CN105158355A - Method for rapidly measuring content of four spermidine ingredients in carthamus tinctorius simultaneously - Google Patents
Method for rapidly measuring content of four spermidine ingredients in carthamus tinctorius simultaneously Download PDFInfo
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Abstract
The invention discloses a method for rapidly measuring content of four spermidine ingredients in carthamus tinctorius simultaneously and belongs to the technical field of measurement of content of effective components in a traditional Chinese medicine. Specifically, HPLC (high performance liquid chromatography) is adopted, and the liquid chromatogram analysis conditions are shown as follows: octadecylsilane chemically bonded silica is adopted as a filling agent, an organic solvent-water solution is adopted as a moving phase, isocratic elution is adopted, the detection wavelength is 210-360 nm, and the column temperature is 30 DEG C. The method is reliable and easy to operate, and a new method is provided for measurement of the content of spermidine. The method has an application prospect in the aspects of extraction, synthesis and analysis of spermidine and quality control during research, development and production of related drugs.
Description
Technical field
The present invention relates to Effective Component of Chinese Medicine assay technical field, specifically belong to the method for four kinds of spermidine component contents in a kind of Rapid Simultaneous Determination safflower.
Background technology
Safflower is the dry tubular flower of composite family safflower platymiscium safflower (CarthamustinctoriusL.), and floral tube is carefully narrow, is about 1.5 centimetres, orange red.Its original name is red indigo plant flower, calls as safflower cherry, safflower, thorn safflower, Flos Carthami, meeting safflower etc.The main place of production is the provinces such as Xinjiang, Henan, Zhejiang and Sichuan, pungent, the micro-hardship of its taste, warm in nature, the thoughts of returning home and Liver Channel, can activating blood to promote menstruation, blood stasis removing analgesic, hypotensive and reducing blood lipid, be used for the treatment of through closing, dysmenorrhoea, angina pectoris, hypertension, traumatic injury, coronary heart disease etc.
Safflower chemical composition has diversity, is therefrom separated so far and obtains comprising the 250 number of chemical compositions such as quinoid chalcone glycoside, flavonoids, sterols, alkaloids, lignanoids, organic acid, alkyl diol class and polyyne class.Spermidine compounds refers to have H
2n (CH
2)
3nH (CH
2)
4nH
2the polyamine compounds of structural unit, from safflower, qualification four kinds of spermidine constituents: N1 are separated at present, N5, N10-(Z)-tri-p-coumaroylspermidine (1), N1, N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (2), N1 (E)-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (3) and N1, N5, N10-(E)-tri-p-coumaroyl-spermidine (4).
The structural formula of four kinds of spermidine compositions:
Spermidine constituents has the biologically active widely such as antiviral, anti-oxidant, antitumor, antibacterial.Qualification spermidine composition N1 is separated from safflower, N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (compound 2 namely in this patent) demonstrates has very strong selectivity inhibit activities to 5-HT re uptake, has Cure of depression prospect.Because spermidine exists numerous isomers, there is many difficulties in analysis, separation, qualification.At present about the existing patent report (Chinese patent: a kind of high speed adverse current chromatogram that uses is separated the method preparing spermidine, application number from safflower: 201310396939.X) of spermidine quick separating preparation in safflower.And spermidine constituents content assaying method has no report in relevant safflower, especially in Rapid Simultaneous Determination safflower, the method for four kinds of spermidine has no report.
Summary of the invention
The object of the present invention is to provide four kinds of spermidine composition N1 in a kind of Rapid Simultaneous Determination safflower, N5, N10-(Z)-tri-p-coumaroyl-spermidine (1), N1, N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (2), N1 (E)-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (3) and N1, the method of N5, N10-(E)-tri-p-coumaroyl-spermidine (4) content.The method should be stablized, reliable, easy to operate, the method for flos carthami quality control can not only be improved, simultaneously can also provide reliable detection method for spermidine class species analysis.
