CN110133138A - The detection method of stauntonvine Content of Chlorogenic Acid - Google Patents
The detection method of stauntonvine Content of Chlorogenic Acid Download PDFInfo
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- CN110133138A CN110133138A CN201910429406.4A CN201910429406A CN110133138A CN 110133138 A CN110133138 A CN 110133138A CN 201910429406 A CN201910429406 A CN 201910429406A CN 110133138 A CN110133138 A CN 110133138A
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- chlorogenic acid
- stauntonvine
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- substance stock
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
Abstract
This application discloses a kind of detection methods of stauntonvine Content of Chlorogenic Acid of field of biotechnology, including 1) stauntonvine product are taken to be ground into powder, and methanol is added, and sample to be tested is obtained by filtration in ultrasonic extraction 30min；2) 1mg/ml chlorogenic acid reference substance stock solution is configured to methanol after taking chlorogenic acid standard items to weigh；3) high effective liquid chromatography for measuring is used, wherein stationary phase is the chromatographic column of Accucore aQ, and mobile phase A is phosphate buffer solution, and Mobile phase B is acetonitrile, and the volume ratio of A:B is 85:15；Flow velocity is 0.6mL/min；Detection wavelength: 325nm；30 DEG C of column temperature；Sample volume is 2 μ L；4) using the peak area of reference substance stock solution as ordinate, using reference substance Stock concentrations as abscissa, the standard curve of chlorogenic acid is drawn, the content of measuring samples Content of Chlorogenic Acid is calculated according to standard curve.The above method can effectively detect the stauntonvine product under low concentration containing chlorogenic acid, and detection time is short, and accuracy is high.
Description
Technical field
The present invention and field of biotechnology, and in particular to the detection method of stauntonvine Content of Chlorogenic Acid, more specifically one
The detection method of stauntonvine product Content of Chlorogenic Acid under kind low concentration.
Background technique
Stauntonvine is a kind of tropical fruit (tree), belongs to Lardizabalaceae, is tropical evergreen dungarunga) also known as iron pin pear, pawpaw it is prominent etc.,
" fruits of hundred benefits " laudatory title is enjoyed, is a kind of natural, pure green, organic edible fruit that name conforms to reality for the endemic plant of China,
It is the optimum leisure of modern, dietotherapy fruit.The ground such as Guizhou, the Chongqing of China's Mainland are distributed in, wherein with zunyi, guizhou open country
Pawpaw is the most famous, and Zun Yi Zhengan County is also referred to as " township of stauntonvine ".
Stauntonvine is preferred with " sour " person, and pawpaw organic acid complicated composition, type are abundant, such as small molecule fatty acid thick grass
Acid, citric acid etc. have anti-inflammatory, antibacterial, analgesic effect.The phenolic acid class such as protocatechuic acid, chlorogenic acid, caffeic acid, cinnamic acid at
Point there are the multiple biological activities such as significant anti-inflammatory, anti-oxidant, antiviral, cholagogue, the triterpenes such as ursolic acid, oleanolic acid acids at
Point have it is anti-inflammatory it is antibacterial, reduce transaminase, prevent the multiple efficacies such as cirrhosis, calmness, hypoglycemic.
For the product quality of effective monitoring stauntonvine, need to contain a variety of Determination of Organic Acids in pawpaw one by one
It is fixed to measure, such as when measuring chlorogenic acid content, a kind of ultra high efficiency liquid phase as disclosed in Publication No. CN105954392A
The method of chromatographic determination chlorogenic acid content, and specifically disclose, it takes sample 2g to be placed in 20ml brown volumetric flask, adds volume ratio 1:1
Methanol and acetonitrile mixture 15ml be ultrasonically treated and make to dissolve for 10 minutes, take out, be evaporated, add acetonitrile to scale, then accurate measure
Subsequent filtrate 2 sets 10ml brown volumetric flask, adds water to scale, shakes up, and draws 2ml sample liquid addition centrifuge tube and is purified, solution
The measurement of ultra high efficiency liquid phase instrument is injected after filter membrane；Chromatographic condition is as follows: chromatographic column be ACQUITYUPLCBEHC182.1mm ×
50mm, 1.7 μm；The column temperature of chromatographic column: 40 DEG C, mobile phase A: 0.1% formic acid solution；Mobile phase B: methanol, using gradient elution,
Flow velocity is 0.3mL/min；Detection wavelength: 297nm；Analysis time: 7min.When gradient elution, gradient are as follows: 0-1min,
99%A, 1%B；1-1.5min, 90%A, 10%B；1.5-5min, 60%A, 40%B；5-7min, 10%A, 90%B；7.0-
7.1min, 1%A, 99%B.Due to needing gradient elution, take long time, and the content accuracy detected is lower.
