg, promptly gets;
Preparation powder 1-3g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, accurate methyl alcohol 20-30ml, the close plug of adding; Claim decide weight, sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 5-10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets.
B. the assay of cinnaldehydrum
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the acetonitrile-water of 30-70:30-70; The detection wavelength is 290nm; Number of theoretical plate calculates by the cinnaldehydrum peak should be not less than 3000;
It is an amount of that the cinnaldehydrum reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 35 μ g, promptly gets;
Preparation powder 1-3g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, accurate methyl alcohol 20-30ml, the close plug of adding; Claim decide weight, sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 5-10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets.
C. the assay of pipering
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the methanol-water of 75-80:20-25; The detection wavelength is 343nm; Number of theoretical plate calculates by the pipering peak should be not less than 1500;
It is an amount of that the pipering reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown measuring bottle, adds absolute ethyl alcohol and processes the solution that every 1ml contains 20 μ g, promptly gets;
Preparation powder 2-4g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate absolute ethyl alcohol 20-30ml that adds; Close plug is claimed to decide weight, and sonicated 30 minutes is put cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up with absolute ethyl alcohol; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 5-10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets.
D. the assay of hydroxyl radical carthamin yellow carthamus A
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is that 1% glacial acetic acid aqueous solution is a moving phase with volume parts than methyl alcohol-volume parts ratio of 25-35:65-75; The detection wavelength is 403nm; Number of theoretical plate calculates by the hydroxyl radical carthamin yellow carthamus A peak should be not less than 3000;
It is an amount of that the hydroxyl radical carthamin yellow carthamus A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown bottle, adds volume parts and processes the solution that every 1ml contains 0.13mg than 25% methanol aqueous solution, promptly gets;
Preparation powder 1-3g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, and the accurate volume parts that adds is than 25% methanol aqueous solution 20-30ml; Close plug is claimed to decide weight, ultrasonic 40 minutes, puts cold; Claim again to decide weight, supply the weight that subtracts mistake than 25% methanol aqueous solution, shake up with volume parts; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 5-10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets.
Said preparation raw material medicine composition is preferably: bonduc 90 weight portions, seed of pomegranate 90 weight portions, Chinese cassia tree 5 weight portions, cardamom 40 weight portions, the Bi roots of grass 5 weight portions, safflower 50 weight portions.
Said preparation is for the Six-element bonduc looses, Six-element bonduc particle, Six-element bonduc ball, Six-element bonduc sheet or Six-element bonduc capsule.This shows that the diffusing bulk drug composition of Six-element bonduc particle, Six-element bonduc ball, Six-element bonduc sheet and Six-element bonduc capsule and Six-element bonduc is identical, therefore can adopt same or analogous quality determining method.
Form or its preparation to above-mentioned preferred feedstock medicine, said quality determining method specifically:
Differentiate:
A. the discriminating of bonduc
Get preparation powder 3g to be detected, add methyl alcohol 20ml, sonicated 30 minutes filters, and filtrating is as need testing solution; Other gets bonduc control medicinal material 1g, is equipped with bonduc control medicinal material solution with legal system; According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw each 3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Is that 60-90 ℃ of petroleum ether-ethyl acetate-glacial acetic acid is developping agent with volume parts than the boiling range of 10:0.3:0.1; Launch, take out, dry; Than 10% ethanol solution of sulfuric acid, it is clear to be heated to the spot colour developing at 105 ℃ with volume parts in spray; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
B. the discriminating of cardamom
Get preparation powder 2g to be detected; Put in the 100ml round-bottomed flask, add water 50ml, extract according to determination of volatile oil Division A League Matches of French Football methods (an appendix X of Chinese Pharmacopoeia version in 2010 D); Jia Shui overflows in the flask water on the top of volatile oil extractor; Add ethyl acetate 1ml again, refluxing extraction 2 hours is obtained acetic acid ethyl fluid as need testing solution; Other gets cardamom control medicinal material 1g, is equipped with cardamom control medicinal material solution with legal system; According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent with volume parts than the boiling range of 8:2; Launch, take out, dry; Spray is with the vanillic aldehyde sulfuric acid solution of mass and size portion rate 1%, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
C. the discriminating of Chinese cassia tree
Get preparation powder 2g to be detected, put in the 100ml round-bottomed flask, add 60ml water; Extract according to determination of volatile oil Division A League Matches of French Football method (an appendix X of Chinese Pharmacopoeia version in 2010 D), Jia Shui overflows in the flask water on the top of volatile oil extractor, adds ethyl acetate 1ml again; Refluxing extraction 1 hour; Be cooled to room temperature, obtain acetic acid ethyl fluid, as need testing solution; Get Chinese cassia tree control medicinal material 1g, be equipped with control medicinal material solution with legal system; According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica G TLCP; Is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent with volume parts than the boiling range of (10:2); Launch, take out, dry; Spray is with mass and size portion rate 1% vanillic aldehyde sulfuric acid solution, and 105 ℃ to be heated to the spot colour developing clear; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Assay:
A. the assay of gallic acid
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase than methyl alcohol-volume parts of 2:98 than 1% glacial acetic acid aqueous solution with volume parts; The detection wavelength is 275nm; Number of theoretical plate calculates by the gallic acid peak should be not less than 2000;
It is an amount of that the gallic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 0.1mg, promptly gets;
Preparation powder 2g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, accurate methyl alcohol 25ml, the close plug of adding; Claim decide weight, sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article gallic acid (C 7H 6O 5) content must not be less than 2.32mg/g.
B. the assay of cinnaldehydrum
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the acetonitrile-water of 36:64; The detection wavelength is 290nm; Number of theoretical plate calculates by the cinnaldehydrum peak should be not less than 3000;
It is an amount of that the cinnaldehydrum reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 35 μ g, promptly gets;
Preparation powder 2g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, accurate methyl alcohol 25ml, the close plug of adding; Claim decide weight, sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article cinnaldehydrum (C 9H 8O) content must not be less than 0.21mg/g.
C. the assay of pipering
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the methanol-water of 78:22; The detection wavelength is 343nm; Number of theoretical plate calculates by the pipering peak should be not less than 1500;
It is an amount of that the pipering reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown measuring bottle, adds absolute ethyl alcohol and processes the solution that every 1ml contains 20 μ g, promptly gets;
Preparation powder 3g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, accurate absolute ethyl alcohol 25ml, the close plug of adding; Claim decide weight, sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with absolute ethyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article pipering (C 17H 19NO 3) content must not be less than 0.13mg/g.
D. the assay of hydroxyl radical carthamin yellow carthamus A
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is that 1% glacial acetic acid aqueous solution is a moving phase with volume parts than methyl alcohol-volume parts ratio of 30:70; The detection wavelength is 403nm; Number of theoretical plate calculates by the hydroxyl radical carthamin yellow carthamus A peak should be not less than 3000;
It is an amount of that the hydroxyl radical carthamin yellow carthamus A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown bottle, adds volume parts and processes the solution that every 1ml contains 0.13mg than 25% methanol aqueous solution, promptly gets;
Preparation powder 2g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, and the accurate volume parts that adds is than 25% methanol aqueous solution 25ml; Close plug is claimed to decide weight, ultrasonic 40 minutes, puts cold; Claim again to decide weight, supply the weight that subtracts mistake than 25% methanol aqueous solution, shake up with volume parts; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article hydroxyl radical carthamin yellow carthamus A (C 27H 30O 15) content must not be less than 1.15mg/g.
Tibetan medicine hospital's fame president, the Tibetan medicine principal expert of Qinghai Province Nima identify that the bonduc control medicinal material is the dry mature seed of legume bonduc Caesalpinia crista L. among the application through Qinghai Province; The cardamom control medicinal material is the dry mature fruit of zingiberaceous plant Amomum cardamomum Amomum kravanh Pierre ex Gagnep..
The unit corresponding relation of weight portion/mass parts described in this instructions and parts by volume is g/ml or kg/L.
The invention provides that a kind of Tibetan medicine composition Six-element bonduc looses and preparation in the thin-layer chromatography qualitative identification method of bonduc, cardamom; The liquid chromatography content assaying method of 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, the hydroxyl radical carthamin yellow carthamus A that safflower contains; Control the quality of said preparation comprehensively; Make said preparation in the treatment disease, guaranteed the safe and effective, quality controllable of medication.
