CN103604877A - Construction method and applications of high performance liquid chromatography (HPLC) fingerprint of alkaloid compounds of dendrobe - Google Patents
Construction method and applications of high performance liquid chromatography (HPLC) fingerprint of alkaloid compounds of dendrobe Download PDFInfo
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- CN103604877A CN103604877A CN201310526400.1A CN201310526400A CN103604877A CN 103604877 A CN103604877 A CN 103604877A CN 201310526400 A CN201310526400 A CN 201310526400A CN 103604877 A CN103604877 A CN 103604877A
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Abstract
The invention discloses a construction method of fingerprint of alkaloid compounds of dendrobe. According to the construction method, thermostatic waterbath reflux extraction technology is adopted; dendrobe powder is immersed in ammonium hydroxide, and is subjected to extraction with chloroform, filtration, condensation and drying so as to obtain a dendrobe total alkaloid crude product; the dendrobe total alkaloid crude product is dissolved in methanol so as to obtain a stock solution; and the stock solution is used for high performance liquid chromatographic analysis so as to obtain the dendrobe alkaloid compound fingerprint. Advantages of the construction method are that: the obtained chromatographic fingerprint is capable of comprehensively reflecting alkaloid compounds contained by dendrobium, reflecting complexity of dendrobium alkaloid compounds, and providing scientific basis for identification of different dendrobium varieties; the constructed HPLC fingerprint possesses relatively high stability and repeatability; and scientific reference is provided for quality control of dendrobium variety.
Description
Technical field
The present invention relates to stem of noble dendrobium alkaloids composition efficient liquid-phase chromatograph finger print atlas construction method, belong to biological products and Chinese drug preparation technique field.
Background technology
Alkaloid is one of medicinal ingredient important in the stem of noble dendrobium, is also separated and carry out the compound of structural confirmation the earliest in Dendrobium Sw.It is generally acknowledged, that the alkaloidal pharmacological action of the stem of noble dendrobium is mainly manifested in is antitumor, to cardiovascular, intestines and stomach inhibiting effect and the pain relieving effects such as strong god of bringing down a fever.Research shows HERBA DENDROBII principal ingredient dendrobine tool pain relieving refrigeration function, can reduce heart rate, blood pressure, slows down breathing, can produce the hyperglycaemia of moderate, and the strong effect of tool also can be separated barbitalism.Yet, composition and the content difference of different stem of noble dendrobium breed organism alkali compositions are larger, Wang Xiankai etc. sum up the research of alkaloid aspect before, find that there is 5 kinds of Dendrobium Sws (D.nobile, D.kildebrandii, D.friedricksianum, D.findlayanum, D.wardianum) in containing dendrobine Alkaloid, 8 kinds of Dendrobium Sws are containing other types and each other without the alkaloid of base core jointly, and dendrobine is only found in (D.nobile, D.findlayanum).In view of the complicacy of stem of noble dendrobium alkaloid component research, be badly in need of a kind of fingerprint spectrum method rapidly and efficiently of exploitation, for discriminating and the stem of noble dendrobium evaluation of medical materials' quality of the different stems of noble dendrobium.At present, in the document of delivering both at home and abroad, only adopt Microscopic Identification, thin-layer chromatography and high performance liquid chromatography and vapor-phase chromatography and mass spectrum are studied some characteristic component in certain stem of noble dendrobium, lacking different stem of noble dendrobium alkaloids total systems studies and compares, obviously, the quality of stem of noble dendrobium alkaloid component can not accurately be differentiated and evaluate to this quality evaluating method.
Summary of the invention
The construction method that the object of this invention is to provide a kind of stem of noble dendrobium alkaloids composition efficient liquid-phase chromatograph finger print atlas.The present invention adopts HPLC technology to measure the different stems of noble dendrobium, sets up alkaloidal finger-print, for the scientific research of stem of noble dendrobium alkaloids composition later provides the ways and means of an express-analysis and detection.
