CN101642481A - Method for establishing Tripterygium wilfordii fingerprint chromatography and standard fingerprint chromatography thereof - Google Patents
Method for establishing Tripterygium wilfordii fingerprint chromatography and standard fingerprint chromatography thereof Download PDFInfo
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- CN101642481A CN101642481A CN 200910169869 CN200910169869A CN101642481A CN 101642481 A CN101642481 A CN 101642481A CN 200910169869 CN200910169869 CN 200910169869 CN 200910169869 A CN200910169869 A CN 200910169869A CN 101642481 A CN101642481 A CN 101642481A
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Abstract
The invention provides a method for establishing high performance liquid chromatography (HPLC) fingerprint chromatography of Tripterygium wilfordii and a standard fingerprint chromatography thereof, and the method comprises the steps of preparing test solution, selecting chromatographic conditions, manufacturing HPLC fingerprint chromatography, determining the standard fingerprint chromatography and the like. By comparing the HPLC Tripterygium wilfordii fingerprint chromatography in 17 batches to determine 24 common characteristic peaks, the common characteristic peaks form the fingerprint characteristics of the Tripterygium wilfordii which can be taken as the standard fingerprint chromatography of the Tripterygium wilfordii. The invention has the advantages of simple method, good repeatability, a lot of characteristic peaks, accuracy and reliability and the like. The established standard fingerprint chromatography can effectively represent quality of the Tripterygium wilfordii.
Description
Technical field
The present invention relates to a kind of method for building up of Chinese crude drug finger printing, the method for building up of tripterygium wilfordii high performance liquid chromatography (HPLC) finger printing especially, and the resulting tripterygium wilfordii HPLC of method standard finger-print thus.Belong to the pharmaceutical analysis technical field.
Background technology
Chinese medicine fingerprint refers in particular to the collection of illustrative plates or the image of each component population characteristic of Chinese medicine that the Chinese medicine sample obtains through modern analytical techniques such as spectrum or chromatographs.At present finger printing has become the discriminating herbal species of generally acknowledging both at home and abroad and has estimated one of effective means of Chinese medicine quality.FDA (Food and Drug Adminstration) is in " FDA is about the guide of vegetable products " formulated in 1996, and requiring provides corresponding finger printing to product and plant amedica product in the middle of plant material, the plant amedica.Medicinal and the fragrant plant association of British Herbal Pharmacopoeia, India herbal medicine allusion quotation and Canada, German medicinal plants association also accept chromatographic fingerprinting.China national drug regulatory department has issued " specification requirement of the finger printing research of Chinese medicine " in 2000, explicitly call for Chinese medicine of newly declaring and the Chinese medicine that has gone on the market are carried out the finger printing standard.
Chinese medicine fingerprint has passed through the research of many decades, many modern analysis means of testing such as comprising ultraviolet, infrared, gas phase, high performance liquid chromatogram, thin layer, nuclear magnetic resonance, NMR, scanning electron microscope, computer image analysis, electrophoretic techniques, isoenzyme analysis method, Protocols in Molecular Biology, cluster analysis technology have been used, for the material of Chinese medicine is identified and component analysis has accumulated lot of data.But, also reach the purpose that reflects effectively and control the total quality of Chinese medicine far away from the angle of Chinese medicine overall quality control.High performance liquid chromatography (HPLC) finger printing obtains approval in the world easily, comprises that many countries of the U.S., Britain, France, Canada, Germany, Japan and India more are ready to accept the HPLC finger printing.Therefore, setting up corresponding HPLC finger printing for Chinese crude drug, is the effective means of present stage control Chinese medicine inherent quality.