The invention discloses that a kind of Tibetan medicine composition Six-element bonduc looses and the quality determining method of preparation.Use thin-layered chromatography (TLC method) that the diffusing middle bonduc of Six-element bonduc, cardamom, Chinese cassia tree have been carried out qualitative identification, the gallic acid that bonduc and seed of pomegranate contained during employing high performance liquid chromatography (HPLC method) was loose to the Six-element bonduc, the cinnaldehydrum that Chinese cassia tree contains, the pipering that the Bi roots of grass contains, the hydroxyl radical carthamin yellow carthamus A that safflower contains have carried out detection by quantitative.The inventive method has been set up the discrimination method of bonduc, cardamom, Chinese cassia tree in the said preparation; Content of effective assay methods such as gallic acid, cinnaldehydrum, pipering, hydroxyl radical carthamin yellow carthamus A; Improved target level of product quality; Guarantee the safe and effective, quality controllable of medication, made said preparation can treat disease more effectively.This law also can be used for other preparations that Tibetan medicine composition Six-element bonduc looses simultaneously, like Six-element bonduc particle, Six-element bonduc ball, Six-element bonduc sheet, Six-element bonduc capsule etc.
Following experimental example and embodiment are used to further specify but are not limited to the present invention.
Experimental example 1: thin layer identification experiment
Tibetan medicine composition Six-element bonduc looses and to scold Tibetan medicine medicine company incorporated company by the Qinghai gold and provide.
A. the discriminating of bonduc
Get Tibetan medicine composition Six-element bonduc divided powder 3g, add methyl alcohol 20ml, sonicated 30 minutes filters, and filtrating is as need testing solution; Get the negative control sample powder 3g that lacks bonduc in the preparation of prescription ratio, lack the negative control solution of bonduc with the method preparation; Other gets bonduc control medicinal material 1g, is equipped with bonduc control medicinal material solution with legal system; According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw each 3 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate; Is that 60-90 ℃ of petroleum ether-ethyl acetate-glacial acetic acid is developping agent with volume parts than the boiling range of 10:0.3:0.1; Launch, take out, dry; Than 10% ethanol solution of sulfuric acid, it is clear to be heated to the spot colour developing at 105 ℃ with volume parts in spray.
Test findings: in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color, negative control does not disturb.The result sees Fig. 1.
Investigated chloroform-methanol-glacial acetic acid respectively, boiling range is that 60-90 ℃ of sherwood oil-acetone-glacial acetic acid, boiling range are the 60-90 ℃ of chromatographic resolution effect under the different developping agent different volumes ratios such as petroleum ether-ethyl acetate-glacial acetic acid; The result is that 60-90 ℃ of petroleum ether-ethyl acetate-glacial acetic acid is under the developping agent condition with volume parts than the boiling range of 10:0.1-0.5:0.1, all has and launches effect preferably.Wherein, it is best to be than the boiling range of 10:0.3:0.1 that 60-90 ℃ of petroleum ether-ethyl acetate-glacial acetic acid is that developping agent launches effect with volume parts.The result shows that this discrimination method specificity is strong, can be used as the thin layer discrimination method of the diffusing middle bonduc of Tibetan medicine composition Six-element bonduc.
B. the discriminating of cardamom
Get Tibetan medicine composition Six-element bonduc divided powder 2g; Put in the 100ml round-bottomed flask, add water 50ml, extract according to determination of volatile oil Division A League Matches of French Football methods (an appendix X of Chinese Pharmacopoeia version in 2010 D); Jia Shui overflows in the flask water on the top of volatile oil extractor; Add ethyl acetate 1ml again, refluxing extraction 2 hours is obtained acetic acid ethyl fluid as need testing solution; Get the negative control sample powder 2g that lacks cardamom in the preparation of prescription ratio, lack the negative control solution of cardamom with the method preparation; Other gets cardamom control medicinal material 1g, is equipped with cardamom control medicinal material solution with legal system; According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate; Is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent with volume parts than the boiling range of 8:2; Launch, take out, dry; Spray is with the vanillic aldehyde sulfuric acid solution of mass and size portion rate 1%, and it is clear to be heated to the spot colour developing at 105 ℃.
Test findings: in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color, negative control does not disturb.The result sees Fig. 2.
Is under the 60-90 ℃ of petroleum ether-ethyl acetate developping agent condition in volume parts than the boiling range of 9-8:1-2, all has and launches effect preferably.Wherein, it is best to be than the boiling range of 8:2 that 60-90 ℃ of petroleum ether-ethyl acetate is that developping agent launches effect with volume parts.The result shows that this discrimination method specificity is strong, can be used as the thin layer discrimination method of the diffusing middle cardamom of Tibetan medicine composition Six-element bonduc.
C. the discriminating of Chinese cassia tree
Get Tibetan medicine composition Six-element bonduc divided powder 2g, porphyrize is put in the 100ml round-bottomed flask; Add water 60ml, extract according to determination of volatile oil Division A League Matches of French Football methods (an appendix X of Chinese Pharmacopoeia version in 2010 D), Jia Shui overflows in the flask water on the top of volatile oil extractor; Add 1ml ethyl acetate again, refluxing extraction 1 hour is cooled to room temperature; Obtain acetic acid ethyl fluid, as need testing solution.Get Chinese cassia tree control medicinal material 1g, be equipped with control medicinal material solution with legal system.Get misrun osmanthus negative control article,, be equipped with negative sample solution with legal system according to the test sample preparation method in the preparation of prescription ratio.According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica G TLCP; Is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent with volume parts than the boiling range of 10:2; Launch, take out, dry; Spray is with 1% vanillic aldehyde sulfuric acid solution, and 105 ℃ to be heated to the spot colour developing clear.
Test findings: in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color, negative control is noiseless.The result sees Fig. 3.
Be under 60-90 ℃ the petroleum ether-ethyl acetate developping agent condition, all to have and launch effect preferably in volume parts than the boiling range of 6-10:1-3.Wherein, be that to launch effect under 60-90 ℃ the petroleum ether-ethyl acetate developping agent condition best with the boiling range of volume ratio 10:2.The result shows that this discrimination method specificity is strong, can be used as the thin layer discrimination method of the diffusing middle Chinese cassia tree of Tibetan medicine composition Six-element bonduc
Experimental example 2: the assay experiment of gallic acid
1. instrument, reagent and confession test agent
Instrument: 1220 type Agilent high performance liquid chromatographs; Tianjin, island AuW220D electronic balance.
Reference substance: gallic acid reference substance (Nat'l Pharmaceutical & Biological Products Control Institute) lot number: 110831-200803.
Sample: the Six-element bonduc looses (the Qinghai gold is scolded Tibetan medicine medicine company incorporated company and provided), and lot number is respectively: 20110516,20110517,20110518.
2. detect the selection of wavelength
Get the gallic acid reference substance solution; In 190 ~ 400nm wavelength coverage, scan; Recording it has absorption maximum at 215nm and 275nm wavelength, compares the chromatogram of sample under 215nm and 275nm wavelength under the identical chromatographic conditions, and the result finds to detect chromatogram gallic acid peak and the adjacent chromatographic peak that wavelength is 275nm and reaches good baseline separation; And impurity disturbs less, and therefore selected 275nm is for detecting wavelength.
3. moving phase is selected
Discover that when being moving phase than methyl alcohol-volume parts of 1-3:99-97 than 1% glacial acetic acid aqueous solution with volume parts, gallic acid all can reach good chromatographic resolution effect.Be that moving phase is optimum than the volume parts of 1% glacial acetic acid aqueous solution than 2:98 wherein with methyl alcohol-volume parts.
4. system suitability test
Under above-mentioned chromatographic condition, accurate respectively reference substance solution, each 10 μ l of need testing solution of drawing inject liquid chromatograph, the record chromatogram.The result shows that the degree of separation that gallic acid is adjacent chromatographic peak in reference substance, the test sample chromatogram is all greater than 1.5.The result sees Fig. 3, Fig. 4.
5. reference substance preparation
It is an amount of to get the gallic acid reference substance, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 0.1mg, promptly gets.
6. test sample preparation
6.1 the investigation of method for distilling
By method under the assay item need testing solution is detected.According to the preparation of the test solution was prepared under the three copies, respectively, ultrasound, reflux, vibration rockers for 30 minutes.Content with gallic acid in every gram medicine is that index is confirmed method for distilling.The result sees table 1.
Table 1 method for distilling is investigated test findings
Figure BDA00001985421500081
The result shows, the content basically identical of gallic acid considers that ultrasonic Extraction is easy, easy to operate in ultrasonic and the every gram medicine of refluxing extraction gained, is ultrasonic Extraction so select method for distilling for use.
6.2 extract the investigation of solvent
By method under the assay item need testing solution is detected.The method for preparing under the item of pressing need testing solution prepares 3 parts, and precision adds entry, ethanol, each 25ml of methyl alcohol respectively.Content with gallic acid in every gram medicine is that index confirms to extract solvent.The result sees table 2.
Table 2 extracts solvent and investigates test findings
Figure BDA00001985421500082
The result shows that the gallic acid content that methyl alcohol is surveyed is the highest, is methyl alcohol so select the extraction solvent for use.
6.3 the investigation of extraction time
By method under the assay item need testing solution is detected.The method for preparing under the item of pressing need testing solution prepares 3 parts, and sonicated is 20 minutes, 30 minutes, 40 minutes respectively.Content with gallic acid in every gram medicine is that index is confirmed extraction time.The result sees table 3.
