CN101732404A - Method for identifying gentiana macrophylla medicine - Google Patents
Method for identifying gentiana macrophylla medicine Download PDFInfo
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- CN101732404A CN101732404A CN200910247876A CN200910247876A CN101732404A CN 101732404 A CN101732404 A CN 101732404A CN 200910247876 A CN200910247876 A CN 200910247876A CN 200910247876 A CN200910247876 A CN 200910247876A CN 101732404 A CN101732404 A CN 101732404A
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Abstract
The invention relates to a qualitative identification method for commonly used medicines. In the method for identifying gentiana macrophylla medicine, roburic acid is used as a reference, and a thin layer chromatography is adopted to perform qualitative identification. The method for identifying gentiana macrophylla medicine comprises the following steps of: (a) preparing a sample solution; (b) preparing a reference solution; and (c) identifying by the thin layer chromatography, wherein step (a) and step (b) are not in sequence. In the method for identifying gentiana macrophylla medicine, roburic acid is selected as the reference, and thin layer identification has the advantages of strong specificity, simple operation, good reproducibility and low cost. The method can be listed into legal standards to be used a specificity identification method for gentiana macrophylla medicine.
Description
Technical field
The present invention relates to a kind of thin layer discrimination method of conventional Chinese medicine material Radix Gentianae Macrophyllae, belong to Chinese crude drug Quality Control Technology field.
Background technology
Radix Gentianae Macrophyllae is a kind of Chinese crude drug commonly used, has the effect of wind-damp dispelling, Shujin bone, clearing away damp-heat, stopping numbness pain, is used for rheumatic arthralgia, muscles and bones and arrests symptoms such as twin and hands and feet caused by paralysis.Radix Gentianae Macrophyllae beginning is stated from Shennong's Herbal, classifies middle product as, thereafter " Tang. Newly Revised Canon of Materia Medica " etc. the successive dynasties book on Chinese herbal medicine argumentation is all arranged.The Pharmacopoeia of the People's Republic of China (version was one one in 2005) regulation Radix Gentianae Macrophyllae is the dry root of gentianaceae plant Radix Gentianae Macrophyllae Gentiana macrophyllia Pall., gentiana straminea maxim Gentiana straminea Maxim., gentiana crassicaulis Duthie Gentianacrassicaulis Duthie ex Burk. or radix gentiane dahuvicae Gentiana dahurica Fisch..Because gentiana macrophylla medicine is mainly from wild, growth cycle is long, and price goes up steadily, excessively excavates to make legal certified products gentiana macrophylla medicine supply falls short of demand day by day.Because of interests drive, market also has the root of the plants such as red Radix Gentianae Macrophyllae Salvia przewalskii, Ranunculaceae aconitum plant Aconitum spp. of Veratrilla baillonii Franch. Veratrilla baillonii Franch. with Gentianaceae Yunnan Scutellaria, Labiatae to come puppet to fill Radix Gentianae Macrophyllae, has great drug safety hidden danger.
The puppet product of Radix Gentianae Macrophyllae and the character of certified products are quite similar, though experienced expert can then not accomplish general pharmaceutical worker and doctor according to the character identification true and false.Relate to the discriminating of Radix Gentianae Macrophyllae in one one of the existing version Chinese Pharmacopoeia, do not have thin-layer method to differentiate, but adopt test-tube reaction to differentiate alkaloidal method, and there is not alkaloid in a large amount of gentiana macrophylla medicines that studies show that itself, thereby this method lacks science and reasonability; Showing under uviol lamp according to the medical material transverse section wherein also lacks specificity at the discriminating of fluorescence color.
From chemical constituent, Radix Gentianae Macrophyllae and its belong to another flavor Chinese medicine Radix Gentianae of (Gentiana Gentiana) together, all contain similar iridoids composition, as gentiopicrin (gentiopicroside).It is that reference substance carries out the discriminating of gentiana macrophylla medicine thin layer chromatography with iridoids compositions such as gentiopicrins that document is arranged, similar with the discriminating of a middle Radix Gentianae of existing version Chinese Pharmacopoeia, lacks specificity.Moreover according to our research, the Veratrilla baillonii Franch. one by one of the pseudo-product of the Radix Gentianae Macrophyllae of above medical material market circulation also contains the gentiopicrin of high level, is that reference substance carries out the thin layer chromatography discriminating and can not discern the false from the genuine with the gentiopicrin.
Summary of the invention
At above problem, the present invention has aimed to provide the discrimination method of the strong gentiana macrophylla medicine of a kind of specificity.
The discrimination method of gentiana macrophylla medicine of the present invention is to be reference substance with roburic acid (roburic acid), adopts thin layer chromatography to carry out qualitative identification.