For another example " Contemporary Chinese medicine " the 10th phase in 2010, the content of the caulis stauntoniae cbinensis tablet Content of Chlorogenic Acid of 114-115 pages of introduction
Measurement, which employs HPLC method, chromatographic column: Hypersil BDS C18 column (4.6mm × 250mm, 5 μm), mobile phase: acetonitrile:
0.1% phosphoric acid (11: 89), flow velocity 1ml/min, measurement wavelength are 327nm.Column temperature: room temperature.As the result is shown: chlorogenic acid exists
Have good linear relationship (r=1.0000) in 10.21~102.1 μ g/ml concentration ranges, the rate of recovery is 98.90% (RSD=
0.8%), but work as chlorogenic acid and be inaccurate in the measurement effect of 10 μ g/ml concentration this method used below, and the prior art
Stauntonvine product such as caulis stauntoniae cbinensis tablet, stauntonvine powder, concentration are all lower and substantially below lower than 10 μ g/ml, it is therefore desirable to
One is re-established for lower than under 10 μ g/ml concentration, and without gradient elution for stauntonvine product Content of Chlorogenic Acid
Measuring method.
Summary of the invention
The detection method of present invention stauntonvine product Content of Chlorogenic Acid to be provided, especially caulis stauntoniae cbinensis tablet and stauntonvine powder
The detection method of Content of Chlorogenic Acid.
The scheme of the invention is: a kind of detection method of stauntonvine Content of Chlorogenic Acid, comprising the following steps:
1) it takes stauntonvine product to be ground into powder, methanol is added, sample to be tested is obtained by filtration in ultrasonic extraction 30min；
2) 1mg/ml chlorogenic acid reference substance stock solution is configured to methanol after taking chlorogenic acid standard items to weigh；
3) high effective liquid chromatography for measuring is used, wherein stationary phase is the chromatographic column of Accucore aQ, and mobile phase A is phosphorus
Hydrochlorate buffer solution, Mobile phase B are acetonitrile, and the volume ratio of A:B is 85:15；Flow velocity is 0.6mL/min；Detection wavelength: 325nm；
30 DEG C of column temperature；Sample volume is 2 μ L；
4) it using the peak area of reference substance stock solution as ordinate, using the concentration of reference substance stock solution as abscissa, draws green
The standard curve of ortho acid calculates the content of measuring samples Content of Chlorogenic Acid according to standard curve.
Method of the invention, use is equally efficient liquid phase chromatographic analysis, using Thermo Fisher
Accucore aQ (150x3mm, 2.6 μm) chromatographic column, wherein mobile phase A phosphate buffer solution, Mobile phase B are acetonitrile, A:B
=85:15；Flow velocity is 0.6mL/min；Detection wavelength: 325nm；30 DEG C of column temperature；Sample volume be 2 μ L, during detection by
In not using gradient elution, using isocratic elution, convenient for operation, detection method is simple, and has good linear relationship, precision
Degree, stability and repeatability, sample recovery rate is high, and testing result is accurate, reliable.In so avoiding gradient elutions due to needing
It wants multi-solvents to mix, it is accurately low to make to form constantly variation bring, it is difficult to the problems such as reappearing, while detection can also be reduced
Time, in addition it can avoid the regeneration treatment to chromatographic column from operating.Standard curve, calibration curve equation are drawn after detection
For Y=103x9.83X-102X2.94, coefficient R2=0.9999, the results showed that, chlorogenic acid is in 0.5~10 linear model of μ g/mL
Enclose that interior linear relationship is good, the content value measured is more accurate.
Preferably, the concentration of the phosphate buffer is 0.04mol/L, and pH value 2.2, the phosphate is di(2-ethylhexyl)phosphate
Hydrogen potassium.
Preferably, when prepared by potassium phosphate buffer, potassium dihydrogen phosphate 5.44g is first weighed, is added in 1L water and dissolves, shake
It is even, add phosphoric acid tune pH to 2.2, filter, ultrasonic dissolution obtains.
Preferably, the stauntonvine product is caulis stauntoniae cbinensis tablet or stauntonvine powder.
Detailed description of the invention
Fig. 1 is chlorogenic acid reference substance stock solution chromatogram；
Fig. 2 is the chromatogram of 1 powder -1 of stauntonvine sample.