Table 3 extraction time investigation test findings
Figure BDA00001985421500091
The result shows, the content basically identical of gallic acid is 30 minutes so select extraction time for use in ultrasonic Extraction 30 minutes and the 40 minutes every gram medicines of gained.
7. the investigation of the preparation of typical curve and linear relationship
Precision is measured gallic acid reference substance stock solution solution (gallic acid content is 251.2 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml; Put respectively in the 10ml volumetric flask, be diluted to scale, shake up with methyl alcohol; Each accurate sample introduction 10 μ l; With peak area (A) reference substance concentration (C) is carried out linear regression, get gallic acid regression equation: A=14.005C+4.1658, related coefficient: R=0.9997.The result shows that gallic acid is in 25.12 μ g/ml ~ 251.2 μ g/ml scopes, and the peak area of gallic acid (A) is good with reference substance concentration (C) linear relationship.The result sees table 4.
Table 4 gallic acid linear relationship is investigated the result
8. precision test
The accurate gallic acid reference substance solution 10 μ l that draw inject liquid chromatograph, each METHOD FOR CONTINUOUS DETERMINATION 6 times, and the record peak area also calculates relative standard deviation.The result shows that instrument precision is good.The result sees table 5.
Table 5 gallic acid Precision test result
9. stability test
After the need testing solution preparation was accomplished, the accurate 10 μ l that draw injected liquid chromatograph, and the record peak area was whenever measured once at a distance from 2 hours later on, investigated 8 hours, and the record peak area also calculates relative standard deviation.The result shows, in 8 hours, to measure the result stable for gallic acid in the need testing solution.The result sees table 6.
Table 6 gallic acid stability test result
10. replica test
Get with the diffusing sample (product batch number: 20110516) 2g of a collection of Tibetan medicine composition Six-element bonduc; The accurate title, decide; Totally 6 parts, prepare need testing solution by the method for preparing under the item of need testing solution, the accurate respectively 10 μ l that draw; Inject liquid chromatograph, the content of gallic acid in the calculation sample.The result shows that this analytical approach repeatability is good.The result sees table 7.
Table 7 gallic acid replica test result
Figure BDA00001985421500102
11. recovery test
Precision takes by weighing looses with a collection of Tibetan medicine composition Six-element bonduc that (product batch number: 20110516) 6 parts, each accurate gallic acid reference substance that adds is measured its content, calculate recovery rate to sample.The result shows that it is accurate that this assay method is measured the result.The result sees table 8.
Table 8 gallic acid recovery test result
Figure BDA00001985421500103
12. sample determination
Get Tibetan medicine composition Six-element bonduc and loose three batches, measure and calculate gallic acid content.The result sees table 9.
Table 9 sample size is measured the result
Figure BDA00001985421500111
Experimental example 3: the assay experiment of cinnaldehydrum
1. instrument, reagent and confession test agent
Instrument: 1220 type Agilent high performance liquid chromatographs; Tianjin, island AuW220D electronic balance.
Reference substance: cinnaldehydrum reference substance (Nat'l Pharmaceutical & Biological Products Control Institute) lot number: 110710-200714.
Sample: the Six-element bonduc looses (the Qinghai gold is scolded Tibetan medicine medicine company incorporated company and provided), and lot number is respectively: 20110516,20110517,20110518.
2. detect the selection of wavelength
Get the cinnaldehydrum reference substance solution, in 190 ~ 400nm wavelength coverage, scan, recording it has absorption maximum in the 290nm wavelength, and therefore selected 290nm is for detecting wavelength.
3. moving phase is selected
Discover that when being moving phase with volume parts than the acetonitrile-water of 30-70:70-40, cinnaldehydrum all can reach good chromatographic resolution effect.Wherein the volume parts with acetonitrile-water is that moving phase is optimum than 36:64.
4. system suitability test and the negative investigation of disturbing
Under above-mentioned chromatographic condition, accurate respectively reference substance solution, need testing solution, each 10 μ l of negative control solution of drawing inject liquid chromatograph, the record chromatogram.The result shows that the degree of separation that cinnaldehydrum is adjacent chromatographic peak in reference substance, the test sample chromatogram is all greater than 1.5, and negative control does not disturb.The result sees Fig. 5, Fig. 6, Fig. 7.
5. reference substance preparation
It is an amount of to get the cinnaldehydrum reference substance, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 35 μ g, promptly gets.
6. test sample preparation
6.1 the investigation of method for distilling
By method under the assay item need testing solution is detected.According to the preparation of the test solution was prepared under the three copies, respectively, ultrasound, reflux, vibration rockers for 30 minutes.Content with cinnaldehydrum in every gram medicine is that index is confirmed method for distilling.The result sees table 10.
Table 10 method for distilling is investigated test findings
Figure BDA00001985421500112
The results showed that ultrasonic extraction medicines obtained per gram of cinnamic aldehyde content was significantly higher than the vibration Oscillating extraction, so the selection of extraction method for the ultrasonic extraction.
6.2 extract the investigation of solvent
By method under the assay item need testing solution is detected.The method for preparing under the item of pressing need testing solution prepares 3 parts, accurate respectively methyl alcohol, ethanol, each 25ml of ethyl acetate of adding.Content with cinnaldehydrum in every gram medicine is that index confirms to extract solvent.The result sees table 11.
Table 11 extracts solvent and investigates test findings
The result shows that the cinnaldehydrum content that methyl alcohol is surveyed is the highest, is methyl alcohol so select the extraction solvent for use.
6.3 the investigation of extraction time
By method under the assay item need testing solution is detected.The method for preparing under the item of pressing need testing solution prepares 3 parts, and sonicated is 20 minutes, 30 minutes, 40 minutes respectively.Content with cinnaldehydrum in every gram medicine is that index is confirmed extraction time.The result sees table 12.
Table 12 extraction time investigation test findings
Figure BDA00001985421500122
The result shows, the content basically identical of cinnaldehydrum is 30 minutes so select extraction time for use in ultrasonic Extraction 30 minutes and the 40 minutes every gram medicines of gained.
7. the investigation of the preparation of typical curve and linear relationship
Precision is measured cinnaldehydrum reference substance stock solution solution (cinnaldehydrum content is 72.4 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml; Put respectively in the 10ml volumetric flask, be diluted to scale, shake up with methyl alcohol; Each accurate sample introduction 10 μ l; With peak area (A) reference substance concentration (C) is carried out linear regression, get cinnaldehydrum regression equation: A=20.391C+2.2135, related coefficient: R=0.9999.The result shows that cinnaldehydrum is in 7.24 μ g/ml ~ 72.4 μ g/ml scopes, and the peak area of cinnaldehydrum (A) is good with reference substance concentration (C) linear relationship.The result sees table 13.
Table 13 cinnaldehydrum linear relationship is investigated the result
Figure BDA00001985421500123
8. precision test
The accurate cinnaldehydrum reference substance solution 10 μ l that draw inject liquid chromatograph, each METHOD FOR CONTINUOUS DETERMINATION 6 times, and the record peak area also calculates relative standard deviation.The result shows that instrument precision is good.The result sees table 14.
Table 14 cinnaldehydrum Precision test result
Figure BDA00001985421500124
Figure BDA00001985421500131
9. stability test
After the need testing solution preparation was accomplished, the accurate 10 μ l that draw injected liquid chromatograph, and the record peak area was whenever measured once at a distance from 2 hours later on, investigated 8 hours, and the record peak area also calculates relative standard deviation.The result shows, in 8 hours, to measure the result stable for cinnaldehydrum in the need testing solution.The result sees table 15.
Table 15 cinnaldehydrum stability test result
Figure BDA00001985421500132
10. replica test
Get with a collection of Tibetan medicine composition Six-element bonduc loose sample (product batch number: 20110516) 2g, accurate claim fixed, totally 6 parts; The method for preparing under the item by need testing solution prepares need testing solution; The accurate respectively 10 μ l that draw inject liquid chromatograph, the content of cinnaldehydrum in the calculation sample.The result shows that this analytical approach repeatability is good.The result sees table 16.
Table 16 cinnaldehydrum replica test result
Figure BDA00001985421500133
11. recovery test
Precision takes by weighing looses with a collection of Tibetan medicine composition Six-element bonduc that (product batch number: 20110516) 6 parts, each accurate cinnaldehydrum reference substance that adds is measured its content, calculate recovery rate to sample.The result shows that it is accurate that this assay method is measured the result.The result sees table 17.
Table 17 cinnaldehydrum recovery test result
12. sample determination
Get Tibetan medicine composition Six-element bonduc and loose three batches, measure and calculate cinnaldehydrum content.The result sees table 18.
Table 18 sample size is measured the result
Figure BDA00001985421500142
Experimental example 4: the assay experiment of pipering
1. instrument, reagent and confession test agent
Instrument: 1220 type Agilent high performance liquid chromatographs; Tianjin, island AuW220D electronic balance.