The discrimination method of gentiana macrophylla medicine of the present invention is made up of following steps:
(a) need testing solution preparation: get gentiana macrophylla medicine powder 0.5g, add methanol 10ml, supersound process 15 minutes filters, and filtrate is as need testing solution;
(b) reference substance solution preparation: get the roburic acid reference substance, add chloroform and make the solution that every 1ml contains 1mg, in contrast product solution;
(c) thin layer chromatography is differentiated: draw above-mentioned need testing solution 2～5 μ l, reference substance solution 1 μ l, put respectively on same silica gel g thin-layer plate, with chloroform-methanol-formic acid (30～80: 1: 0.5) or with the chloroform is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, be put into 105 ℃ of baking ovens or with the electric wind blowing hot-air blow to colour developing suitably, under natural light or ultra-violet lamp (366nm), inspect, in the test sample chromatograph, with reference substance chromatograph relevant position on, show the speckle of same color.
Wherein step (a) with (b) do not have sequencing.
Gentiana macrophylla medicine thin layer chromatography Study on Identification
The need testing solution preparation: investigated the granularity of gentiana macrophylla medicine powder, the powder of crossing No. two sieves, No. three sieves, No. four sieves does not all have marked difference.So preferably used the powder of No. three sieves, so that the powder size during with the middle Radix Gentianae Macrophyllae assay of one of existing version Chinese Pharmacopoeia is consistent.Investigated the extraction solvent,, shown that methanol is better as methanol, 80% methanol, chloroform, dichloromethane, chloroform and acetone (1: 1), chloroform and methanol (1: 1), 80% ethanol.With methanol is solvent, investigated extracting method, as merceration (6 hours, 12 hours, 24 hours), reflux (1 hour, 2 hours, 3 hours), supersound process (15 minutes, 30 minutes, 45 minutes), discovery was handled simple in 15 minutes with supersound process and is saved time.Tried out the supersound extraction instrument of two kinds of power and frequency, the result all can detect, do not have difference.
Reference substance: roburic acid (C
30H
48O
2) extraction separation from gentiana macrophylla medicine obtains for us, structure is through spectrographic method and chromatography is identified and compare purity 〉=95% with document.
Lamellae: investigated different lamellaes, as homemade precoated plate (Yantai Chemical Industry Research Inst., friendly silica gel development corporation, Ltd. converges in Yantai), import precoated plate (MN plate, Merck plate) and from making sheet (silica gel is that Haiyang Chemical Plant, Qingdao produces), all can better detect.For reaching repeatability preferably, preferably select precoated plate for use, especially the MN plate of good cost performance.
Point sample and expansion: point sample amount, reference substance solution have been tried out 1 μ l, and need testing solution has been tried out 2 μ l, 3 μ l, 5 μ l.Chloroform has been investigated in developing solvent, chloroform-methanol-formic acid (80: 1: 0.5), chloroform-methanol-formic acid (50: 1: 0.5), chloroform-methanol-formic acid (30: 1: 0.5), petroleum ether-ethyl acetate (4: 1), dichloromethane etc.Lamellae is investigated presaturation 15 minutes, 30 minutes and 45 minutes in the expansion cylinder of tool developing solvent.Ascending development, exhibition is apart from 6～8cm.Show sample point sample amount 2～5 μ l, chloroform-methanol-formic acid system, especially chloroform-methanol-formic acid (50: 1: 0.5) is selected in developing solvent for use, and presaturation is in the time of 30 minutes, and the separating degree of speckle, Rf value are all better; When single chloroform was selected in developing solvent for use, development system was simplified, and just Rf value is low slightly.
Colour developing: roburic acid is a triterpenoid compound, spray is with 10% sulphuric acid ethanol, blows or 105 ℃ baking oven colour developing suitably, observes down at natural light or ultraviolet light (366nm) with the hair dryer hot blast, in the test sample chromatograph, with the speckle of reference substance corresponding position demonstration same color.
Temperature and humidity influence: investigated different room temperature condition (18 ℃ of air-conditioning controls, 22 ℃, 25 ℃, 30 ℃), (the dehumidifier control 40%, 50%, 60% of relative humidity condition, 70%), show temperature at 18～22 ℃, relative humidity is more satisfactory at 40～50% environment.This is because the polarity of development system is less than normal,, all is unfavorable for launching than big and the higher environment of temperature at relative humidity.
Owing to adopted above technical scheme, to compare with existing gentiana macrophylla medicine authentication technique, the discrimination method of gentiana macrophylla medicine of the present invention is the accuracy height not only, and specificity is strong, favorable reproducibility, advantage such as simple to operate, with low cost.Can list them in statutory standards, as gentiana macrophylla medicine specificity discrimination method.
