CN105158355B - 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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- CN105158355B CN105158355B CN201510492420.0A CN201510492420A CN105158355B CN 105158355 B CN105158355 B CN 105158355B CN 201510492420 A CN201510492420 A CN 201510492420A CN 105158355 B CN105158355 B CN 105158355B
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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, particularly belong in a kind of Rapid Simultaneous Determination Flos Carthami
The method of four kinds of spermidine component contents.
Background technology
Flos Carthami is the dry tubular flower of Compositae Flos Carthami platymiscium Flos Carthami (carthamus tinctorius l.), and floral tube is narrow
Carefully, it is about 1.5 centimetres, orange red.Its original name is red blue flower, calls red for Flos Carthami cherry, Flos Carthami, thorn safflower, Flos Carthami, meeting
Flower etc..The main place of production is that Xinjiang, Henan, Zhejiang and Sichuan etc. save, and its acrid in the mouth, slight bitter, warm in nature, GUIXIN and Liver Channel can be invigorated blood circulation
Stimulate the menstrual flow, eliminating stasis to stop pain, blood pressure lowering and blood fat reducing, for treating amenorrhea, dysmenorrhea, angina pectoriss, hypertension, traumatic injury, coronary heart disease
Deng.
The great multiformity of Flos Carthami chemical composition, has therefrom separately obtained so far including quinoid chalcone glycoside, flavonoid, steroid
More than the 250 kind of chemical composition such as alcohols, alkaloidss, lignanoids, organic acid, alkyl diol class and polyacetylene.Spermidine
Class compound refers to h2n(ch2)3nh(ch2)4nh2The polyamine compounds of construction unit, separate from Flos Carthami at present and reflect
Fixed four kinds of spermidine constituents: n1, 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 have the widely biological activity such as antiviral, antioxidation, antitumor, antibacterial.Divide from Flos Carthami
From identification spermidine ingredient n 1, n5- (z)-n10- (e)-tri-p-coumaroylspermidine (i.e. chemical combination in this patent
Thing 2) show there is very strong selectivity inhibitory activity to 5-ht reuptake, there is treatment depression prospect.Because spermidine exists
Numerous isomerss, exist many difficult in analysis, separation, identification.At present with regard to spermidine sharp separation system in Flos Carthami
A kind of standby existing patent report (Chinese patent: utilization high speed adverse current chromatogram separates the method preparing spermidine, application from Flos Carthami
Number: 201310396939.x).And in relevant Flos Carthami, spermidine constituents content assaying method has no report, especially while quickly
The method measuring four kinds of spermidine in Flos Carthami has no report.
Content of the invention
It is an object of the invention to provide four kinds of spermidine ingredient n 1, n5, n10- in a kind of Rapid Simultaneous Determination Flos Carthami
(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-
The method of coumaroyl-spermidine (4) content.The method should stablize, reliable, easy to operate, can not only improve flos carthami
The method of quality control, can also provide reliable detection method for spermidine class species analysis simultaneously.
The method of four kinds of spermidine component contents in a kind of Rapid Simultaneous Determination Flos Carthami that the present invention provides, walks including following
Rapid:
1) weigh flos carthami powder (crossing 60 mesh sieves) 1.0000g, be placed in the conical flask with cover of 100ml brown, add
15ml methanol, weighed weight, supersound process (supersonic frequency 40khz, power ratio 91%, 25 DEG C of ultrasonic temperature) 10min, after letting cool
Weigh again, supply the weight of less loss with methanol, shake up, filter, obtain Flos Carthami need testing solution, after 0.45 μm of microporous filter membrane
To be measured afterwards；
2) use agilent hplc determination, chromatographic condition: analytical column is agilent c18Chromatographic column (250mm ×
4.6mm, 5 μm), mobile phase is organic solvent/water isocratic elution, and elution flow rate is 1.0ml/min, 30 DEG C of column temperature, Detection wavelength
210-360nm, sampling volume is 10 μ l；
3) peak area corresponding to four kinds of spermidine compositions is brought into below equation respectively and calculate its content；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 methanol, and gradient is 47% methanol.
Described four kinds of spermidine compositions (1), (2), (3), Detection wavelengths of (4) correspond to 270nm, 280nm respectively,
290nm,300nm.
The present invention measures four kinds of spermidine effective constituents contents in Flos Carthami using hplc method simultaneously, and method is reliable easily to be grasped
Make, there is provided the new method that a kind of spermidine measures.The method is in the extraction of spermidine, synthesis, analysis and Related Drug
In thing research and development and production, quality control aspect has application prospect.