Reference substance: pipering reference substance (Nat'l Pharmaceutical & Biological Products Control Institute) lot number: 0775-200203.
Sample: the Six-element bonduc looses (the Qinghai gold is scolded Tibetan medicine medicine company incorporated company and provided), and lot number is respectively: 20110516,20110517,20110518.
2. detect the selection of wavelength
Get the pipering reference substance solution, in 190 ~ 400nm wavelength coverage, scan, pipering has absorption maximum in the 343nm wavelength, so be the detection wavelength according to the selected 343nm of ultraviolet absorpting spectrum.
3. moving phase is selected
Discover that when being moving phase with volume parts than the methanol-water of 75-80:20-25, pipering all can reach good chromatographic resolution effect.Wherein the volume parts with methanol-water is that moving phase is optimum than 78:22.
4. system suitability test and the negative investigation of disturbing
Under above-mentioned chromatographic condition, accurate respectively reference substance solution, need testing solution, each 10 μ l of negative control solution of drawing inject liquid chromatograph, the record chromatogram.The result shows that the degree of separation that pipering is adjacent chromatographic peak in reference substance, the test sample chromatogram is all greater than 1.5, and negative control does not disturb.The result sees Fig. 8, Fig. 9, Figure 10.
5. reference substance preparation
It is an amount of to get the pipering reference substance, and accurate the title decides, and puts in the brown measuring bottle, adds absolute ethyl alcohol and processes the solution that every 1ml contains 20 μ g, promptly gets.
6. test sample preparation
6.1 the investigation of method for distilling
By method under the assay item need testing solution is detected.According to the preparation of the test solution was prepared under the three copies, respectively, ultrasound, reflux, vibration rockers for 30 minutes.Content with pipering in every gram medicine is that index is confirmed method for distilling.The result sees table 19.
Table 19 method for distilling is investigated test findings
Figure BDA00001985421500143
The result shows, the content basically identical of pipering considers that ultrasonic Extraction is easy, easy to operate in ultrasonic and the every gram medicine of refluxing extraction gained, is ultrasonic Extraction so select method for distilling for use.
6.2 extract the investigation of solvent
By method under the assay item need testing solution is detected.The method for preparing under the item of pressing need testing solution prepares 3 parts, and accurate respectively adding methyl alcohol, absolute ethyl alcohol, volume parts are than each 25ml of 80% ethanol water.Content with pipering in every gram medicine is that index confirms to extract solvent.The result sees table 20.
Table 20 extracts solvent and investigates test findings
Figure BDA00001985421500151
The result shows, methyl alcohol, absolute ethyl alcohol, volume parts are more suitable basically than three kinds of content that solvent is measured of 80% ethanol water, but the test sample solvent that extracts with absolute ethyl alcohol, impurity peaks is minimum, is absolute ethyl alcohol so select the extraction solvent for use.
6.3 the investigation of extraction time
By method under the assay item need testing solution is detected.The method for preparing under the item of pressing need testing solution prepares 3 parts, and sonicated is 20 minutes, 30 minutes, 40 minutes respectively.Content with pipering in every gram medicine is that index is confirmed extraction time.The result sees table 21.
Table 21 extraction time investigation test findings
Figure BDA00001985421500152
The result shows, the content basically identical of pipering is 30 minutes so select extraction time for use in ultrasonic Extraction 30 minutes and the 40 minutes every gram medicines of gained.
7. the investigation of the preparation of typical curve and linear relationship
Precision is measured pipering reference substance stock solution solution (content of piperine is 42.8 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml; Put respectively in the 10ml volumetric flask, absolute ethyl alcohol is diluted to scale, shakes up; Each accurate sample introduction 10 μ l; With peak area (A) reference substance concentration (C) is carried out linear regression, get pipering regression equation: A=22.850C+0.6620, related coefficient: R=0.9998.The result shows that pipering is in 4.28 μ g/ml ~ 42.8 μ g/ml scopes, and the peak area of pipering (A) is good with reference substance concentration (C) linear relationship.The result sees table 22.
Table 22 pipering linear relationship is investigated the result
8. precision test
The accurate pipering reference substance solution 10 μ l that draw inject liquid chromatograph, each METHOD FOR CONTINUOUS DETERMINATION 6 times, and the record peak area also calculates relative standard deviation.The result shows that instrument precision is good.The result sees table 23.
Table 23 pipering Precision test result
Figure BDA00001985421500154
Figure BDA00001985421500161
9. stability test
After the need testing solution preparation was accomplished, the accurate 10 μ l that draw injected liquid chromatograph, and the record peak area was whenever measured once at a distance from 2 hours later on, investigated 8 hours, and the record peak area also calculates relative standard deviation.The result shows, in 8 hours, to measure the result stable for pipering in the need testing solution.The result sees table 24.
Table 24 pipering stability test result
10. replica test
Get with a collection of Tibetan medicine composition Six-element bonduc loose sample (product batch number: 20110516) 3g, accurate claim fixed, totally 6 parts; The method for preparing under the item by need testing solution prepares need testing solution; The accurate respectively 10 μ l that draw inject liquid chromatograph, the content of pipering in the calculation sample.The result shows that this analytical approach repeatability is good.The result sees table 25.
Table 25 pipering replica test result
11. recovery test
Precision takes by weighing looses with a collection of Tibetan medicine composition Six-element bonduc that (product batch number: 20110516) 6 parts, each accurate pipering reference substance that adds is measured its content, calculate recovery rate to sample.The result shows that it is accurate that this assay method is measured the result.The result sees table 26.
Table 26 pipering recovery test result
Figure BDA00001985421500164
Figure BDA00001985421500171
12. sample determination
Get Tibetan medicine composition Six-element bonduc and loose three batches, measure and calculate content of piperine.The result sees table 27.
Table 27 sample size is measured the result
Figure BDA00001985421500172
Experimental example 5: the assay experiment of hydroxyl radical carthamin yellow carthamus A
1. instrument, reagent and confession test agent
Instrument: 1220 type Agilent high performance liquid chromatographs; Tianjin, island AuW220D electronic balance.
Reference substance: hydroxyl radical carthamin yellow carthamus A reference substance (Nat'l Pharmaceutical & Biological Products Control Institute), lot number: 111637-200905.
Sample: the Six-element bonduc looses (the Qinghai gold is scolded Tibetan medicine medicine company incorporated company and provided), and lot number is respectively: 20110516,20110517,20110518.
2. detect the selection of wavelength
Get the hydroxyl radical carthamin yellow carthamus A reference substance solution, in 190 ~ 500nm wavelength coverage, scan, hydroxyl radical carthamin yellow carthamus A has absorption maximum in the 403nm wavelength, so be the detection wavelength according to the selected 403nm of ultraviolet absorpting spectrum.
3. moving phase is selected
Discovering, is 1% glacial acetic acid aqueous solution when being moving phase with volume parts than methyl alcohol-volume parts ratio of 25-35:65-75, and hydroxyl radical carthamin yellow carthamus A all can reach good chromatographic resolution effect.Be that moving phase is optimum than the volume parts that is 1% glacial acetic acid aqueous solution than 30:70 wherein with methyl alcohol-volume parts.
4. system suitability test and the negative investigation of disturbing
Under above-mentioned chromatographic condition, accurate respectively reference substance solution, need testing solution, each 10 μ l of negative control solution of drawing inject liquid chromatograph, the record chromatogram.The result shows that the degree of separation that hydroxyl radical carthamin yellow carthamus A is adjacent chromatographic peak in reference substance, the test sample chromatogram is all greater than 1.5, and negative control does not disturb.The result sees Figure 11, Figure 12, Figure 13.
5. reference substance preparation
It is an amount of to get hydroxyl safflower anthocyanidin A reference substance, and accurate the title decides, and puts in the brown bottle, adds volume parts and processes the solution that every 1ml contains 0.13mg than 25% methanol aqueous solution, promptly gets.
6. test sample preparation
6.1 the investigation of method for distilling
By method under the assay item need testing solution is detected.According to the preparation of the test solution was prepared under the three copies, respectively, ultrasound, reflux, vibration rockers for 40 minutes.Content with hydroxyl radical carthamin yellow carthamus A in every gram medicine is that index is confirmed method for distilling.The result sees table 28.
Table 28 method for distilling is investigated test findings
Figure BDA00001985421500181
The result shows that the content of hydroxyl radical carthamin yellow carthamus A is the highest in the every gram medicine of ultrasonic Extraction gained, is ultrasonic Extraction so select method for distilling for use.
6.2 extract the investigation of solvent
By method under the assay item need testing solution is detected.The method for preparing under the item of pressing need testing solution prepares 3 parts, and precision adds entry, volume parts than 25% methanol aqueous solution, each 25ml of methyl alcohol respectively.Content with hydroxyl radical carthamin yellow carthamus A in every gram medicine is that index confirms to extract solvent.The result sees table 29.