Radix Tripterygii Wilfordii is Celastraceae Thunder God Calamus fallen leaves sprawling shrubs, another name has the imperial root of shake, steam the dragon grass, Folium illicii Lanceolati, the Caulis Fibraureae grass, Gelsemium elegans Benth., mountain arsenicum etc., with root, leaf, flower and fruit are used as medicine, main product is in Zhejiang, Anhui, Jiangxi, the Hunan, Guangdong, Guangxi, Fujian, Taiwan, ground such as Yunnan, its active component is very complicated, comprise wilfordine (wilfordine), wilforine (wilforine), wilfortrine skin amide (celacinnine), Celastrus orbiculatus Thunb. furancarboxylic acid amine (celafurine), Celastrus orbiculatus Thunb. benzyl amide (celagbenzine), Triptolide (wilforlide), triptonoterpenol (triptonoterpenol), 16-hydroxytriptolide (16-hydroxytripto-lide), triptolide (triptolide), table Triptolide triol (epitriptriolide), thunder kaurene lactone (tripterifordine), mapping-Lei kaurene lactone (antriptolactone), wilfordic acid (tripterygic acid), straight wedge oxalic acid (orthosphenic acid), cupreol (β-sitosterol), daucosterol (daucosterol), triptotriterpenoidal lactone A (triptoterpenoidlactone A), Triptolide A, B, tripterine (tripterine), big sub-Salacia prinoides (Willd.) DC. acid (salaspermic acid), Orthosphenic acid (triptotriterpenic acid) A, B, C, straight wedge oxalic acid, tripterygone (tripterygone), Radix Tripterygii Wilfordii chlorine lactone liquor-saturated (tripchlorolide), Triptolide triol (triptriolide) and linolenic acid (linolenic acid), 8,9-octadecadienoic acid (8,9-octadecadienoic acid), oleic acid (oleic acid), palmitoleic acid (9-hexadecenoicacid), Palmic acid (palmitic acid), stearic acid (stearic acid) etc.Its pharmacological action mainly contains immunomodulating, antitumor, microcirculation improvement, antiinflammatory, sterilization and antipyretic-antalgic etc.; Can be used for treating lepra reaction, rheumatoid arthritis, pulmonary tuberculosis and other chronic diseases etc. clinically.At present, existing Radix Tripterygii Wilfordii tablet, the tripterygium glycosides sheet, several formulations productions such as Radix Tripterygii Wilfordii syrup, electuary and mixture can be used for multiple treatment of diseases.
Radix Tripterygii Wilfordii is the poisonous plants that include in Chinese Plants spectrum data storehouse, and its toxicity is that Herb is poisonous.Qing Dynasty's ZHAO Xue-Min at supplementary Amplifications of the Compendium of Materia Medica write up its toxicity: " the malicious fish of adopting, all freshwater mussel spiral shells are also dead, its property is the strongest, does not then give birth to its careless cigarette Xun silkworm egg ".Radix Tripterygii Wilfordii is very big to people's toxicity, and often there is the generation of poisoning in rural area, southern each province.Someone wrongly takes its leaf 2-3 sheet and can poison, 7 of tender shoots of clothes or can cause death more than the root bark 30g, even Shiqi nectar also can be poisoned.It mainly acts on animal cardiac muscle, causes decreased heart rate and blood pressure drops, makes the excited earlier back of heart suppress dead to stopping to beat.The general poisoning symptom of clinical findings has dizziness, cardiopalmus, unable, nauseating, vomiting, stomachache, diarrhoea; Liver and kidney district pain, bloody stool etc.Therefore, very careful to the medicinal needs of Radix Tripterygii Wilfordii.
At present, the quality evaluation of tripterygium wilfordii is mainly concentrated on certain effective ingredient or the index composition carries out qualitative, quantitative analysis, lack the standard control device of reflection tripterygium wilfordii quality comprehensively.
Summary of the invention
The method for building up that the purpose of this invention is to provide a kind of tripterygium wilfordii HPLC finger printing, and the resulting tripterygium wilfordii standard finger-print of method thus.For realizing this purpose, the present invention is by the research to the tripterygium wilfordii efficient liquid-phase chromatograph finger print atlas, proposed a kind of tripterygium wilfordii method of quality control, remedied the deficiency of existing Quality Control Technology, the quality control of tripterygium wilfordii is improved and science more.
The method for building up of tripterygium wilfordii HPLC finger printing of the present invention comprises the steps:
(1) preparation of need testing solution: precision takes by weighing exsiccant medicinal powder 10g, place tool plug container, methanol 80～the 120mL of adding 60～80%, ultrasonic concussion 60min, move in the apparatus,Soxhlet's, reflux, extract, 5h is concentrated into extracting solution and is less than 10mL, with 0.45 μ m micro-pore-film filtration, filtrate is settled to 10mL as need testing solution.
(2) efficient liquid phase chromatographic analysis: the chromatographic isolation column packing is 5 μ m Kromasil-C18,250mm * 4.6mm; Mobile phase is water (0.4% phosphoric acid+0.5% acetic acid)-acetonitrile; Adopt the gradient elution mode: 0min → 10min → 30min → 50min → 65min → 85min → 95min, acetonitrile 80% → 65% → 45% → 40% → 35% → 15% → 10%; Flow velocity 0.8～1.2mL/min; Detect wavelength 254nm; 25～35 ℃ of column temperatures.Analyze need testing solution with this understanding, obtain the finger printing of tripterygium wilfordii.