In a kind of Rapid Simultaneous Determination safflower provided by the invention, the method for four kinds of spermidine component contents, comprises the steps:
1) flos carthami powder (crossing 60 mesh sieves) 1.0000g is taken, be placed in the tool plug conical flask that 100ml is brown, add 15ml methyl alcohol, weighed weight, ultrasonic process (ultrasonic frequency 40KHz, power ratio 91%, ultrasonic temperature 25 DEG C) 10min, weighs after letting cool again, the weight of less loss is supplied with methyl alcohol, shake up, filter, obtain safflower need testing solution, to be measured after 0.45 μm of miillpore filter;
2) Agilent hplc determination is used, chromatographic condition: analytical column is AgilentC
18chromatographic column (250mm × 4.6mm, 5 μm), mobile phase is organic solvent/water isocratic elution, and elution flow rate is 1.0ml/min, column temperature 30 DEG C, determined wavelength 210-360nm, and sampling volume is 10 μ l;
3) peak area corresponding to four kinds of spermidine compositions is brought into following formulae discovery its content respectively; Y=40317x-51.630, Y=35750x-29.313, Y=38854x-50.026, Y=48600x-67.042;
Four kinds of described spermidine composition: N1, N5, N10-(Z)-tri-p-coumaroyl-spermidine (1), N1, N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (2), N1 (E)-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (3) and N1, N5, N10-(E)-tri-p-coumaroyl-spermidine (4).
Described organic solvent is methyl alcohol, and gradient is 47% methyl alcohol.
The determined wavelength of described four kinds of spermidine compositions (1), (2), (3), (4) corresponds to 270nm, 280nm, 290nm, 300nm respectively.
The present invention adopts four kinds of spermidine effective constituents content in HPLC method Simultaneously test safflower, and method is reliably easy to operate, provides the new method that a kind of spermidine measures.The method is in the extraction of spermidine, synthesis, analysis and related drugs research and development and have application prospect in quality control in producing.
Accompanying drawing explanation
Four kinds of spermidine composition N1 in safflower under Fig. 1 different wave length, N5, N10-(Z)-tri-p-coumaroyl-spermidine (1), N1, N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (2), N1 (E)-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (3) and N1, N5, N10-(E)-tri-p-coumaroyl-spermidine (4) mixes the HPLC figure of reference substance.
The HPLC figure of flos carthami under Fig. 2 different wave length.
Embodiment
Embodiment 1:
Liquid-phase chromatographic analysis condition: analytical column is AgilentC
18chromatographic column (250mm × 4.6mm, 5 μm), mobile phase is 47% methanol/water isocratic elution, and elution flow rate is 1.0ml/min, column temperature 30 DEG C, and determined wavelength is 270nm, 280nm, 290nm, 300nm multi-wavelength detection, and sampling volume is 10 μ l.
The preparation of reference substance solution:
The preparation of mixing reference substance stock solution: precision takes spermidine compounds 1 standard items 4.16mg respectively, spermidine compounds 2 standard items 5.12mg, spermidine compounds 3 standard items 5.40mg, spermidine compounds 4 standard items 10.04mg, in the brown volumetric flask of 100ml, by methanol constant volume to scale, obtain mixing reference substance solution stock solution (each standard items are prepared by this laboratory).
The preparation of mixing reference substance solution: draw reference substance stock solution 0.5ml with pipette is accurate respectively, 0.8ml, 2.0ml, 3.0ml, 4.0ml 5.0ml is placed in the brown volumetric flask of 10ml respectively, by methanol constant volume to scale, be mixed with the mixing reference substance solution of variable concentrations, miillpore filter (0.22 μm) filters and get final product.
The preparation of need testing solution:
Precision takes flos carthami powder (crossing 60 mesh sieves) 1.0000g, be placed in the tool plug conical flask that 100ml is brown, precision adds 15ml methyl alcohol, weighed weight, ultrasonic process (ultrasonic frequency 40KHz, power ratio 91%, ultrasonic temperature 25 DEG C) 10min, again weighs after letting cool, supplies the weight of less loss with methyl alcohol, shake up, filter, after the safflower need testing solution getting gained crosses 0.45 μm of miillpore filter and get final product.
Draw mixing reference substance solution, each 10 μ l of need testing solution respectively, injection liquid chromatography, measure, to obtain final product.
The method can measure four kinds of spermidine composition N1 in safflower accurately and effectively, N5, N10-(Z)-tri-p-coumaroyl-spermidine (1), N1, N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (2), N1 (E)-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (3) and N1, N5, N10-(E)-tri-p-coumaroyl-spermidine (4) content.
1. linear relationship is investigated
According to above-mentioned liquid-phase chromatographic analysis condition, get above-mentioned prepared mixing reference substance solution sample introduction analysis respectively, parallel sample introduction twice, with the mass concentration of spermidine compounds (mg/ml) for horizontal ordinate, as follows with the typical curve that each spermidine compounds peak area draws 4 spermidine compounds for ordinate.
