CN100410662C - Tripterygium glycosides and their preparation wilforlide A content determining method - Google Patents
Tripterygium glycosides and their preparation wilforlide A content determining method Download PDFInfo
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- CN100410662C CN100410662C CNB2006102004271A CN200610200427A CN100410662C CN 100410662 C CN100410662 C CN 100410662C CN B2006102004271 A CNB2006102004271 A CN B2006102004271A CN 200610200427 A CN200610200427 A CN 200610200427A CN 100410662 C CN100410662 C CN 100410662C
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Abstract
The present invention provides a method for detecting the contents of tripterygium glycosides and wilforlide A in preparation thereof, which is a high-efficiency liquid chromatography using wilforlide A reference substances as reference and using water and acetonitrile as mobile phase, wherein the water takes up 10 to 40 percent, and the acetonitrile takes up 90 to 60 percent. Compared with the prior art, the content detecting method of the present invention adopts the high-efficiency liquid chromatography, which has the advantages of high exclusive performance, good recovery rate and accurate detecting result, the quality of the tripterygium glycosides can be effectively controlled, and thereby, the efficacy and the safety of clinical medication are ensured.
Description
Technical field:
The present invention relates to the content assaying method of wilforlide A in a kind of tripterygium glycosides and the preparation thereof, belong to the technical field of medicine being carried out quality control.
Background technology:
Tripterygium glycosides is a Celastraceae plant tripterygium root through extraction, purifying, the refining active component that forms, and is a good immunodepressant.It not only has immunosuppressive action, also has antiinflammatory action simultaneously, is the choice drug of treatment rheumatoid arthritis.Treat rheumatismal medicine in the market and also quite lack, mainly contain NSAID (non-steroidal anti-inflammatory drug), steroid hormone class, chronic effect antirheumatic drug.The curative effect of NSAID (non-steroidal anti-inflammatory drug), chronic effect antirheumatic drug is unsatisfactory, and main alleviating pain symptom can not effectively be prevented and treated the development of the state of an illness; Though and the hormone medicine curative effect is good, the spinoff that it is special determines it not use for a long time, can only make the impact treatment of short-term.And Chinese medicine has special advantages, and its spinoff is little, and is also non-stimulated to stomach, and be the cause of disease in heresy is gone into beyond the rheumatism, and traditional Chinese medical science strengthening vital QI to eliminate pathogenic factors can directly be effected a permanent cure.Therefore, National Drug Administration's medicine is evaluated the center and has been held special meeting in Dec calendar year 2001 at the thunder godvine preparation, the expert is consistent though attend a meeting thinks that the thunder godvine preparation has certain toxicity, but it is very sure that it treats rheumatismal curative effect, wishes to have the more exploitation of the thunder godvine preparation of " safe and effective, steady quality ".
Tripterygium glycosides is owing to removed main poisonous part and impurity, and the purity of active princlple improves, and spinoff and toxicity descend than crude drug preparation, and safe range improves.But from the statistical figure of clinical research, also has certain spinoff: after about 1/5 patient takes medicine gastrointestinal discomfort is arranged.Therefore for guaranteeing the validity and the security of clinical application, set up an effective quality control method and seem particularly important.
Wilforlide A is one of effective constituent in the tripterygium glycosides, and through consulting domestic and international data of literatures, the assay method of wilforlide A is less, and useful thin layer chromatography scanning is measured, but the thin layer chromatography scanning complex operation, error is bigger.
Summary of the invention:
The objective of the invention is to: the content assaying method that wilforlide A in a kind of tripterygium glycosides and the preparation thereof is provided, the present invention is directed to the deficiencies in the prior art, set up the high performance liquid chromatography that specificity is strong, precision good, the recovery is high, the quality of tripterygium glycosides be can effectively control, thereby the validity and the security of clinical application guaranteed.
Tripterygium glycosides of the present invention is to get tripterygium root to pulverize, and alcohol extracting is collected active princlple and got through column chromatography for separation, and its preparation is the preparation made from tripterygium glycosides.The inventive method is not only applicable to tripterygium glycosides and preparation thereof, also can be used for the pharmaceutical preparation for preparing after the combination of active principles of tripterygium wilfordii or tripterygium glycosides and other medicinal materials or medicinal material.
The present invention constitutes like this: it is to be contrast with the wilforlide A reference substance, and with water: acetonitrile=10～40: 90～60 is the high performance liquid chromatography of moving phase.