Technical solution problem of the present invention adopts following technical scheme:
An efficient liquid-phase chromatograph finger print atlas construction method for stem of noble dendrobium alkaloids composition, its feature is, described method comprises:
(1) material is collected
Collect the fresh seedling of the different cultivars stem of noble dendrobium in the different places of production
(2) preparation of standard stem of noble dendrobium sample solution: the fresh seedling of each stem of noble dendrobium of the getting step 1) 15～20min that completes in the baking oven of 105 ℃, constant temperature drying in 60 ℃ of baking ovens, pulverizes, cross 60 mesh sieves, obtain sample meal, sample thief meal 0.5g, adds 5ml ammoniacal liquor, the close plug of rinse 30min, add chloroform 50ml to weigh, 65 ℃ of backflow 3h of water-bath, cooling after, chloroform is supplied weightlessness, obtains extract; Described extract rotary evaporated to dryness, adds methyl alcohol 5ml and dissolves, and with 0.22 μ m filtering with microporous membrane, gained filtrate is as standard stem of noble dendrobium sample solution;
(2) chromatographic condition: chromatographic column is C18 reverse-phase chromatographic column; Mobile phase: acetonitrile and water gradient elution; Volumetric flow rate: 1ml/min; Column temperature: 30 ℃; Detect wavelength: 220nm; Sample size: 5-10ul; Analysis time: 62min;
Gradient elution program is as follows
| Time (min) | Acetonitrile (%) | Water (%) |
| 0 | 20 | 80 |
| 50 | 80 | 20 |
| 52 | 20 | 80 |
| 62 | 20 | 80 |
In described mobile phase, be added with final concentration 0.005% ammoniacal liquor;
(3) acquisition of standard finger-print, by the standard stem of noble dendrobium sample solution described in step 1) by step 2) described chromatographic condition carries out efficient liquid phase chromatographic analysis, obtains the alkaloids standard finger-print of corresponding stem of noble dendrobium sample.
The application of fingerprint map construction method, its feature is, described method comprises: the efficient liquid-phase chromatograph finger print atlas that builds stem of noble dendrobium alkaloids composition to be measured by as above method, the standard finger-print of described finger-print and above-mentioned obtained counter sample is contrasted, alkaloid component in the stem of noble dendrobium more to be measured and standard stem of noble dendrobium sample, thus the quality of kind, the place of production and the stem of noble dendrobium alkaloid component of the stem of noble dendrobium to be measured differentiated fast.
The present invention compares and has advantages of with method with existing technology: the extracting method alkaloid of (1) alkaloids of the present invention extracts completely, and method is easy.(2) based on Waters XTerra MS C
18post is inorganic/organic hybrid granule technology, mode with chemosynthesis embeds the formed brand-new particle of inorganic poly-silica network by very stable organic carbon bond (Si-C) structure of chemical property, it integrates the advantage of silica gel and polymer filler, there is separation efficiency high, peak shape is good, column life is long, and the feature of pH wide ranges (1～12), therefore the HPLC finger-print of setting up has larger stability and reappearance; (3) mobile phase is preferentially selected not only good separating effect of 0.005% ammoniacal liquor, and can extend the serviceable life of chromatographic column; Because ammoniacal liquor is volatile alkali, so plant liquid-phase chromatography method, be also applicable to LC-MS analysis.(4) dendrobine is that end absorbs, and from 190-220nm uv absorption, reduces gradually, and 240nm does not have uv absorption later substantially; But 190nm place solvent background interference is larger, therefore detect the selected 220nm of wavelength.(5) liquid-phase chromatography method of the present invention is applicable to the constituent analysis of Dendrobidium huoshanness group training seedling total alkaloids, and a kind of quality assessment means of Dendrobidium huoshanness are provided.
Accompanying drawing explanation
Fig. 1 be take the high-efficient liquid phase chromatogram that acetonitrile solution is blank test.
Fig. 2 be take the high-efficient liquid phase chromatogram that dendrobine solution is control test.
Fig. 3 is the alkaloids high-efficient liquid phase chromatogram of group training seedling 1.（Fe55ul）
Fig. 4 is the alkaloids high-efficient liquid phase chromatogram of group training seedling 2.（Cu55ul）
Fig. 5 is the alkaloids high-efficient liquid phase chromatogram of group training seedling 3.（M55ul）
embodiment
By embodiment, technical scheme of the present invention is further detailed and is described below
(1) material is collected
Take dendrobium candidum, Dendrobium Moniliforme, Dendrobidium huoshanness as supplying examination material; Dendrobium candidum, Dendrobium Moniliforme, Dendrobidium huoshanness are carried out to the tissue of conventional method and cultivate, group training condition of culture is: 25 ℃ ± 2 ℃, 12h illumination 12h is dark, through 5 subcultures, cultivates, and subculture cycle 45d left and right, must organize training seedling 1 after cultivation, group training seedling 2, group training seedling 3.