Table 29 extracts solvent and investigates test findings
Figure BDA00001985421500182
The result shows, the hydroxyl radical carthamin yellow carthamus A content basically identical that water, volume parts are surveyed than 25% methanol aqueous solution and methyl alcohol extracts the solvent filter difficulty owing to use water as, and pure methyl alcohol toxicity is big, is that volume parts is than 25% methanol aqueous solution so select the extraction solvent for use.
6.3 the investigation of extraction time
By method under the assay item need testing solution is detected.The method for preparing under the item of pressing need testing solution prepares 3 parts, and sonicated is 30 minutes, 40 minutes, 50 minutes respectively.Content with hydroxyl radical carthamin yellow carthamus A in every gram medicine is that index is confirmed extraction time.The result sees table 30.
Table 30 extraction time investigation test findings
Figure BDA00001985421500183
The result shows, the content basically identical of hydroxyl radical carthamin yellow carthamus A is 40 minutes so select extraction time for use in ultrasonic Extraction 40 minutes and the 50 minutes every gram medicines of gained.
7. the investigation of the preparation of typical curve and linear relationship
Precision is measured 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 respectively in the 10ml volumetric flask, volume parts is diluted to scale than 25% methanol aqueous solution, shakes up; Each accurate sample introduction 10 μ l; With peak area (A) reference substance concentration (C) is carried out linear regression, get hydroxyl radical carthamin yellow carthamus A regression equation: A=22.933C+7.4763, related coefficient: R=0.9998.The result shows that hydroxyl radical carthamin yellow carthamus A is in 24.57 μ g/ml ~ 245.7 μ g/ml scopes, and the peak area of hydroxyl radical carthamin yellow carthamus A (A) is good with reference substance concentration (C) linear relationship.The result sees table 31.
Table 31 hydroxyl radical carthamin yellow carthamus A linear relationship is investigated the result
Figure BDA00001985421500184
Figure BDA00001985421500191
8. precision test
The accurate hydroxyl radical carthamin yellow carthamus A reference substance solution 10 μ l that draw inject liquid chromatograph, each METHOD FOR CONTINUOUS DETERMINATION 6 times, and the record peak area also calculates relative standard deviation.The result shows that instrument precision is good.The result sees table 32.
Table 32 hydroxyl radical carthamin yellow carthamus A Precision test result
Figure BDA00001985421500192
9. stability test
After the need testing solution preparation was accomplished, the accurate 10 μ l that draw injected liquid chromatograph, and the record peak area was whenever measured once at a distance from 2 hours later on, investigated 8 hours, and the record peak area also calculates relative standard deviation.The result shows, in 8 hours, to measure the result stable for hydroxyl radical carthamin yellow carthamus A in the need testing solution.The result sees table 33.
Table 33 hydroxyl radical carthamin yellow carthamus A stability test result
10. replica test
Get with the diffusing sample (product batch number: 20110516) 2g of a collection of Tibetan medicine composition Six-element bonduc; The accurate title, decide; Totally 6 parts, prepare need testing solution by the method for preparing under the item of need testing solution, the accurate respectively 10 μ l that draw; Inject liquid chromatograph, the content of hydroxyl radical carthamin yellow carthamus A in the calculation sample.The result shows that this analytical approach repeatability is good.The result sees table 34.
Table 34 hydroxyl radical carthamin yellow carthamus A replica test result
Figure BDA00001985421500194
Figure BDA00001985421500201
11. recovery test
Precision takes by weighing looses with a collection of Tibetan medicine composition Six-element bonduc that (product batch number: 20110516) 6 parts, each accurate hydroxyl radical carthamin yellow carthamus A reference substance that adds is measured its content, calculate recovery rate to sample.The result shows that it is accurate that this assay method is measured the result.The result sees table 35.
Table 35 hydroxyl radical carthamin yellow carthamus A recovery test result
Figure BDA00001985421500202
12. sample determination
Get Tibetan medicine composition Six-element bonduc and loose three batches, measure and calculate hydroxyl radical carthamin yellow carthamus A content.The result sees table 36.
Table 36 sample size is measured the result
Figure BDA00001985421500203
Following embodiment all can realize the described effect of above-mentioned experimental example.
Description of drawings
Fig. 1 is the thin layer identification color spectrogram of bonduc;
Fig. 2 is the thin layer identification color spectrogram of cardamom;
Fig. 3 is the thin layer identification color spectrogram of Chinese cassia tree;
Fig. 4 is a gallic acid contrast high-efficient liquid phase chromatogram;
Fig. 5 is that gallic acid content is measured the test sample high-efficient liquid phase chromatogram;
Fig. 6 is a cinnaldehydrum contrast high-efficient liquid phase chromatogram;
Fig. 7 is a cinnaldehydrum assay test sample high-efficient liquid phase chromatogram;
Fig. 8 is a misrun osmanthus negative control high-efficient liquid phase chromatogram;
Fig. 9 is a pipering contrast high-efficient liquid phase chromatogram;
Figure 10 is that content of piperine is measured the test sample high-efficient liquid phase chromatogram;
Figure 11 lacks Bi roots of grass negative control high-efficient liquid phase chromatogram;
Figure 12 is a hydroxyl radical carthamin yellow carthamus A contrast high-efficient liquid phase chromatogram;
Figure 13 is a hydroxyl radical carthamin yellow carthamus A assay test sample high-efficient liquid phase chromatogram;
Figure 14 lacks safflower negative control high-efficient liquid phase chromatogram;
Wherein, a is a control medicinal material among Fig. 1, Fig. 2, Fig. 3, and b is a test sample, and c is a 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 the present invention is done detailed elaboration, but be not limited to the embodiment of these concrete records.The Tibetan medicine composition Six-element bonduc that is detected looses and to scold Tibetan medicine medicine company incorporated company product for the Qinghai gold and sell.
Embodiment 1: the quality determining method that the Six-element bonduc looses
Differentiate:
A. the discriminating of bonduc
Get Tibetan medicine composition Six-element bonduc divided powder 3g, add methyl alcohol 20ml, sonicated 30 minutes filters, and filtrating is as need testing solution; Other gets bonduc control medicinal material 1g, is equipped with bonduc control medicinal material solution with legal system; According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw each 3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Is that 60-90 ℃ of petroleum ether-ethyl acetate-glacial acetic acid is developping agent with volume parts than the boiling range of 10:0.3:0.1; Launch, take out, dry; Than 10% ethanol solution of sulfuric acid, it is clear to be heated to the spot colour developing at 105 ℃ with volume parts in spray; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
B. the discriminating of cardamom
Get Tibetan medicine composition Six-element bonduc divided powder 2g; Put in the 100ml round-bottomed flask, add water 50ml, extract according to determination of volatile oil Division A League Matches of French Football methods (an appendix X of Chinese Pharmacopoeia version in 2010 D); Jia Shui overflows in the flask water on the top of volatile oil extractor; Add ethyl acetate 1ml again, refluxing extraction 2 hours is obtained acetic acid ethyl fluid as need testing solution; Other gets cardamom control medicinal material 1g, is equipped with cardamom control medicinal material solution with legal system; According to thin-layered chromatography (" appendix VIB of Chinese pharmacopoeia version in 2010) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent with volume parts than the boiling range of 8:2; Launch, take out, dry; Spray is with the vanillic aldehyde sulfuric acid solution of mass and size portion rate 1%, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
C. the discriminating of Chinese cassia tree
Get Tibetan medicine composition Six-element bonduc divided powder 2g, put in the 100ml round-bottomed flask, add 60ml water; Extract according to determination of volatile oil Division A League Matches of French Football method (an appendix X of Chinese Pharmacopoeia version in 2010 D), Jia Shui overflows in the flask water on the top of volatile oil extractor, adds ethyl acetate 1ml again; Refluxing extraction 1 hour; Be cooled to room temperature, obtain acetic acid ethyl fluid, as need testing solution; Get Chinese cassia tree control medicinal material 1g, be equipped with control medicinal material solution with legal system; According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica G TLCP; Is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent with volume parts than the boiling range of (10:2); Launch, take out, dry; Spray is with mass and size portion rate 1% vanillic aldehyde sulfuric acid solution, and 105 ℃ to be heated to the spot colour developing clear; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Assay:
A. the assay of gallic acid
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase than methyl alcohol-volume parts of 2:98 than 1% glacial acetic acid aqueous solution with volume parts; The detection wavelength is 275nm; Number of theoretical plate calculates by the gallic acid peak should be not less than 2000;
It is an amount of that the gallic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 0.1mg, promptly gets;
Tibetan medicine composition Six-element bonduc divided powder 2g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate methyl alcohol 25ml that adds; Close plug is claimed to decide weight, and sonicated 30 minutes is put cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article gallic acid (C 7H 6O 5) content must not be less than 2.32mg/g.