Concrete assay method is:
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; With water: acetonitrile=10～40: 90～60 is moving phase, and flow velocity is 1ml/min; The detection wavelength is 210nm; Number of theoretical plate calculates by wilforlide A should be not less than 3000;
It is an amount of that the wilforlide A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and makes the solution that every 1ml contains 30 μ g, promptly;
The preparation of need testing solution is got and is equivalent to the about 0.12g of tripterygium glycosides fine powder, the accurate title, decide, put in the 20ml measuring bottle, add the chloroform dissolving, and be diluted to scale, shake up the accurate supernatant 5ml that draws, be added on 100～200 orders, 6g, internal diameter 1cm, wet method dress post, the neutral alumina post with an amount of chloroform prewashing, water content 2～4%, with ethyl acetate: acetone=2～4: solution 20～30ml of 1, wash-out under 30～50 ℃ of constant temperatures is collected eluent, evaporate to dryness, the residue dissolve with methanol is transferred in the 2ml measuring bottle, adds methyl alcohol to scale, shake up, promptly;
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly;
Contain wilforlide A in the tripterygium glycosides and must not be less than 1mg/g.
Assay method is more specifically:
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; With water: acetonitrile=25: 75 is a moving phase, and flow velocity is 1ml/min; The detection wavelength is 210nm; Number of theoretical plate calculates by wilforlide A should be not less than 3000;
It is an amount of that the wilforlide A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and makes the solution that every 1ml contains 30 μ g, promptly;
The preparation of need testing solution is got and is equivalent to the about 0.12g of tripterygium glycosides fine powder, the accurate title, decide, put in the 20ml measuring bottle, add the chloroform dissolving, and be diluted to scale, shake up the accurate supernatant 5ml that draws, be added on 100～200 orders, 6g, internal diameter 1cm, wet method dress post, the neutral alumina post with an amount of chloroform prewashing, water content 3%, with ethyl acetate: the solution 25ml of acetone=3: 1, wash-out under 38～40 ℃ of constant temperatures is collected eluent, evaporate to dryness, the residue dissolve with methanol is transferred in the 2ml measuring bottle, adds methyl alcohol to scale, shake up, promptly;
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly;
Contain wilforlide A in the tripterygium glycosides and must not be less than 1mg/g.
In order to ensure content assaying method science of the present invention, reasonable, feasible, the applicant has carried out following experimental study, to determine relevant technical conditions, data or the like:
The test example:
1, the selection of moving phase
With water-acetonitrile, methyl alcohol-acetonitrile-water and 0.1%H
2PO
4The mixed solution of the different proportion of-acetonitrile is that moving phase is tested, relatively, and the result shows that (10～40: be that the separating effect of moving phase is better 90～60), particularly under water-acetonitrile (25: 75) moving phase condition, separating effect is best with water-acetonitrile.
2, the test of chromatographic condition and system suitability is a filling agent with the octadecylsilane chemically bonded silica; (10～40: 90～60) be moving phase, flow velocity is 1ml/min with water-acetonitrile; The detection wavelength is 210nm.Number of theoretical plate calculates by triptolide should be not less than 3000.
3, sample solution preparation
It is an amount of that the wilforlide A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and makes the solution that every 1ml contains 30 μ g, promptly.
The preparation of need testing solution is got and is equivalent to the about 0.12g of tripterygium glycosides fine powder, and accurate the title decides, and puts in the 20ml measuring bottle, add the chloroform dissolving, and be diluted to scale, shake up the accurate supernatant 5ml that draws, be added to neutral alumina post (100～200 orders that are ready to water content 2～4%, 6g, internal diameter 1cm, wet method dress post, with an amount of chloroform prewashing) on, with ethyl acetate-acetone (2～4: 1) 20～30ml, wash-out under 30～50 ℃ of constant temperatures, collection eluent, evaporate to dryness, the residue dissolve with methanol is transferred in the 2ml measuring bottle, adds methyl alcohol to scale, shake up, promptly.
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.
4, the range of linearity
It is an amount of that precision takes by weighing the wilforlide A reference substance, adds dissolve with methanol dilution and make and contain the solution that wilforlide A is 6 μ g/ml, 12 μ g/ml, 24 μ g/ml, 48 μ g/ml, 72 μ g/ml, 120 μ g/ml approximately.Measure according to chromatographic condition and system suitability test condition, (A) makes straight line to sample concentration with peak area, gets regression equation y=4638.3X+5760.3R=0.9997
The result shows: wilforlide A is good linear relationship in 6～120 μ g/ml scopes.
5, interference test
According to a blank solution that does not add sample of " preparation methods of 3 following need testing solutions " preparation, and with method mensuration, the result does not see in the relevant position chromatographic peak, shows blank noiseless.