The specific embodiment
Below in conjunction with the specific embodiment the present invention is elaborated, but do not show the present invention is carried out any restriction.
Embodiment 1:
Get Radix Gentianae Macrophyllae sample powder (crossing sieve No. three) 0.5g, put in the tool plug conical flask, add methanol 10ml, supersound process 15 minutes (power 250W, frequency 40KHZ) filters, and filtrate is made need testing solution.It is an amount of that other gets the roburic acid reference substance, adds chloroform and make the solution that every 1ml contains 1mg, in contrast product solution.According to thin layer chromatography (" an appendix VI of Chinese pharmacopoeia version in 2005 B) test.With full-automatic point sample instrument (Automatic TLC Sampler-4) band application, get above-mentioned need testing solution 3 μ l and reference substance solution 1 μ l respectively, point is on the efficient lamellae of same MN.With chloroform-methanol-formic acid (50: 1: 0.5) is developing solvent, and ascending development takes out to 6cm, volatilizes.Evenly spray is with 10% sulphuric acid ethanol with lamellae, and it is suitable 105 ℃ baking oven colour developing to put into temperature, inspects under natural light and under the ultra-violet lamp (366nm).In the test sample chromatograph, with reference substance chromatograph relevant position on, show the same color speckle, be gentiana macrophylla medicine.
Embodiment 2:
Get Radix Gentianae Macrophyllae sample powder (crossing sieve No. three) 0.5g, put in the tool plug conical flask, add methanol 10ml, supersound process 15 minutes (power 300W, frequency 20KHZ) filters, and filtrate is made need testing solution.It is an amount of that other gets the roburic acid reference substance, adds chloroform and make the solution that every 1ml contains 1mg, in contrast product solution.According to thin layer chromatography (" an appendix VI of Chinese pharmacopoeia version in 2005 B) test.Draw above-mentioned need testing solution 5 μ l and reference substance solution 1 μ l respectively with the point sample capillary tube, point is on same homemade prefabricated lamellae.With chloroform-methanol-formic acid (80: 1: 0.5) is developing solvent, and ascending development takes out to 8cm, volatilizes.Evenly spray is with 10% sulphuric acid ethanol with lamellae, and it is suitable 105 ℃ baking oven colour developing to put into temperature, inspects under natural light and under the ultra-violet lamp (366nm).In the test sample chromatograph, with reference substance chromatograph relevant position on, show the same color speckle, be gentiana macrophylla medicine.
Embodiment 3:
Get Radix Gentianae Macrophyllae sample powder (crossing sieve No. three) 0.5g, put in the tool plug conical flask, add methanol 10ml, supersound process 15 minutes (power 250W, frequency 40KHZ) filters, and filtrate is made need testing solution.It is an amount of that other gets the roburic acid reference substance, adds chloroform and make the solution that every 1ml contains 1mg, in contrast product solution.According to thin layer chromatography (" an appendix VI of Chinese pharmacopoeia version in 2005 B) test.Draw above-mentioned need testing solution 5 μ l and reference substance solution 1 μ l respectively with the point sample capillary tube, point is on the self-control lamellae.With chloroform-methanol-formic acid (30: 1: 0.5) is developing solvent, and ascending development takes out to 8cm, volatilizes.Evenly spray is with 10% sulphuric acid ethanol with lamellae, and it is suitable to blow to colour developing with the hair dryer hot blast, inspects under natural light and under the ultra-violet lamp (366nm).In the test sample chromatograph, with reference substance chromatograph relevant position on, show the same color speckle, be gentiana macrophylla medicine.
Embodiment 4:
Sample thief (Radix Gentianae Macrophyllae certified products: Radix Gentianae Macrophyllae, gentiana straminea maxim, gentiana crassicaulis Duthie, radix gentiane dahuvicae; The pseudo-product of Radix Gentianae Macrophyllae: Veratrilla baillonii Franch., red Radix Gentianae Macrophyllae, Aconitum certain; Radix Gentianae certified products: Radix Gentianae and Gentiana rigescens Franch) powder (crossing sieve No. three) 0.5g puts in the tool plug conical flask, adds methanol 10ml, and supersound process 15 minutes (power 250W, frequency 40KHZ) filters, and filtrate is made need testing solution.It is an amount of that other gets the roburic acid reference substance, adds chloroform and make the solution that every 1ml contains 1mg, in contrast product solution.According to thin layer chromatography (" an appendix VI of Chinese pharmacopoeia version in 2005 B) test.With semi-automatic point sample instrument (Camag Autosampler 3) band application, get above-mentioned need testing solution 2 μ l and reference substance solution 1 μ l respectively, point is on the efficient lamellae of same Merck.It is the saturated 30 minutes expansion cylinder of chloroform with developing solvent in advance that lamellae is put into, and ascending development takes out to 6cm, volatilizes.Evenly spray is with 10% sulphuric acid ethanol with lamellae, and it is suitable 105 ℃ baking oven colour developing to put into temperature, inspects under natural light and under the ultra-violet lamp (366nm).In the test sample chromatograph, with reference substance chromatograph relevant position on, show the gentiana macrophylla medicine that is of same color speckle; With reference substance chromatograph relevant position on, be speckless or the pseudo-product of Radix Gentianae medical material or Radix Gentianae Macrophyllae that are of different colours speckle arranged.