(2) preparation of need testing solution: the fresh seedling of getting the stem of noble dendrobium is some, the 15～20min that completes in the baking oven of 105 ℃, dries in 60 ℃ of baking ovens, pulverizes, and crosses 60 mesh sieves.Get several sample dry powder 0.5g, add respectively 5ml ammoniacal liquor, the close plug of rinse 30min, precision adds chloroform 50ml to weigh, 65 ℃ of backflow 3h of water-bath, extract is flung to partial solvent, cooling after, chloroform is supplied weightlessness.Extract rotary evaporated to dryness adds methyl alcohol 5ml sample dissolution, and with 0.22 μ m filtering with microporous membrane, filtrate is as need testing solution.
(3) preparation of reference substance solution: accurately take dendrobine reference substance 10mg, add acetonitrile dissolving and be settled to 10ml, shake up, be made into 1mg/ml, standby.(purchased from Man Site bio tech ltd, Chengdu, content is greater than 98%)
(4) chromatographic condition: chromatographic column: Waters XTerra MS C
18post (250*4.6mm, 5 μ m); Mobile phase: acetonitrile and water (being added with 0.005% ammoniacal liquor) gradient elution; Volumetric flow rate: 1ml/min; Column temperature: 30 ℃; Detect wavelength: 220nm; Sample size: 5-10ul; Analysis time: 62min.Gradient elution program is in Table 1.
Table 1 gradient elution program
| Time (min) | Acetonitrile (%) | Water (%) |
| 0 | 20 | 80 |
| 50 | 80 | 20 |
| 52 | 20 | 80 |
| 62 | 20 | 80 |
(5) methodological study:
Blank test: draw acetonitrile blank solution 10ul, inject high performance liquid chromatograph by chromatographic condition, record chromatogram, result as shown in Figure 1.
Control test: draw dendrobine solution 10ul, inject high performance liquid chromatograph by chromatographic condition, record chromatogram, result as shown in Figure 2.
Precision test: get with a collection of need testing solution, by chromatographic condition continuous sample introduction 5 times, calculate respectively the relative standard deviation (RSD) of retention time and peak area, result is as shown in table 2.
Table 2
Stability test: get with a collection of need testing solution, at room temperature place, carry out according to chromatographic condition, analyze respectively at 0,2,4,6,8,12,24h sample introduction, calculate respectively the relative standard deviation (RSD) of retention time and peak area, result is as shown in table 3.
Table 3
(6) test sample is measured: according to chromatographic condition, measure several test sample HPLC chromatograms, and spectral data is analyzed, obtain stem of noble dendrobium alkaloids high-efficient liquid phase chromatogram (sample introduction 5ul), result as in Figure 3-5, as can be seen from the figure, three kinds of dendrobe tissue culture seedlings have 10 total peaks.
The present embodiment gained chromatographic fingerprinting can reflect the contained alkaloids composition of the stem of noble dendrobium more comprehensively, embodies the complicacy of stem of noble dendrobium alkaloids, can differentiate scientific basis is provided for the different cultivars stem of noble dendrobium; The HPLC finger-print of setting up has larger stability and reappearance, and this research is controlled science reference is provided for stem of noble dendrobium variety and quality.
Claims (2)
1. an efficient liquid-phase chromatograph finger print atlas construction method for stem of noble dendrobium alkaloids composition, is characterized in that, described method comprises:
(1) material is collected
Collect different stem of noble dendrobium kinds, and cultivation group training seedling
(2) preparation of standard stem of noble dendrobium sample solution: each dendrobe tissue culture seedling of getting step 1) 15～20min that completes in the baking oven of 105 ℃, constant temperature drying in 60 ℃ of baking ovens, pulverizes, cross 60 mesh sieves, obtain sample meal, sample thief meal 0.5g, adds 5ml ammoniacal liquor, the close plug of rinse 30min, add chloroform 50ml to weigh, 65 ℃ of backflow 3h of water-bath, cooling after, chloroform is supplied weightlessness, obtains extract; Described extract rotary evaporated to dryness, adds methyl alcohol 5ml and dissolves, and with 0.22 μ m filtering with microporous membrane, gained filtrate is as standard stem of noble dendrobium sample solution;
(2) chromatographic condition: chromatographic column is C18 reverse-phase chromatographic column; Mobile phase: acetonitrile and water gradient elution; Volumetric flow rate: 1ml/min; Column temperature: 30 ℃; Detect wavelength: 220nm; Sample size: 5-10ul; Analysis time: 62min;
Gradient elution program is as follows
In described mobile phase, be added with final concentration 0.005% ammoniacal liquor;
(3) acquisition of stem of noble dendrobium alkaloids ingredients fingerprint, injects efficient liquid phase chromatographic analysis instrument by standard stem of noble dendrobium sample solution described in step 1), by step 2) described chromatographic condition measures, the standard finger-print of acquisition counter sample.