6, precision test
6.1 the about 0.12g of replica test sample thief fine powder, six parts, the accurate title, decide, and by " preparation methods of 3 following need testing solutions " preparation need testing solution, measures in accordance with the law, the results are shown in following table:
6.2 the different personnel of middle precision are at the about 0.12g of same date sample thief fine powder not, and six parts, accurately claim surely, by " preparation methods of 3 following need testing solutions " preparation need testing solution, mensuration in accordance with the law the results are shown in following table:
7, stability test
The about 0.12g of sample thief fine powder, the accurate title, decide, by " preparation methods of 3 following need testing solutions " preparation need testing solution, according to the form below timing 6 times, its peak area such as following table:
| Time h | 0 | 1 | 2 | 4 | 8 | 24 | RSD％ |
| Peak area | 243721 | 246352 | 246367 | 250594 | 246965 | 244957 | 0.93 |
8, recovery test
Get the test liquid solution of the processing under 3, press the assay method of wilforlide A and measure content, calculate its recovery.The results are shown in following table:
This method wilforlide A average recovery rate is 98.98%, and relative average debiation is 1.99%, and as seen this method recovery is more satisfactory.
9, assay
By " preparation methods of 3 following need testing solutions " preparation need testing solution, measure ten batch samples of different year in accordance with the law, the results are shown in following table:
| Lot number | 0202002 | 0210705 | 0301701 | 0312701 | 0403701 |
| Content % | 0.413 | 0.64 | 0.368 | 0.477 | 0.366 |
| Lot number | 0411701 | 0505701 | 0507701 | 0601701 | 0601702 |
| Content % | 0.458 | 0.238 | 0.415 | 0.271 | 0.339 |
According to the assay result of wilforlide A in above ten batch samples, contain wilforlide A in the tentative tripterygium glycosides and must not be less than 1mg/g.
Compared with prior art, assay of the present invention adopts high performance liquid chromatography, and its specificity is strong, precision good, the recovery is high, and measurement result is accurate, can effectively control the quality of tripterygium glycosides, thereby guarantees the validity and the security of clinical application.
Embodiment:
Embodiments of the invention 1: the content assaying method of wilforlide A is in the tripterygium glycosides:
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; With water: acetonitrile=25: 75 is a moving phase, and flow velocity is 1ml/min; The detection wavelength is 210nm; Number of theoretical plate calculates by wilforlide A should be not less than 3000.
It is an amount of that the wilforlide A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and makes the solution that every 1ml contains 30 μ g, promptly.
The about 0.12g of tripterygium glycosides fine powder is got in the preparation of need testing solution, the accurate title, decide, put in the 20ml measuring bottle, add the chloroform dissolving, and be diluted to scale, shake up the accurate supernatant 5ml that draws, be added on 100～200 orders, 6g, internal diameter 1cm, wet method dress post, the neutral alumina post with an amount of chloroform prewashing, water content 3%, with ethyl acetate: the solution 25ml of acetone=3: 1, wash-out under 38～40 ℃ of constant temperatures is collected eluent, evaporate to dryness, the residue dissolve with methanol is transferred in the 2ml measuring bottle, adds methyl alcohol to scale, shake up, promptly.
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.
Contain wilforlide A in the tripterygium glycosides and must not be less than 1mg/g.
Embodiments of the invention 2: described content assaying method also can for:
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; With water: acetonitrile=10: 90 is a moving phase, and flow velocity is 1ml/min; The detection wavelength is 210nm; Number of theoretical plate calculates by wilforlide A should be not less than 3000.
It is an amount of that the wilforlide A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and makes the solution that every 1ml contains 30 μ g, promptly.
The about 0.12g of tripterygium glycosides fine powder is got in the preparation of need testing solution, the accurate title, decide, put in the 20ml measuring bottle, add the chloroform dissolving, and be diluted to scale, shake up the accurate supernatant 5ml that draws, be added on 100～200 orders, 6g, internal diameter 1cm, wet method dress post, the neutral alumina post with an amount of chloroform prewashing, water content 2%, with ethyl acetate: the solution 20ml of acetone=2: 1, wash-out under 30～38 ℃ of constant temperatures is collected eluent, evaporate to dryness, the residue dissolve with methanol is transferred in the 2ml measuring bottle, adds methyl alcohol to scale, shake up, promptly.
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.
Contain wilforlide A in the tripterygium glycosides and must not be less than 1mg/g.
Embodiments of the invention 3: described content assaying method can also for:
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; With water: acetonitrile=40: 60 is a moving phase, and flow velocity is 1ml/min; The detection wavelength is 210nm; Number of theoretical plate calculates by wilforlide A should be not less than 3000.
It is an amount of that the wilforlide A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and makes the solution that every 1ml contains 30 μ g, promptly.