Table 1 spermidine compounds 1-4 regression equation and the range of linearity
2. precision is investigated
According to above-mentioned prepared safflower need testing solution, draw same need testing solution and carry out efficient liquid phase chromatographic analysis according to above-mentioned liquid-phase chromatographic analysis condition, continuous sample introduction 6 times, by the RSD value of the retention time and peak area that calculate each spermidine compounds, carry out the investigation of the precision of method, investigate and the results are shown in Table 2.
Table 2 precision investigates result
As can be seen from Table 2, in the investigation of precision, the retention time of each spermidine compounds and the RSD value of reservation peak area are all less than 5%, and the precision of illustration method is good, and instrument is comparatively stable.
3 repeatability are investigated
Get with a collection of flos carthami powder, according to above-mentioned prepared need testing solution, repeat preparation 6 parts, efficient liquid phase chromatographic analysis is carried out according to above-mentioned liquid-phase chromatographic analysis condition, by the RSD value of the retention time and peak area that calculate each spermidine compounds, carry out the investigation of the repeatability of method.
Table 3 reappearance investigates result
As can be seen from Table 3, the RSD value of each spermidine compounds its retention time and reservation peak area in reappearance is investigated all is less than 5%, and result shows that the reappearance of method is good.
4 study on the stability
According to above-mentioned prepared need testing solution, efficient liquid phase chromatographic analysis is carried out respectively at 0h, 2h, 4h, 6h, 12h, the 24h after the preparation of safflower need testing solution according to above-mentioned liquid-phase chromatographic analysis condition, by the RSD value of the retention time and peak area that calculate each spermidine compounds, carry out the investigation of the stability of method, investigate and the results are shown in Table 4.
Table 4 study on the stability result
As can be seen from Table 4, the RSD value of each spermidine compounds its retention time and reservation peak area in study on the stability is all less than 5%, and result shows that safflower need testing solution is good at 24 hours internal stabilities.
5 accuracy are investigated
Adopt the method that application of sample reclaims, precision takes the flos carthami powder 0.5000g of known each spermidine kind compound content, parallel 9 parts, respectively according to 50% of spermidine kind compound content each in safflower sample, 100%, 150% adds reference substance solution, each concentration 3 parts, according to above-mentioned prepared need testing solution, then sample introduction analysis is carried out according to above-mentioned liquid-phase chromatographic analysis condition, according to the typical curve of peak area and each spermidine compounds, calculate the content of 4 spermidine compounds in need testing solution, and calculate average recovery and its RSD value of each spermidine compounds, carry out the investigation of the accuracy of method, the results are shown in Table 5-8.
Table 5 spermidine compounds 1 average recovery
Table 6 spermidine compounds 2 average recovery
Table 7 spermidine compounds 3 average recovery
Table 8 spermidine compounds 4 average recovery
As can be seen from table 5-8, the average recovery of 4 spermidine compounds is all in the scope of 95%-105%, and its RSD value is all less than 5%, shows that the accuracy of the method is good.
Embodiment 2
1) flos carthami powder (crossing 60 mesh sieves) 1.0000g is taken, be placed in the tool plug conical flask that 100ml is brown, add 15ml methyl alcohol, weighed weight, ultrasonic process (ultrasonic frequency 40KHz, power ratio 91%, ultrasonic temperature 25 DEG C) 10min, weighs after letting cool again, the weight of less loss is supplied with methyl alcohol, shake up, filter, obtain safflower need testing solution, to be measured after 0.45 μm of miillpore filter;
2) Agilent hplc determination is used, chromatographic condition: analytical column is AgilentC
18chromatographic column (250mm × 4.6mm, 5 μm), mobile phase is 47% methanol/water isocratic elution, and elution flow rate is 1.0ml/min, column temperature 30 DEG C, and determined wavelength is 270nm, 280nm, 290nm, 300nm, and sampling volume is 10 μ l; Obtain the peak area corresponding to four spermidine compositions;
3) peak area corresponding to four kinds of spermidine compositions is brought into following formulae discovery its content respectively; Y=40317x-51.630, Y=35750x-29.313, Y=38854x-50.026, Y=48600x-67.042; Spermidine compounds 1 i.e. N1 in result display safflower, N5, the content of N10-(Z)-tri-p-coumaroylspermidine is 0.0165%, spermidine compounds 2 i.e. N1-(E)-N5, the content of N10-(Z)-tri-p-coumaroylspermidine is 0.0330%, the content of spermidine compounds 3 i.e. N1 (E)-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine is 0.0405%, spermidine compounds 4 i.e. N1, the content of N5, N10-(E)-tri-p-coumaroylspermidine is 0.1140%.