Specific embodiment
It is further described below by specific embodiment:
1 materials and methods
1.1 materials and reagent
Chlorogenic acid standard items are purchased from Aladdin company, potassium dihydrogen phosphate (KH2PO4), phosphoric acid be purchased from Guangzhou brilliance science and technology share
Co., Ltd；Methanol, acetonitrile are purchased from Aladdin company；Used water is ultrapure water, is made by oneself by this laboratory.
1.2 instrument and equipment
Waters H-Class Ultra Performance Liquid Chromatography instrument (is equipped with Waters PDA detector, empower chromatography management system
System)；METTLER TOLEDO a ten thousandth balance；METTLER TOLEDO pH meter；Eppendorf liquid-transfering gun；ELGAChorus ultra-pure-water treatment system；The new sesame ultrasonic washing instrument in Ningbo.
1.3 method
1.3.1 chromatographic condition
Thermo Fisher Accucore aQ (150x3mm, 2.6 μm) chromatographic column, mobile phase A 0.04mol/L,
The phosphate buffer solution of pH2.2, Mobile phase B are acetonitrile, A:B=85:15；Flow velocity is 0.6mL/min；Detection wavelength:
325nm；30 DEG C of column temperature；Sample volume is 2 μ L.
1.3.2 the preparation of mobile phase A
Weigh KH2PO4About 5.44g is added in 1L water and dissolves, shakes up, add phosphoric acid tune pH to 2.2 or so, filters, ultrasound, i.e.,
?.
1.3.3 the preparation of chlorogenic acid reference substance stock solution
It is configured to 1mg/mL chlorogenic acid reference substance stock solution with methanol after chlorogenic acid standard items precise, it is spare.
1.3.4 sample pre-treatments
Caulis stauntoniae cbinensis tablet (mortar is ground into powder), each 100mg of stauntonvine powder are weighed, adds 5ml methanol, ultrasonic extraction 30 minutes, mistake
Filter, both.
2 results and analysis
The foundation of 2.1 chlorogenic acid standard curves
It is appropriate that above-mentioned 1mg/mL chlorogenic acid reference substance stock solution is drawn respectively, and is diluted to 0.5,1,2.5,5,10 μ
The serial chlorogenic acid reference substance stock solution of g/mL.The chlorogenic acid reference substance stock solution 2 of 0.5,1,2.5,5,10 μ g/mL is drawn respectively
μ L, by above-mentioned chromatographic condition sample introduction.Linear regression is carried out to peak area Y with chlorogenic acid reference substance stock solution mass concentration X, is obtained
Standard curve, calibration curve equation Y=103x9.83X-102X2.94, coefficient R2=0.9999, the results showed that, it is green
Ortho acid linear relationship in 0.5~10 μ g/mL range of linearity is good.Chlorogenic acid reference substance stock solution chromatogram is as shown in Figure 1, open country
The chromatogram of 1 powder -1 of pawpaw sample detected as shown in Fig. 2, complex chart 1 and Fig. 2 can be seen that the time in 1.5mins
Substance be exactly chlorogenic acid, calculated by the way that peak area to be updated in calibration curve equation, obtain the concentration value of chlorogenic acid.
2.2 caulis stauntoniae cbinensis tablets, stauntonvine powder Content of Chlorogenic Acid measurement result
Sample is handled by above-mentioned sample-pretreating method, and using above-mentioned chromatographic condition to treated sample into
Sample analysis, measures caulis stauntoniae cbinensis tablet, stauntonvine powder Content of Chlorogenic Acid, and each sample is measured in parallel 2 times, as a result see the table below.
Table 1 is the content table of stauntonvine powder, caulis stauntoniae cbinensis tablet Content of Chlorogenic Acid
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, although referring to aforementioned reality
Applying example, invention is explained in detail, for those skilled in the art, still can be to aforementioned each implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features.It is all in essence of the invention
Within mind and principle, any modification, equivalent substitution, improvement and etc. done be should all be included in the protection scope of the present invention.
Claims (4)
1. the detection method of stauntonvine Content of Chlorogenic Acid, it is characterised in that: the following steps are included:
1) it takes stauntonvine product to be ground into powder, methanol is added, sample to be tested is obtained by filtration in ultrasonic extraction 30min；
2) 1mg/ml chlorogenic acid reference substance stock solution is configured to methanol after taking chlorogenic acid standard items to weigh；
3) high effective liquid chromatography for measuring is used, wherein stationary phase is the chromatographic column of Accucore aQ, and mobile phase A is phosphate
Buffer solution, Mobile phase B are acetonitrile, and the volume ratio of A:B is 85:15；Flow velocity is 0.6mL/min；Detection wavelength: 325nm；Column temperature
30℃；Sample volume is 2 μ L；
4) using the peak area of reference substance stock solution as ordinate, using the concentration of reference substance stock solution as abscissa, chlorogenic acid is drawn
Standard curve, according to standard curve calculate measuring samples Content of Chlorogenic Acid content.