Brief description
Four kinds of spermidine ingredient n 1, n5, n10- (z)-tri-p-coumaroyl- in Flos Carthami under Fig. 1 different wave length
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.
Specific embodiment
Embodiment 1:
Liquid-phase chromatographic analysis condition: analytical column is agilent c18Chromatographic column (250mm × 4.6mm, 5 μm), mobile phase is
47% methanol/water isocratic elution, elution flow rate is 1.0ml/min, 30 DEG C of column temperature, and Detection wavelength is 270nm, 280nm, 290nm,
300nm multi-wavelength detection, sampling volume is 10 μ l.
The preparation of reference substance solution:
The preparation of mixing reference substance stock solution: precision weighs spermidine class compound 1 standard substance 4.16mg, spermidine respectively
Class compound 2 standard substance 5.12mg, spermidine class compound 3 standard substance 5.40mg, spermidine class compound 4 standard substance
10.04mg, in 100ml brown volumetric flask, with methanol constant volume to scale, obtains mixed reference substance solution stock solution (each standard
Product are by prepared by this laboratory).
The preparation of mixed reference substance solution: draw reference substance stock solution 0.5ml, 0.8ml with pipette is accurate respectively,
2.0ml, 3.0ml, 4.0ml, 5.0ml are respectively placed in 10ml brown volumetric flask, with methanol constant volume to scale, are configured to different dense
The mixed reference substance solution of degree, (0.22 μm) filtration of microporous filter membrane obtains final product.
The preparation of need testing solution:
Precision weighs flos carthami powder (crossing 60 mesh sieves) 1.0000g, is placed in the conical flask with cover of 100ml brown, essence
Close addition 15ml methanol, weighed weight, supersound process (supersonic frequency 40khz, power ratio 91%, 25 DEG C of ultrasonic temperature) 10min,
Weigh again after letting cool, supply the weight of less loss with methanol, shake up, filter, take the Flos Carthami need testing solution of gained to cross 0.45 μm
Obtain final product after microporous filter membrane.
Draw mixed reference substance solution, each 10 μ l of need testing solution respectively, inject chromatograph of liquid, measure, obtain final product.
The method can accurately and effectively measure four kinds of spermidine ingredient n 1, n5, n10- (z)-tri-p- in Flos Carthami
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, take above-mentioned prepared mixed reference substance solution sample introduction to analyze respectively, put down
Traveling sample twice, with the mass concentration (mg/ml) of spermidine class compound as abscissa, with each spermidine class compound peaks area
The standard curve drawing 4 spermidine class compounds for vertical coordinate is as follows.
Table 1 spermidine class compound 1-4 regression equation and the range of linearity
2. precision is investigated
According to above-mentioned prepared Flos Carthami need testing solution, draw same need testing solution and divide according to above-mentioned liquid chromatograph
Analysis condition carries out efficient liquid phase chromatographic analysis, continuous sample introduction 6 times, by calculate each spermidine class compound retention time and
The rsd value of peak area, carries out the investigation of the precision of method, investigates and the results are shown in Table 2.
Table 2 precision investigates result
As can be seen from Table 2, the retention time of each spermidine class compound and reservation peak area in the investigation of precision
Rsd value be respectively less than 5%, the precision of illustration method is good, and instrument is relatively stable.
3 repeatability are investigated
Take with a collection of flos carthami powder, according to above-mentioned prepared need testing solution, repeat to prepare 6 parts, according to above-mentioned
Liquid-phase chromatographic analysis condition carries out efficient liquid phase chromatographic analysis, by calculating retention time and the peak of each spermidine class compound
The rsd value of area, carries out the investigation of the repeatability of method.
Table 3 repeatability investigates result
As can be seen from Table 3, each spermidine class compound its retention time and retain peak area in repeatability investigation
Rsd value is respectively less than 5%, and result shows that the repeatability of method is good.
4 study on the stability
According to above-mentioned prepared need testing solution, molten respectively at Flos Carthami test sample according to above-mentioned liquid-phase chromatographic analysis condition
0h, 2h, 4h, 6h, 12h, 24h after liquid preparation carry out efficient liquid phase chromatographic analysis, by calculating each spermidine class compound
The rsd value of retention time and peak area, carries out the investigation of the stability of method, investigates and the results are shown in Table 4.
Table 4 study on the stability result
As can be seen from Table 4, each spermidine class compound its retention time and retain peak area in study on the stability
Rsd value is respectively less than 5%, and result shows that Flos Carthami need testing solution is good in 24 hours internal stabilities.