Claims (3)
1. the method for four kinds of spermidine component contents in Rapid Simultaneous Determination safflower, it is characterized in that, step comprises:
1) flos carthami powder 1.0000g is taken, be placed in the tool plug conical flask that 100ml is brown, add 15ml methyl alcohol, weighed weight, ultrasonic process 10min, weighs after letting cool again, the weight of less loss is supplied with methyl alcohol, shake up, filter, obtain safflower need testing solution, to be measured after 0.45 μm of miillpore filter;
2) Agilent hplc determination is used, chromatographic condition: analytical column is AgilentC
18chromatographic column, its specification is 250mm × 4.6mm, 5 μm, and mobile phase is organic solvent/water isocratic elution, and elution flow rate is 1.0ml/min, column temperature 30 DEG C, determined wavelength 210-360nm, and sampling volume is 10 μ l;
3) peak area corresponding to four kinds of spermidine compositions is brought into following formulae discovery its content respectively; Y=40317x-51.630, Y=35750x-29.313, Y=38854x-50.026, Y=48600x-67.042;
Four kinds of described spermidine composition: N1, N5, N10-(Z)-tri-p-coumaroyl-spermidine (1), N1, N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (2), N1 (E)-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (3) and N1, N5, N10-(E)-tri-p-coumaroyl-spermidine (4).
2. the method measuring four kinds of spermidine component contents in safflower as claimed in claim 1, it is characterized in that, described organic solvent is methyl alcohol, and gradient is 47% methyl alcohol.
3. measure the method for four kinds of spermidine component contents in safflower as claimed in claim 1, it is characterized in that described four kinds of spermidine compositions (1), (2), (3), (4) determined wavelength correspond to 270nm respectively, 280nm, 290nm, 300nm.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110279738A (en) * | 2019-07-03 | 2019-09-27 | 山西大学 | A kind of extracting method of antidepressant spermidine active component and the purposes of spermidine effective part extract |
| CN110794060A (en) * | 2019-11-12 | 2020-02-14 | 华润三九（雅安）药业有限公司 | Method for determining spermidine content in safflower medicinal material and method for enriching spermidine |
| CN112433017A (en) * | 2020-12-09 | 2021-03-02 | 宁夏农林科学院枸杞科学研究所 | Method for detecting specific metabolites of spermidine lycium barbarum |
| CN114324633A (en) * | 2021-12-14 | 2022-04-12 | 南昌大学 | Method for identifying authenticity of Chinese tallow tree honey |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103508912A (en) * | 2013-09-04 | 2014-01-15 | 中国科学院西北高原生物研究所 | Method for separation and preparation of spermidines from safflower by employing high-speed counter-current chromatography |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103508912A (en) * | 2013-09-04 | 2014-01-15 | 中国科学院西北高原生物研究所 | Method for separation and preparation of spermidines from safflower by employing high-speed counter-current chromatography |
Non-Patent Citations (3)
| Title |
|---|
| LI, WEN-CONG; WANG, XIAO-YAN; LIN, PENG-CHENG: "Preparative separation and purification of four cis-trans isomers of coumaroylspermidine analogs from safflower by high-speed counter-current chromatography", 《JOURNAL OF CHROMATOGRAPHY》 * |
| 扈晓佳 等: "红花的化学成分及其药理活性研究进展", 《药学实践杂志》 * |
| 袁茂叶 等: "红花中亚精胺衍生物分离纯化及其抑制［3H-5-HT再摄取的作用", 《山西医科大学学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110279738A (en) * | 2019-07-03 | 2019-09-27 | 山西大学 | A kind of extracting method of antidepressant spermidine active component and the purposes of spermidine effective part extract |
| CN110794060A (en) * | 2019-11-12 | 2020-02-14 | 华润三九（雅安）药业有限公司 | Method for determining spermidine content in safflower medicinal material and method for enriching spermidine |
| CN112433017A (en) * | 2020-12-09 | 2021-03-02 | 宁夏农林科学院枸杞科学研究所 | Method for detecting specific metabolites of spermidine lycium barbarum |
| CN112433017B (en) * | 2020-12-09 | 2022-10-18 | 宁夏农林科学院枸杞科学研究所 | Method for detecting specific metabolites of spermidine lycium barbarum |
| CN114324633A (en) * | 2021-12-14 | 2022-04-12 | 南昌大学 | Method for identifying authenticity of Chinese tallow tree honey |
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