The about 0.12g of tripterygium glycosides fine powder is got in the preparation of need testing solution, the accurate title, decide, put in the 20ml measuring bottle, add the chloroform dissolving, and be diluted to scale, shake up the accurate supernatant 5ml that draws, be added on 100～200 orders, 6g, internal diameter 1cm, wet method dress post, the neutral alumina post with an amount of chloroform prewashing, water content 4%, with ethyl acetate: the solution 30ml of acetone=4: 1, wash-out under 40～50 ℃ of constant temperatures is collected eluent, evaporate to dryness, the residue dissolve with methanol is transferred in the 2ml measuring bottle, adds methyl alcohol to scale, shake up, promptly.
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.
Contain wilforlide A in the tripterygium glycosides and must not be less than 1mg/g.
Claims (2)
1. the content assaying method of wilforlide A in tripterygium glycosides and the preparation thereof is characterized in that: it is to adopt high performance liquid chromatography, measures according to following concrete steps:
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; With water: acetonitrile=10～40: 90～60 is moving phase, and flow velocity is 1ml/min; The detection wavelength is 210nm; Number of theoretical plate calculates by wilforlide A should be not less than 3000;
It is an amount of that the wilforlide A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and makes the solution that every 1ml contains 30 μ g, promptly;
The preparation of need testing solution is got and is equivalent to tripterygium glycosides fine powder 0.12g, the accurate title, decide, put in the 20ml measuring bottle, add the chloroform dissolving, and be diluted to scale, shake up the accurate supernatant 5ml that draws, be added on 100～200 orders, 6g, internal diameter 1cm, wet method dress post, the neutral alumina post with an amount of chloroform prewashing, water content 2～4%, with ethyl acetate: acetone=2～4: solution 20～30ml of 1, wash-out under 30～50 ℃ of constant temperatures is collected eluent, evaporate to dryness, the residue dissolve with methanol is transferred in the 2ml measuring bottle, adds methyl alcohol to scale, shake up, promptly;
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly;
Contain wilforlide A in the tripterygium glycosides and must not be less than 1mg/g.
2. according to the content assaying method of wilforlide A in described tripterygium glycosides of claim 1 and the preparation thereof, it is characterized in that: assay method is more specifically:
Chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica; With water: acetonitrile=25: 75 is a moving phase, and flow velocity is 1ml/min; The detection wavelength is 210nm; Number of theoretical plate calculates by wilforlide A should be not less than 3000;
It is an amount of that the wilforlide A reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds methyl alcohol and makes the solution that every 1ml contains 30 μ g, promptly;
The preparation of need testing solution is got and is equivalent to tripterygium glycosides fine powder 0.12g, the accurate title, decide, put in the 20ml measuring bottle, add the chloroform dissolving, and be diluted to scale, shake up the accurate supernatant 5ml that draws, be added on 100～200 orders, 6g, internal diameter 1cm, wet method dress post, the neutral alumina post with an amount of chloroform prewashing, water content 3%, with ethyl acetate: the solution 25ml of acetone=3: 1, wash-out under 38～40 ℃ of constant temperatures is collected eluent, evaporate to dryness, the residue dissolve with methanol is transferred in the 2ml measuring bottle, adds methyl alcohol to scale, shake up, promptly;
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly;
Contain wilforlide A in the tripterygium glycosides and must not be less than 1mg/g.
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102048795B (en) * | 2010-12-27 | 2014-06-25 | 浙江农林大学 | Method for simultaneously measuring content of multiple active ingredients in common threewingnut root medicinal materials |
| CN104991020B (en) * | 2015-06-29 | 2017-03-15 | 公安部物证鉴定中心 | The Liquid Chromatography-Tandem Mass Spectrometry method of inspection of wilforlide A, Tripterygium wilfordii lactone ketone, triptolide and tripterine |
| CN110687224B (en) * | 2019-09-30 | 2022-04-19 | 浙江工业大学 | Method for measuring triptolide A in tripterygium wilfordii medicinal material and tripterygium wilfordii multi-glycoside tablet prepared from tripterygium wilfordii medicinal material |
Citations (2)
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|---|---|---|---|---|
| US4005108A (en) * | 1973-04-03 | 1977-01-25 | Research Corporation | Novel anti-leukemic diterpenoid triepoxides |
| CN1062531A (en) * | 1991-12-23 | 1992-07-08 | 中国医学科学院皮肤病研究所 | The synthetic method of Triptonide |
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2006
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4005108A (en) * | 1973-04-03 | 1977-01-25 | Research Corporation | Novel anti-leukemic diterpenoid triepoxides |
| CN1062531A (en) * | 1991-12-23 | 1992-07-08 | 中国医学科学院皮肤病研究所 | The synthetic method of Triptonide |
Non-Patent Citations (4)
| Title |
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| 雷公藤内酯甲含量测定方法的改进. 阙慧卿等.中药材，第28卷第2期. 2005 |
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