5 accuracy are investigated
The method being reclaimed using sample-adding, precision weighs the flos carthami powder of known each spermidine kind compound content
0.5000g, parallel 9 parts, respectively according to 50%, 100%, 150% addition of spermidine kind compound content each in Flos Carthami sample
Reference substance solution, 3 parts of each concentration, according to above-mentioned prepared need testing solution, then according to above-mentioned liquid-phase chromatographic analysis bar
Part carries out sample introduction analysis, according to the standard curve of peak area and each spermidine class compound, calculates 4 sub- essences in need testing solution
The content of aminated compoundss, and calculate average recovery and its rsd value of each spermidine class compound, carry out the accurate of method
The investigation of degree, the results are shown in Table 5-8.
Table 5 spermidine class compound 1 average recovery
Table 6 spermidine class compound 2 average recovery
Table 7 spermidine class compound 3 average recovery
Table 8 spermidine class compound 4 average recovery
The average recovery of 4 spermidine class compounds be can be seen that all in the range of 95%-105% by table 5-8,
And its rsd value is respectively less than 5%, shows that the accuracy of the method is good.
Embodiment 2
1) weigh flos carthami powder (crossing 60 mesh sieves) 1.0000g, be placed in the conical flask with cover of 100ml brown, add
15ml methanol, weighed weight, supersound process (supersonic frequency 40khz, power ratio 91%, 25 DEG C of ultrasonic temperature) 10min, after letting cool
Weigh again, supply the weight of less loss with methanol, shake up, filter, obtain Flos Carthami need testing solution, after 0.45 μm of microporous filter membrane
To be measured afterwards；
2) use agilent hplc determination, chromatographic condition: analytical column is agilent c18Chromatographic column (250mm ×
4.6mm, 5 μm), mobile phase is 47% methanol/water isocratic elution, and elution flow rate is 1.0ml/min, 30 DEG C of column temperature, Detection wavelength
For 270nm, 280nm, 290nm, 300nm, 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 below equation respectively and calculate its content；Y=40317x-
51.630, y=35750x-29.313, y=38854x-50.026, y=48600x-67.042；Result display Flos Carthami Central Asia essence
Aminated compoundss 1 are n1, n5, and the content of n10- (z)-tri-p-coumaroylspermidine is 0.0165%, spermidine class
Compound 2 is n1- (e)-n5, and the content of n10- (z)-tri-p-coumaroylspermidine is 0.0330%, spermidine class
Compound 3 is the content of n1 (e)-n5- (z)-n10- (e)-tri-p-coumaroylspermidine is 0.0405%, sub- essence
Aminated compoundss 4 are n1, n5, and the content of n10- (e)-tri-p-coumaroylspermidine is 0.1140%.
Claims (1)
1. in a kind of Rapid Simultaneous Determination Flos Carthami the method for four kinds of spermidine component contents it is characterised in that step includes:
1) weigh flos carthami powder 1.0000g, be placed in the conical flask with cover of 100ml brown, add 15ml methanol, weighed heavy
Amount, supersound process 10min, weigh again after letting cool, supply the weight of less loss with methanol, shake up, filter, obtain Flos Carthami test sample
Solution, to be measured after 0.45 μm of microporous filter membrane；
2) use agilent hplc determination, chromatographic condition: analytical column is agilent c18Chromatographic column, its specification is 250mm
× 4.6mm, 5 μm, mobile phase is methanol/water=47/53 eluting, and elution flow rate is 1.0ml/min, 30 DEG C of column temperature, four kinds of spermidines
Composition (1), (2), (3), the Detection wavelength of (4) correspond to 270nm, 280nm, 290nm, 300nm respectively, and sampling volume is 10 μ
l；
3) peak area corresponding to four kinds of spermidine compositions is brought into below equation respectively and calculate its content；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).
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| 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 |
| CN110794060B (en) * | 2019-11-12 | 2022-11-18 | 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 |
| 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 |
| CN114324633B (en) * | 2021-12-14 | 2022-09-13 | 南昌大学 | Method for identifying authenticity of sapium sebiferum honey |
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| 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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| 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》.2013,第938卷(第75期), * |
| 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;袁茂叶 等;《山西医科大学学报》;20150531;第46卷(第5期);第1.2.3节 * |
| 红花的化学成分及其药理活性研究进展;扈晓佳 等;《药学实践杂志》;20130525;第31卷(第3期);第1.4节和图5 * |
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