2. the detection method of stauntonvine Content of Chlorogenic Acid according to claim 1, it is characterised in that: the phosphate is slow
The concentration of fliud flushing is 0.04mol/L, and pH value 2.2, the phosphate is potassium dihydrogen phosphate.
3. the detection method of stauntonvine Content of Chlorogenic Acid according to claim 2, it is characterised in that: potassium dihydrogen phosphate is slow
When prepared by fliud flushing, potassium dihydrogen phosphate 5.44g is first weighed, is added in 1L water and dissolves, shake up, add phosphoric acid tune pH to 2.2, is filtered, is surpassed
Sound dissolution obtains.
4. the detection method of stauntonvine Content of Chlorogenic Acid according to claim 3, it is characterised in that: the stauntonvine produces
Product are caulis stauntoniae cbinensis tablet or stauntonvine powder.
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| CN201910429406.4A CN110133138A (en) | 2019-05-22 | 2019-05-22 | The detection method of stauntonvine Content of Chlorogenic Acid |
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| CN201910429406.4A CN110133138A (en) | 2019-05-22 | 2019-05-22 | The detection method of stauntonvine Content of Chlorogenic Acid |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175790A (en) * | 2011-01-07 | 2011-09-07 | 山东省农业科学院中心实验室 | HPLC (high performance liquid chromatography) method for synchronously detecting five polyphenols in apples and distinguishing varieties |
| CN102608250A (en) * | 2012-03-13 | 2012-07-25 | 承德燕峰药业有限责任公司 | Rapid detection method for Jinfangganmao granules |
| CN103308615A (en) * | 2013-05-30 | 2013-09-18 | 哈药集团中药二厂 | Method for content determination of multiple components in traditional Chinese medicinal preparation Shuanghuanglian for injection |
| CN103592390A (en) * | 2013-11-22 | 2014-02-19 | 中国人民解放军总医院第一附属医院 | Quality detection method of chlorogenic acid in Yinmu liver-relieving granules |
| CN104880528A (en) * | 2015-06-03 | 2015-09-02 | 新乡医学院 | Construction method for finger print of Shuanghuanglian oral liquid |
| CN105954392A (en) * | 2016-04-25 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Method for detecting content of chlorogenic acid with ultra-high performance liquid chromatography |
-
2019
- 2019-05-22 CN CN201910429406.4A patent/CN110133138A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175790A (en) * | 2011-01-07 | 2011-09-07 | 山东省农业科学院中心实验室 | HPLC (high performance liquid chromatography) method for synchronously detecting five polyphenols in apples and distinguishing varieties |
| CN102608250A (en) * | 2012-03-13 | 2012-07-25 | 承德燕峰药业有限责任公司 | Rapid detection method for Jinfangganmao granules |
| CN103308615A (en) * | 2013-05-30 | 2013-09-18 | 哈药集团中药二厂 | Method for content determination of multiple components in traditional Chinese medicinal preparation Shuanghuanglian for injection |
| CN103592390A (en) * | 2013-11-22 | 2014-02-19 | 中国人民解放军总医院第一附属医院 | Quality detection method of chlorogenic acid in Yinmu liver-relieving granules |
| CN104880528A (en) * | 2015-06-03 | 2015-09-02 | 新乡医学院 | Construction method for finger print of Shuanghuanglian oral liquid |
| CN105954392A (en) * | 2016-04-25 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Method for detecting content of chlorogenic acid with ultra-high performance liquid chromatography |
Non-Patent Citations (4)
| Title |
|---|
| GULSEN KENDIR 等: "Ultra-Performance Liquid Chromatography for the Simultaneous Quantification of Rutin and Chlorogenic Acid in Leaves of Ribes L. Species by Conventional and Chemometric Calibration Approaches", 《JOURNAL OF CHROMATOGRAPHIC SCIENCE》 * |
| 余良忠 等: "高效液相色谱法测定野木瓜中绿原酸的含量", 《医药导报》 * |
| 蔡荣华 等: "超高效液相色谱—紫外检测法同时测定绿咖啡豆提取物中的绿原酸和咖啡因含量", 《中国食品添加剂》 * |
| 韩煜 等: "不同产地木瓜中绿原酸、原儿茶酸和总酚的含量测定", 《中国药房》 * |
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Application publication date: 20190816 |