B. the assay of cinnaldehydrum
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the acetonitrile-water of 36:64; The detection wavelength is 290nm; Number of theoretical plate calculates by the cinnaldehydrum peak should be not less than 3000;
It is an amount of that the cinnaldehydrum reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 35 μ g, promptly gets;
Tibetan medicine composition Six-element bonduc divided powder 2g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate methyl alcohol 25ml that adds; Close plug is claimed to decide weight, and sonicated 30 minutes is put cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article cinnaldehydrum (C 9H 8O) content must not be less than 0.21mg/g.
C. the assay of pipering
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the methanol-water of 78:22; The detection wavelength is 343nm; Number of theoretical plate calculates by the pipering peak should be not less than 1500;
It is an amount of that the pipering reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown measuring bottle, adds absolute ethyl alcohol and processes the solution that every 1ml contains 20 μ g, promptly gets;
Tibetan medicine composition Six-element bonduc divided powder 3g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate absolute ethyl alcohol 25ml that adds; Close plug is claimed to decide weight, and sonicated 30 minutes is put cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up with absolute ethyl alcohol; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article pipering (C 17H 19NO 3) content must not be less than 0.13mg/g.
D. the assay of hydroxyl radical carthamin yellow carthamus A
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is that 1% glacial acetic acid aqueous solution is a moving phase with volume parts than methyl alcohol-volume parts ratio of 30:70; The detection wavelength is 403nm; Number of theoretical plate calculates by the hydroxyl radical carthamin yellow carthamus A peak should be not less than 3000;
It is an amount of that the hydroxyl radical carthamin yellow carthamus A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown bottle, adds volume parts and processes the solution that every 1ml contains 0.13mg than 25% methanol aqueous solution, promptly gets;
Tibetan medicine composition Six-element bonduc divided powder 2g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, and the accurate volume parts that adds is than 25% methanol aqueous solution 25ml; Close plug is claimed to decide weight, ultrasonic 40 minutes, puts cold; Claim again to decide weight, supply the weight that subtracts mistake than 25% methanol aqueous solution, shake up with volume parts; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article hydroxyl radical carthamin yellow carthamus A (C 27H 30O 15) content must not be less than 1.15mg/g.
Embodiment 2: the quality determining method of Six-element bonduc particle
Differentiate:
A. the discriminating of bonduc
Get Tibetan medicine composition Six-element bonduc particle powder 3g, add methyl alcohol 20ml, sonicated 30 minutes filters, and filtrating is as need testing solution; Other gets bonduc control medicinal material 1g, is equipped with bonduc control medicinal material solution with legal system; According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw each 3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Is that 60-90 ℃ of petroleum ether-ethyl acetate-glacial acetic acid is developping agent with volume parts than the boiling range of 10:0.3:0.1; Launch, take out, dry; Than 10% ethanol solution of sulfuric acid, it is clear to be heated to the spot colour developing at 105 ℃ with volume parts in spray; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
B. the discriminating of cardamom
Get Tibetan medicine composition Six-element bonduc particle powder 2g; Put in the 100ml round-bottomed flask, add water 50ml, extract according to determination of volatile oil Division A League Matches of French Football methods (an appendix X of Chinese Pharmacopoeia version in 2010 D); Jia Shui overflows in the flask water on the top of volatile oil extractor; Add ethyl acetate 1ml again, refluxing extraction 2 hours is obtained acetic acid ethyl fluid as need testing solution; Other gets cardamom control medicinal material 1g, is equipped with cardamom control medicinal material solution with legal system; According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent with volume parts than the boiling range of 8:2; Launch, take out, dry; Spray is with the vanillic aldehyde sulfuric acid solution of mass and size portion rate 1%, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
C. the discriminating of Chinese cassia tree
Get Tibetan medicine composition Six-element bonduc particle powder 2g, put in the 100ml round-bottomed flask, add 60ml water; Extract according to determination of volatile oil Division A League Matches of French Football method (an appendix X of Chinese Pharmacopoeia version in 2010 D), Jia Shui overflows in the flask water on the top of volatile oil extractor, adds ethyl acetate 1ml again; Refluxing extraction 1 hour; Be cooled to room temperature, obtain acetic acid ethyl fluid, as need testing solution; Get Chinese cassia tree control medicinal material 1g, be equipped with control medicinal material solution with legal system; According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica G TLCP; Is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent with volume parts than the boiling range of (10:2); Launch, take out, dry; Spray is with mass and size portion rate 1% vanillic aldehyde sulfuric acid solution, and 105 ℃ to be heated to the spot colour developing clear; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Assay:
A. the assay of gallic acid
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase than methyl alcohol-volume parts of 2:98 than 1% glacial acetic acid aqueous solution with volume parts; The detection wavelength is 275nm; Number of theoretical plate calculates by the gallic acid peak should be not less than 2000;
It is an amount of that the gallic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 0.1mg, promptly gets;
Tibetan medicine composition Six-element bonduc particle powder 2g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate methyl alcohol 25ml that adds; Close plug is claimed to decide weight, and sonicated 30 minutes is put cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article gallic acid (C 7H 6O 5) content must not be less than 2.32mg/g.
B. the assay of cinnaldehydrum
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the acetonitrile-water of 36:64; The detection wavelength is 290nm; Number of theoretical plate calculates by the cinnaldehydrum peak should be not less than 3000;
It is an amount of that the cinnaldehydrum reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 35 μ g, promptly gets;
Tibetan medicine composition Six-element bonduc particle powder 2g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate methyl alcohol 25ml that adds; Close plug is claimed to decide weight, and sonicated 30 minutes is put cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article cinnaldehydrum (C 9H 8O) content must not be less than 0.21mg/g.
C. the assay of pipering
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the methanol-water of 78:22; The detection wavelength is 343nm; Number of theoretical plate calculates by the pipering peak should be not less than 1500;
It is an amount of that the pipering reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown measuring bottle, adds absolute ethyl alcohol and processes the solution that every 1ml contains 20 μ g, promptly gets;
Tibetan medicine composition Six-element bonduc particle powder 3g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate absolute ethyl alcohol 25ml that adds; Close plug is claimed to decide weight, and sonicated 30 minutes is put cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up with absolute ethyl alcohol; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article pipering (C 17H 19NO 3) content must not be less than 0.13mg/g.
D. the assay of hydroxyl radical carthamin yellow carthamus A
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is that 1% glacial acetic acid aqueous solution is a moving phase with volume parts than methyl alcohol-volume parts ratio of 30:70; The detection wavelength is 403nm; Number of theoretical plate calculates by the hydroxyl radical carthamin yellow carthamus A peak should be not less than 3000;
It is an amount of that the hydroxyl radical carthamin yellow carthamus A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown bottle, adds volume parts and processes the solution that every 1ml contains 0.13mg than 25% methanol aqueous solution, promptly gets;
Tibetan medicine composition Six-element bonduc particle powder 2g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, and the accurate volume parts that adds is than 25% methanol aqueous solution 25ml; Close plug is claimed to decide weight, ultrasonic 40 minutes, puts cold; Claim again to decide weight, supply the weight that subtracts mistake than 25% methanol aqueous solution, shake up with volume parts; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article hydroxyl radical carthamin yellow carthamus A (C 27H 30O 15) content must not be less than 1.15mg/g.
Described Tibetan medicine composition Six-element bonduc particle is meant with the diffusing bulk drug prescription of Six-element bonduc bonduc 90g, seed of pomegranate 90g, Chinese cassia tree 5g, cardamom 40g, Bi roots of grass 5g, safflower 50g; Add conventional auxiliary material, according to the granule of common process preparation.