(3) determining of standard finger-print: according to method provided by the invention, to the different places of production, the different qualities or the tripterygium wilfordii in different storage times have been set up the HPLC finger printing, by analyzing relatively, 24 common characteristic peaks have been determined, retention time under its a kind of chromatographic condition is respectively: 2.2min, 7.6min, 8.6min, 14.2min, 20.0min, 22.4min, 26.8min, 31.4min, 35.0min, 38.4min, 41.6min, 43.0min, 45.4min, 48.8min, 55.6min, 57.6min, 61.4min, 67.2min, 69.6min, 76.6min, 79.8min, 87.4min, 89.0min, 90.2min, these common characteristic peaks have constituted the fingerprint characteristic of tripterygium wilfordii, can be used as the standard finger-print of tripterygium wilfordii.
The present invention has following significant advantage and purposes:
(1) tripterygium wilfordii is done as a whole treating, can be found out nuance between the different medical materials by its total peak relatively;
(2) test sample is simple for production, and chromatographic condition is realized easily;
(3) stability of method and repeatability are all relatively good;
(4) be suitable for discriminating and control to the tripterygium wilfordii true and false, the place of production and quality.
Description of drawings
Fig. 1 is the standard finger-print of tripterygium wilfordii provided by the invention.Its 24 common characteristic peaks have from left to right been marked respectively.
The specific embodiment
The preferred embodiments of the present invention are as follows:
(1) instrument and medicine: use Tianjin, island LC10A high performance liquid chromatograph; Totally 17 batches of tripterygium wilfordiis are all purchased in the Chinese herbal medicine shop; Other reagent is chromatographically pure or analytical pure.
(2) preparation of need testing solution: precision takes by weighing exsiccant medicinal powder 10g, place tool plug container, methanol 80～the 120mL of adding 60～80%, ultrasonic concussion 60min, move in the apparatus,Soxhlet's, reflux, extract, 5h is concentrated into extracting solution and is less than 10mL, with 0.45 μ m micro-pore-film filtration, filtrate is settled to 10mL as need testing solution.
(3) efficient liquid phase chromatographic analysis: the chromatographic isolation column packing is 5 μ m Kromasil-C18,250mm * 4.6mm; Mobile phase is water (0.4% phosphoric acid+0.5% acetic acid)-acetonitrile; Adopt the gradient elution mode: 0min → 10min → 30min → 50min → 65min → 85min → 95min, acetonitrile 80% → 65% → 45% → 40% → 35% → 15% → 10%; Flow velocity 1.0mL/min; Detect wavelength 254nm; 30 ℃ of column temperatures.Analyze need testing solution with this understanding, obtain the finger printing of tripterygium wilfordii.
(4) according to method provided by the invention, to the different places of production, different qualities or 17 batches of tripterygium wilfordiis in different storage times have been set up the HPLC finger printing, by analyzing relatively, 24 common characteristic peaks have been determined, retention time under its a kind of chromatographic condition is respectively: 2.2min, 7.6min, 8.6min, 14.2min, 20.0min, 22.4min, 26.8min, 31.4min, 35.0min, 38.4min, 41.6min, 43.0min, 45.4min, 48.8min, 55.6min, 57.6min, 61.4min, 67.2min, 69.6min, 76.6min, 79.8min, 87.4min, 89.0min, 90.2min, these common characteristic peaks have constituted the fingerprint characteristic of tripterygium wilfordii, can be used as the standard finger-print of tripterygium wilfordii.
Claims (4)
1, a kind of method for building up of tripterygium wilfordii efficient liquid-phase chromatograph finger print atlas, its feature comprise preparation, the making of finger printing and the determining of standard finger-print of need testing solution.
2, the method for building up of tripterygium wilfordii efficient liquid-phase chromatograph finger print atlas according to claim 1, the preparation method that it is characterized in that described need testing solution is: precision takes by weighing exsiccant medicinal powder 10g, place tool plug container, methanol 80～the 120mL of adding 60～80%, ultrasonic concussion 60min moves in the apparatus,Soxhlet's, reflux, extract, 5h, extracting solution is concentrated into is less than 10mL, with 0.45 μ m micro-pore-film filtration, filtrate is settled to 10mL as need testing solution.