2. the application of fingerprint map construction method described in claim 1, it is characterized in that: the efficient liquid-phase chromatograph finger print atlas that builds stem of noble dendrobium alkaloids composition to be measured by the method for claim 1, the standard finger-print of the counter sample obtaining in described finger-print and claim 1 is contrasted, alkaloid component in the stem of noble dendrobium more to be measured and standard stem of noble dendrobium sample, thus the kind of the stem of noble dendrobium to be measured and the quality of stem of noble dendrobium alkaloid component differentiated fast.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104713979A (en) * | 2014-09-23 | 2015-06-17 | 上海中医药大学 | Thin-layer identification method for Dendrobium officinale |
| CN105424850A (en) * | 2015-11-05 | 2016-03-23 | 北京蓝标一成科技有限公司 | Quality evaluation method for dendrobe medicinal material |
| CN106501437A (en) * | 2016-12-27 | 2017-03-15 | 安徽农业大学 | Based on the method that UPLC/LTQ Orbitrap Velos MS detect stem of noble dendrobium alkaloid component |
| CN108645802A (en) * | 2018-05-16 | 2018-10-12 | 安徽农业大学 | A kind of detection method of dendrobium candidum total alkaloid content |
| CN109283269A (en) * | 2018-09-29 | 2019-01-29 | 合肥工业大学 | The analysis method of feature alkaloid composition in a kind of fresh Huoshan rice dry measure used in former times |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101491644A (en) * | 2007-04-18 | 2009-07-29 | 北京和润创新医药科技发展有限公司 | Method for separating dendrobium stem total alkaloids in dendrobium stem extract |
| CN101726547A (en) * | 2008-10-15 | 2010-06-09 | 中山市中智药业集团有限公司 | Dendrobe chromatogram finger print measuring method |
| WO2010070590A1 (en) * | 2008-12-16 | 2010-06-24 | Sea Marconi Technologies Di Vander Tumiatti S.A.S. | The corrosity of sulfur in insulating oils is determined with a copper strip under heated and stirred conditions |
| CN103076294A (en) * | 2012-12-29 | 2013-05-01 | 安徽农业大学 | Method for testing content of alkaloid in dendrobe |
-
2013
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101491644A (en) * | 2007-04-18 | 2009-07-29 | 北京和润创新医药科技发展有限公司 | Method for separating dendrobium stem total alkaloids in dendrobium stem extract |
| CN101726547A (en) * | 2008-10-15 | 2010-06-09 | 中山市中智药业集团有限公司 | Dendrobe chromatogram finger print measuring method |
| WO2010070590A1 (en) * | 2008-12-16 | 2010-06-24 | Sea Marconi Technologies Di Vander Tumiatti S.A.S. | The corrosity of sulfur in insulating oils is determined with a copper strip under heated and stirred conditions |
| CN103076294A (en) * | 2012-12-29 | 2013-05-01 | 安徽农业大学 | Method for testing content of alkaloid in dendrobe |
Non-Patent Citations (1)
| Title |
|---|
| 高巍等: "黔产金钗石斛生物碱组分的HPLC指纹图谱建立", 《安徽农业科学》, vol. 37, no. 28, 10 October 2009 (2009-10-10), pages 13610 - 13611 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104713979A (en) * | 2014-09-23 | 2015-06-17 | 上海中医药大学 | Thin-layer identification method for Dendrobium officinale |
| CN104713979B (en) * | 2014-09-23 | 2016-08-31 | 上海中医药大学 | A kind of thin-layer identification method of dendrobium candidum |
| CN105424850A (en) * | 2015-11-05 | 2016-03-23 | 北京蓝标一成科技有限公司 | Quality evaluation method for dendrobe medicinal material |
| CN106501437A (en) * | 2016-12-27 | 2017-03-15 | 安徽农业大学 | Based on the method that UPLC/LTQ Orbitrap Velos MS detect stem of noble dendrobium alkaloid component |
| CN108645802A (en) * | 2018-05-16 | 2018-10-12 | 安徽农业大学 | A kind of detection method of dendrobium candidum total alkaloid content |
| CN108645802B (en) * | 2018-05-16 | 2020-01-03 | 安徽农业大学 | Detection method for content of total alkaloids in dendrobium officinale |
| CN109283269A (en) * | 2018-09-29 | 2019-01-29 | 合肥工业大学 | The analysis method of feature alkaloid composition in a kind of fresh Huoshan rice dry measure used in former times |
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