Among the above embodiment, each reagent is analytical pure, roburic acid purity 〉=95%.
Claims (2)
1. the discrimination method of gentiana macrophylla medicine is characterized in that with the roburic acid being reference substance, adopts thin layer chromatography to carry out qualitative identification.
2. according to the discrimination method of right 1, it is characterized in that comprising the steps:
(a) need testing solution preparation: get gentiana macrophylla medicine powder 0.5g, add methanol 10ml, supersound process 15 minutes filters, and filtrate is as need testing solution;
(b) reference substance solution preparation: get the roburic acid reference substance, add chloroform and make the solution that every 1ml contains 1mg, in contrast product solution;
(c) thin layer chromatography is differentiated: draw above-mentioned need testing solution 2～5 μ l, reference substance solution 1 μ l, put respectively on same silica gel g thin-layer plate, with chloroform-methanol-formic acid (30～80: 1: 0.5) or with the chloroform is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, be put into 105 ℃ of baking ovens or electric wind blow with hot blast blow to colour developing suitably, under natural light or ultra-violet lamp (366nm), inspect, in the test sample chromatograph, with reference substance chromatograph relevant position on, show the speckle of same color;
Wherein step (a) with (b) do not have sequencing.
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| CN2009102478765A CN101732404B (en) | 2009-12-31 | 2009-12-31 | Method for identifying gentiana macrophylla medicine |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770771A (en) * | 2016-12-27 | 2017-05-31 | 中南民族大学 | A kind of method for building up and its finger-print of yellow gentiana macrophylla medicine finger-print |
| CN110068547A (en) * | 2019-05-06 | 2019-07-30 | 中国科学院西北高原生物研究所 | A kind of method of discrimination of Huang pipe gentianae macrophyllae |
| CN113049732A (en) * | 2021-03-26 | 2021-06-29 | 正大青春宝药业有限公司 | Rapid thin-layer identification method for astragalus root and poria cocos granules with one detection and multiple medicines |
| KR20210092971A (en) * | 2020-01-17 | 2021-07-27 | (주)메모리얼 | Molecular marker for distinguishing Gentina macrophylla, Aconitum pseudolaeve and Aconitum longecassidatum |
-
2009
- 2009-12-31 CN CN2009102478765A patent/CN101732404B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770771A (en) * | 2016-12-27 | 2017-05-31 | 中南民族大学 | A kind of method for building up and its finger-print of yellow gentiana macrophylla medicine finger-print |
| CN106770771B (en) * | 2016-12-27 | 2020-01-21 | 中南民族大学 | Method for establishing fingerprint of gentiana macrophylla medicine and fingerprint thereof |
| CN110068547A (en) * | 2019-05-06 | 2019-07-30 | 中国科学院西北高原生物研究所 | A kind of method of discrimination of Huang pipe gentianae macrophyllae |
| CN110068547B (en) * | 2019-05-06 | 2021-07-09 | 中国科学院西北高原生物研究所 | Method for distinguishing gentiana macrophylla pall |
| KR20210092971A (en) * | 2020-01-17 | 2021-07-27 | (주)메모리얼 | Molecular marker for distinguishing Gentina macrophylla, Aconitum pseudolaeve and Aconitum longecassidatum |
| KR102313439B1 (en) | 2020-01-17 | 2021-10-15 | (주)메모리얼 | Molecular marker for distinguishing Gentina macrophylla, Aconitum pseudolaeve and Aconitum longecassidatum |
| CN113049732A (en) * | 2021-03-26 | 2021-06-29 | 正大青春宝药业有限公司 | Rapid thin-layer identification method for astragalus root and poria cocos granules with one detection and multiple medicines |
| CN113049732B (en) * | 2021-03-26 | 2022-04-08 | 正大青春宝药业有限公司 | Rapid thin-layer identification method for astragalus root and poria cocos granules with one detection and multiple medicines |
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| CN101732404B (en) | 2012-04-25 |
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