Embodiment 3: the quality determining method of Six-element bonduc ball
Differentiate:
A. the discriminating of bonduc
Get Tibetan medicine composition Six-element bonduc ball powder 3g, add methyl alcohol 20ml, sonicated 30 minutes filters, and filtrating is as need testing solution; Other gets bonduc control medicinal material 1g, is equipped with bonduc control medicinal material solution with legal system; According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 B) test, draw each 3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Is that 60-90 ℃ of petroleum ether-ethyl acetate-glacial acetic acid is developping agent with volume parts than the boiling range of 10:0.3:0.1; Launch, take out, dry; Than 10% ethanol solution of sulfuric acid, it is clear to be heated to the spot colour developing at 105 ℃ with volume parts in spray; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
B. the discriminating of cardamom
Get Tibetan medicine composition Six-element bonduc ball powder 2g; Put in the 100ml round-bottomed flask, add water 50ml, extract according to determination of volatile oil Division A League Matches of French Football methods (an appendix X of Chinese Pharmacopoeia version in 2010 D); Jia Shui overflows in the flask water on the top of volatile oil extractor; Add ethyl acetate 1ml again, refluxing extraction 2 hours is obtained acetic acid ethyl fluid as need testing solution; Other gets cardamom control medicinal material 1g, is equipped with cardamom control medicinal material solution with legal system; According to thin-layered chromatography (" appendix VIB of Chinese pharmacopoeia version in 2010) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent with volume parts than the boiling range of 8:2; Launch, take out, dry; Spray is with the vanillic aldehyde sulfuric acid solution of mass and size portion rate 1%, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
C. the discriminating of Chinese cassia tree
Get Tibetan medicine composition Six-element bonduc ball powder 2g, put in the 100ml round-bottomed flask, add 60ml water; Extract according to determination of volatile oil Division A League Matches of French Football method (an appendix X of Chinese Pharmacopoeia version in 2010 D), Jia Shui overflows in the flask water on the top of volatile oil extractor, adds ethyl acetate 1ml again; Refluxing extraction 1 hour; Be cooled to room temperature, obtain acetic acid ethyl fluid, as need testing solution; Get Chinese cassia tree control medicinal material 1g, be equipped with control medicinal material solution with legal system; According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica G TLCP; Is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent with volume parts than the boiling range of (10:2); Launch, take out, dry; Spray is with mass and size portion rate 1% vanillic aldehyde sulfuric acid solution, and 105 ℃ to be heated to the spot colour developing clear; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Assay:
A. the assay of gallic acid
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase than methyl alcohol-volume parts of 2:98 than 1% glacial acetic acid aqueous solution with volume parts; The detection wavelength is 275nm; Number of theoretical plate calculates by the gallic acid peak should be not less than 2000;
It is an amount of that the gallic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 0.1mg, promptly gets;
Tibetan medicine composition Six-element bonduc ball powder 2g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate methyl alcohol 25ml that adds; Close plug is claimed to decide weight, and sonicated 30 minutes is put cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article gallic acid (C 7H 6O 5) content must not be less than 2.32mg/g.
B. the assay of cinnaldehydrum
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the acetonitrile-water of 36:64; The detection wavelength is 290nm; Number of theoretical plate calculates by the cinnaldehydrum peak should be not less than 3000;
It is an amount of that the cinnaldehydrum reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 35 μ g, promptly gets;
Tibetan medicine composition Six-element bonduc ball powder 2g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate methyl alcohol 25ml that adds; Close plug is claimed to decide weight, and sonicated 30 minutes is put cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article cinnaldehydrum (C 9H 8O) content must not be less than 0.21mg/g.
C. the assay of pipering
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the methanol-water of 78:22; The detection wavelength is 343nm; Number of theoretical plate calculates by the pipering peak should be not less than 1500;
It is an amount of that the pipering reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown measuring bottle, adds absolute ethyl alcohol and processes the solution that every 1ml contains 20 μ g, promptly gets;
Tibetan medicine composition Six-element bonduc ball powder 3g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate absolute ethyl alcohol 25ml that adds; Close plug is claimed to decide weight, and sonicated 30 minutes is put cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up with absolute ethyl alcohol; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article pipering (C 17H 19NO 3) content must not be less than 0.13mg/g.
D. the assay of hydroxyl radical carthamin yellow carthamus A
Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2010 D);
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is that 1% glacial acetic acid aqueous solution is a moving phase with volume parts than methyl alcohol-volume parts ratio of 30:70; The detection wavelength is 403nm; Number of theoretical plate calculates by the hydroxyl radical carthamin yellow carthamus A peak should be not less than 3000;
It is an amount of that the hydroxyl radical carthamin yellow carthamus A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown bottle, adds volume parts and processes the solution that every 1ml contains 0.13mg than 25% methanol aqueous solution, promptly gets;
Tibetan medicine composition Six-element bonduc ball powder 2g is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, and the accurate volume parts that adds is than 25% methanol aqueous solution 25ml; Close plug is claimed to decide weight, ultrasonic 40 minutes, puts cold; Claim again to decide weight, supply the weight that subtracts mistake than 25% methanol aqueous solution, shake up with volume parts; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
These article hydroxyl radical carthamin yellow carthamus A (C 27H 30O 15) content must not be less than 1.15mg/g.
Described Tibetan medicine composition Six-element bonduc ball is meant with the diffusing bulk drug prescription of Six-element bonduc bonduc 90g, seed of pomegranate 90g, Chinese cassia tree 5g, cardamom 40g, Bi roots of grass 5g, safflower 50g; Add conventional auxiliary material, according to the pill of common process preparation.

Claims (7)

1. the quality determining method of a Six-element bonduc preparation; The various preparations of said preparation for processing by the pharmacy conventional method by bonduc, seed of pomegranate, Chinese cassia tree, cardamom, the Bi roots of grass, safflower; It is characterized in that; Quality determining method comprises the thin-layer chromatography qualitative identification method to bonduc, cardamom, Chinese cassia tree in the preparation, one or more in the liquid chromatography content assaying method of the gallic acid that bonduc and seed of pomegranate are contained, the cinnaldehydrum that Chinese cassia tree contains, pipering that the Bi roots of grass contains, hydroxyl radical carthamin yellow carthamus A that safflower contains.
2. quality determining method as claimed in claim 1 is characterized in that, quality determining method is one or more in the following method:
Differentiate:
A. the discriminating of bonduc: it is to be contrast with the bonduc control medicinal material; With volume parts than 10: (0.1-0.5): 0.1 boiling range is that 60-90 ℃ of petroleum ether-ethyl acetate-glacial acetic acid is that developping agent launches on silica gel g thin-layer plate, is the thin-layered chromatography of developer with volume parts than 10% ethanol solution of sulfuric acid;
B. the discriminating of cardamom: it is to be contrast with the cardamom control medicinal material; With volume parts than (8-9): boiling range (1-2) is that 60-90 ℃ of petroleum ether-ethyl acetate is that developping agent launches on silica gel g thin-layer plate, is the thin-layered chromatography of developer with the vanillic aldehyde sulfuric acid solution of mass and size portion rate 1%;
C. the discriminating of Chinese cassia tree: it is to be contrast with the Chinese cassia tree control medicinal material; With volume parts than (6-10): boiling range (1-3) is that 60-90 ℃ of petroleum ether-ethyl acetate is that developping agent launches on silica gel g thin-layer plate, is the thin-layered chromatography of developer with the vanillic aldehyde sulfuric acid solution of mass and size portion rate 1-5%;
Assay:
A. the assay of gallic acid: it is to be contrast with the gallic acid reference substance, is filling agent with the octadecylsilane chemically bonded silica; With volume parts than (1-3): methyl alcohol-volume parts (97-99) is a moving phase than 1% glacial acetic acid aqueous solution, and detecting wavelength is the high performance liquid chromatography of 275nm;
B. the assay of cinnaldehydrum: it is to be contrast with the cinnaldehydrum reference substance, is filling agent with the octadecylsilane chemically bonded silica, is moving phase with volume parts than the acetonitrile-water of 30-70:30-70, and detecting wavelength is that 290nm is a high performance liquid chromatography;
C. the assay of pipering: it is to be contrast with the pipering reference substance, is filling agent with the octadecylsilane chemically bonded silica, is moving phase with volume parts than the methanol-water of 75-80:20-25, and detecting wavelength is the high performance liquid chromatography of 343nm;
D. the assay of hydroxyl radical carthamin yellow carthamus A: it is to be contrast with the hydroxyl radical carthamin yellow carthamus A reference substance; With the octadecylsilane chemically bonded silica is filling agent; Is that 1% glacial acetic acid aqueous solution is a moving phase with volume parts than methyl alcohol-volume parts ratio of 25-35:65-75, and detecting wavelength is the high performance liquid chromatography of 403nm.