3, according to the method for building up of claim 1 and 2 described tripterygium wilfordii efficient liquid-phase chromatograph finger print atlas, the manufacturing process that it is characterized in that described finger printing is: with 5 μ m Kromasil-C18 as the chromatography column (filler of 250mm * 4.6mm), mobile phase is water (0.4% phosphoric acid+0.5% acetic acid)-acetonitrile, the gradient elution mode is 0min → 10min → 30min → 50min → 65min → 85min → 95min corresponding acetonitrile 80% → 65% → 45% → 40% → 35% → 15% → 10% respectively, flow velocity 0.8～1.2mL/min; Detect wavelength 254nm; 25～35 ℃ of column temperatures; Under this high-efficient liquid phase chromatogram condition, analyze need testing solution, obtain the finger printing of tripterygium wilfordii.
4, according to claim 1, the method for building up of 2 and 3 described tripterygium wilfordii efficient liquid-phase chromatograph finger print atlas, the definite result who it is characterized in that described standard finger-print is: 17 batches of tripterygium wilfordiis have been set up the HPLC finger printing, 24 common characteristic peaks have been determined, retention time under its a kind of chromatographic condition is respectively: 2.2min, 7.6min, 8.6min, 14.2min, 20.0min, 22.4min, 26.8min, 31.4min, 35.0min, 38.4min, 41.6min, 43.0min, 45.4min, 48.8min, 55.6min, 57.6min, 61.4min, 67.2min, 69.6min, 76.6min, 79.8min, 87.4min, 89.0min, 90.2min, these common characteristic peaks have constituted the fingerprint characteristic of tripterygium wilfordii, can be used as the standard finger-print of tripterygium wilfordii.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103033570A (en) * | 2011-09-29 | 2013-04-10 | 广州陈李济药厂有限公司 | Quality detection method for tripterygii hypoglauci medicinal materials |
| CN108318599A (en) * | 2018-02-06 | 2018-07-24 | 赖旭宇 | A kind of efficient liquid-phase chromatography method of separation triptolide and 2- table triptolides |
| CN108508112A (en) * | 2018-04-10 | 2018-09-07 | 吉林师范大学 | The detection method of wilforlide A in celastrus orbiculatus, Wiofolide B content |
| CN110241247A (en) * | 2019-07-12 | 2019-09-17 | 华润三九医药股份有限公司 | A kind of molecular labeling, primer pair and discrimination method identified for tripterygium wilfordii |
| CN113533604A (en) * | 2021-06-25 | 2021-10-22 | 深圳海王医药科技研究院有限公司 | Method for establishing HPLC fingerprint of traditional Chinese medicine celastrus orbiculatus |
-
2009
- 2009-09-07 CN CN 200910169869 patent/CN101642481A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103033570A (en) * | 2011-09-29 | 2013-04-10 | 广州陈李济药厂有限公司 | Quality detection method for tripterygii hypoglauci medicinal materials |
| CN103033570B (en) * | 2011-09-29 | 2014-08-20 | 广州白云山陈李济药厂有限公司 | Quality detection method for tripterygii hypoglauci medicinal materials |
| CN108318599A (en) * | 2018-02-06 | 2018-07-24 | 赖旭宇 | A kind of efficient liquid-phase chromatography method of separation triptolide and 2- table triptolides |
| CN108508112A (en) * | 2018-04-10 | 2018-09-07 | 吉林师范大学 | The detection method of wilforlide A in celastrus orbiculatus, Wiofolide B content |
| CN108508112B (en) * | 2018-04-10 | 2020-10-09 | 吉林师范大学 | Method for detecting content of triptolide A and triptolide B in celastrus orbiculatus |
| CN110241247A (en) * | 2019-07-12 | 2019-09-17 | 华润三九医药股份有限公司 | A kind of molecular labeling, primer pair and discrimination method identified for tripterygium wilfordii |
| CN110241247B (en) * | 2019-07-12 | 2023-06-20 | 华润三九医药股份有限公司 | Molecular marker and primer pair for identifying tripterygium wilfordii medicinal materials and identification method |
| CN113533604A (en) * | 2021-06-25 | 2021-10-22 | 深圳海王医药科技研究院有限公司 | Method for establishing HPLC fingerprint of traditional Chinese medicine celastrus orbiculatus |
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Application publication date: 20100210 |