3. quality determining method as claimed in claim 1 is characterized in that, quality determining method is one or more in the following method:
Differentiate:
A. the discriminating of bonduc
Get preparation powder 2-4g to be detected, add methyl alcohol 10-30ml, sonicated 20-40 minute, filter, filtrating is as need testing solution; Other gets bonduc control medicinal material 0.5-1g, is equipped with bonduc control medicinal material solution with legal system; According to the thin-layered chromatography test, draw each 2-5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With volume parts than 10: (0.1-0.5): 0.1 boiling range is that 60-90 ℃ of petroleum ether-ethyl acetate-glacial acetic acid is developping agent; Launch, take out, dry; Than 10% ethanol solution of sulfuric acid, it is clear to be heated to the spot colour developing at 105 ℃ with volume parts in spray; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
B. the discriminating of cardamom
Get preparation powder 1-3g to be detected; Put in the 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 an appendix X of Chinese Pharmacopoeia version in 2010 D; Jia Shui overflows in the flask water on the top of volatile oil extractor; Add ethyl acetate 1ml again, refluxing extraction 1-3 hour, obtain acetic acid ethyl fluid as need testing solution; Other gets cardamom control medicinal material 0.5-1g, is equipped with cardamom control medicinal material solution with legal system; According to the thin-layered chromatography test, draw each 5-10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With volume parts than (8-9): boiling range (1-2) is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent; Launch, take out, dry; Spray is with the vanillic aldehyde sulfuric acid solution of mass and size portion rate 1%, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
C. Chinese cassia tree is differentiated
Get preparation powder 1-5g to be detected, put in the 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 an appendix X of Chinese Pharmacopoeia version in 2010 D, Jia Shui overflows in the flask water on the top of volatile oil extractor, adds ethyl acetate 1-2ml again; Refluxing extraction 1-3 hour; Be cooled to room temperature, obtain acetic acid ethyl fluid, as need testing solution.Get Chinese cassia tree control medicinal material 0.5-2g, be equipped with control medicinal material solution with legal system; According to the thin-layered chromatography test, draw each 5-10 μ l of above-mentioned two kinds of solution, put respectively on same silica G TLCP; With volume parts than (6～10): the boiling range of (1～3) is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent; Launch, take out, dry; Spray is with mass and size portion rate 1-5% vanillic aldehyde sulfuric acid solution, and 105 ℃ to be heated to the spot colour developing clear; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
Assay:
A. the assay of gallic acid
According to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; With volume parts than (1-3): methyl alcohol-volume parts (97-99) is a moving phase than 1% glacial acetic acid aqueous solution; The detection wavelength is 275nm; Number of theoretical plate calculates by the gallic acid peak should be not less than 2000;
It is an amount of that the gallic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 0.1mg, promptly gets;
Preparation powder 1-3g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, accurate methyl alcohol 20-30ml, the close plug of adding; Claim decide weight, sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 5-10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
B. the assay of cinnaldehydrum
According to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the acetonitrile-water of 30-70:30-70; The detection wavelength is 290nm; Number of theoretical plate calculates by the cinnaldehydrum peak should be not less than 3000;
It is an amount of that the cinnaldehydrum reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 35 μ g, promptly gets;
Preparation powder 1-3g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, accurate methyl alcohol 20-30ml, the close plug of adding; Claim decide weight, sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 5-10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
C. the assay of pipering
According to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the methanol-water of 75-80:20-25; The detection wavelength is 343nm; Number of theoretical plate calculates by the pipering peak should be not less than 1500;
It is an amount of that the pipering reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown measuring bottle, adds absolute ethyl alcohol and processes the solution that every 1ml contains 20 μ g, promptly gets;
Preparation powder 2-4g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate absolute ethyl alcohol 20-30ml that adds; Close plug is claimed to decide weight, and sonicated 30 minutes is put cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up with absolute ethyl alcohol; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 5-10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
D. the assay of hydroxyl radical carthamin yellow carthamus A
According to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is that 1% glacial acetic acid aqueous solution is a moving phase with volume parts than methyl alcohol-volume parts ratio of 25-35:65-75; The detection wavelength is 403nm; Number of theoretical plate calculates by the hydroxyl radical carthamin yellow carthamus A peak should be not less than 3000;
It is an amount of that the hydroxyl radical carthamin yellow carthamus A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown bottle, adds volume parts and processes the solution that every 1ml contains 0.13mg than 25% methanol aqueous solution, promptly gets;
Preparation powder 1-3g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, and the accurate volume parts that adds is than 25% methanol aqueous solution 20-30ml; Close plug is claimed to decide weight, ultrasonic 40 minutes, puts cold; Claim again to decide weight, supply the weight that subtracts mistake than 25% methanol aqueous solution, shake up with volume parts; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 5-10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets.
4. quality determining method as claimed in claim 1 is characterized in that, said preparation raw material medicine consists of: bonduc 90 weight portions, seed of pomegranate 90 weight portions, Chinese cassia tree 5 weight portions, cardamom 40 weight portions, the Bi roots of grass 5 weight portions, safflower 50 weight portions.
5. quality determining method as claimed in claim 4 is characterized in that, said preparation is for the Six-element bonduc looses, Six-element bonduc particle, Six-element bonduc ball, Six-element bonduc sheet or Six-element bonduc capsule.
6. like claim 4 or 5 described quality determining methods, it is characterized in that, said quality determining method specifically:
Differentiate:
A. the discriminating of bonduc
Get preparation powder 3g to be detected, add methyl alcohol 20ml, sonicated 30 minutes filters, and filtrating is as need testing solution; Other gets bonduc control medicinal material 1g, is equipped with bonduc control medicinal material solution with legal system; According to the thin-layered chromatography test, draw each 3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Is that 60-90 ℃ of petroleum ether-ethyl acetate-glacial acetic acid is developping agent with volume parts than the boiling range of 10:0.3:0.1; Launch, take out, dry; Than 10% ethanol solution of sulfuric acid, it is clear to be heated to the spot colour developing at 105 ℃ with volume parts in spray; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
B. the discriminating of cardamom
Get preparation powder 2g to be detected; Put in the 100ml round-bottomed flask, add water 50ml, according to extracting according to the determination of volatile oil Division A League Matches of French Football method on an appendix X of Chinese Pharmacopoeia version in 2010 D; Jia Shui overflows in the flask water on the top of volatile oil extractor; Add ethyl acetate 1ml again, refluxing extraction 2 hours is obtained acetic acid ethyl fluid as need testing solution; Other gets cardamom control medicinal material 1g, is equipped with cardamom control medicinal material solution with legal system; According to the thin-layered chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent with volume parts than the boiling range of 8:2; Launch, take out, dry; Spray is with the vanillic aldehyde sulfuric acid solution of mass and size portion rate 1%, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
C. the discriminating of Chinese cassia tree
Get preparation powder 2g to be detected, put in the 100ml round-bottomed flask, add 60ml water; According to extracting according to the determination of volatile oil Division A League Matches of French Football method on an appendix X of Chinese Pharmacopoeia version in 2010 D, Jia Shui overflows in the flask water on the top of volatile oil extractor, adds ethyl acetate 1ml again; Refluxing extraction 1 hour; Be cooled to room temperature, obtain acetic acid ethyl fluid, as need testing solution; Get Chinese cassia tree control medicinal material 1g, be equipped with control medicinal material solution with legal system; According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2010 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica G TLCP; Is that 60-90 ℃ of petroleum ether-ethyl acetate is developping agent with volume parts than the boiling range of (10:2); Launch, take out, dry; Spray is with mass and size portion rate 1% vanillic aldehyde sulfuric acid solution, and 105 ℃ to be heated to the spot colour developing clear; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
Assay:
A. the assay of gallic acid
According to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase than methyl alcohol-volume parts of 2:98 than 1% glacial acetic acid aqueous solution with volume parts; The detection wavelength is 275nm; Number of theoretical plate calculates by the gallic acid peak should be not less than 2000;
It is an amount of that the gallic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 0.1mg, promptly gets;
Preparation powder 2g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, accurate methyl alcohol 25ml, the close plug of adding; Claim decide weight, sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
B. the assay of cinnaldehydrum
According to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the acetonitrile-water of 36:64; The detection wavelength is 290nm; Number of theoretical plate calculates by the cinnaldehydrum peak should be not less than 3000;
It is an amount of that the cinnaldehydrum reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains 35 μ g, promptly gets;
Preparation powder 2g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, accurate methyl alcohol 25ml, the close plug of adding; Claim decide weight, sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
C. the assay of pipering
According to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is moving phase with volume parts than the methanol-water of 78:22; The detection wavelength is 343nm; Number of theoretical plate calculates by the pipering peak should be not less than 1500;
It is an amount of that the pipering reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown measuring bottle, adds absolute ethyl alcohol and processes the solution that every 1ml contains 20 μ g, promptly gets;
Preparation powder 3g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, accurate absolute ethyl alcohol 25ml, the close plug of adding; Claim decide weight, sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with absolute ethyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets;
D. the assay of hydroxyl radical carthamin yellow carthamus A
According to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; Is that 1% glacial acetic acid aqueous solution is a moving phase with volume parts than methyl alcohol-volume parts ratio of 30:70; The detection wavelength is 403nm; Number of theoretical plate calculates by the hydroxyl radical carthamin yellow carthamus A peak should be not less than 3000;
It is an amount of that the hydroxyl radical carthamin yellow carthamus A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown bottle, adds volume parts and processes the solution that every 1ml contains 0.13mg than 25% methanol aqueous solution, promptly gets;
Preparation powder 2g to be detected is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, and the accurate volume parts that adds is than 25% methanol aqueous solution 25ml; Close plug is claimed to decide weight, ultrasonic 40 minutes, puts cold; Claim again to decide weight, supply the weight that subtracts mistake than 25% methanol aqueous solution, shake up with volume parts; Filter, get subsequent filtrate, promptly get;
Determination method is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets.
7. quality determining method as claimed in claim 1 is characterized in that, nutgall in the assay project of said gallic acid
Acid content must not be less than 2.32mg/g, and cinnaldehydrum content must not be less than 0.21mg/g in the assay project of said cinnaldehydrum,
Content of piperine must not be less than 0.13mg/g, said hydroxyl radical carthamin yellow carthamus A in the assay project of said pipering
Hydroxyl radical carthamin yellow carthamus A content must not be less than 1.15mg/g in the assay project.
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