CN101419200A - HPLC fingerprint identification method for origin ginseng protection - Google Patents
HPLC fingerprint identification method for origin ginseng protection Download PDFInfo
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- CN101419200A CN101419200A CNA2008100797916A CN200810079791A CN101419200A CN 101419200 A CN101419200 A CN 101419200A CN A2008100797916 A CNA2008100797916 A CN A2008100797916A CN 200810079791 A CN200810079791 A CN 200810079791A CN 101419200 A CN101419200 A CN 101419200A
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Abstract
The invention provides a method for identifying HPLC fingerprints for protecting ginseng of origin, which is characterized by comprising the following steps: A, establishing liquid phase chromatograph fingerprints of the ginseng of origin, namely using the ginseng in the same habitat as a standard, analyzing the ginseng by liquid phase chromatograph and establishing fingerprints; and B, detecting a sample, namely taking a ginseng sample to be detected, detecting the sample under the same conditions as the standard respectively to obtain a spectrogram of the detected sample, and analyzing the fingerprints of the detected sample and the standard by a direct observational method or fingerprints software as qualitative basis. The method has simple pretreatment for fingerprints of the ginseng medicinal material established by adopting the liquid phase chromatograph, multiple peak numbers and better separation degree, therefore, contents of main chemical compositions of the ginseng medicinal material have large difference caused by different growing geographic environments and climates. The HPLC fingerprints of the ginseng can provide reference for distinguishing high-end ginseng health-care products from origin places and non-origin places, and provide reference for establishing fingerprints of other traditional Chinese medicinal materials. The research of methodology shows that all the precision, the stability and the repeatability have better application prospect.
Description
Technical field
The present invention relates to a kind of HPLC fingerprint discrimination method that is used for origin ginseng protection, belong to the liquid-phase chromatographic analysis technical field.
Background technology
Because nutritive value and pharmacological action that genseng is abundant, the ginseng crude drug receives much concern always.A lot of high-end made health food and health medicines of ginseng crude drug's extract have appearred in market abroad.The ginseng crude drug also becomes one of autonomic drug preparation the most salable on the American-European natural drug market.And the genseng in the different places of production, because condition effect such as amblent air temperature water and soil, the composition that it contains is not quite similar thereby has also just caused the difference of clinical efficacy.Therefore, a lot of illegal retailers adulterate with some artificial gensengs, mix the spurious with the genuine, and have greatly jeopardized consumer's physical and mental health.Ginseng crude drug's preparation of many fake and forged commodity also is flooded with high-end health-product market.This original producton location for the ginseng crude drug is differentiated and has just been proposed certain requirement.
At present, there has been certain progress in China for the research of ginseng crude drug's finger-print.Some worker has summed up some actual working experiences, is applied to ginseng crude drug's scientific research.At present, it is simple that the conventional component of high-efficient liquid phase chromatogram technique measuring genseng has moving phase, precision height, good separating effect, characteristics such as accuracy height.Gaoyang, Yuan Yongyue etc. once utilized high performance liquid chromatography to indicate 18 total peaks in ginseng crude drug's finger-print and measured its similarity.Zhang Xuexiang etc. utilize the HPLC method to measure the content that monomer such as multiple saponin(e in 3 kinds of red ginsengs is produced in Jilin.Zhang Xiaoyong etc. also adopt the HPLC method that monomer saponin Rb1 and Re among Jilin Fusong County product gen-seng haulms powder and the Radix Ginseng extractum have been carried out separation determination respectively.The method is suitable for the content analysis of ginseng saponins.Shi Wei etc. use high performance liquid chromatography-evaporative light-scattering detection system and microwave abstracting auxiliary extraction technology, take the method for gradient elution to study the extraction and the separation determination of ginsenoside in the ginseng.Also have some scientific research personnel to utilize high performance liquid chromatography that the composition in American Ginseng, gen-seng and the medicine materical crude slice has been carried out separation determination simultaneously.But the research of the domestic feature identification factor for high-end health products genseng also is in developing stage.
Summary of the invention
The object of the present invention is to provide a kind of not examined person's subjectivity influence short, the detection method of utilizing the HPLC method to set up the genseng finger-print and differentiate original producton location and the high-end health products genseng in non-original producton location.
Technical scheme of the present invention is achieved in that this HPLC fingerprint discrimination method that is used for origin ginseng protection, comprises the steps:
A, set up the liquid-phase chromatograph finger print atlas of origin ginseng
Genseng with same original producton location is standard items, sets up its finger-print by liquid-phase chromatographic analysis;
B, test sample
Get samples of Ginseng to be detected and use the condition identical to detect respectively, draw the spectrogram of test sample with standard items, with both finger-print with direct observational method or finger-print software analysis as qualitative foundation.
The described HPLC fingerprint discrimination method that is used for origin ginseng protection specifically comprises the steps:
The preparation of A, reference substance solution
It is an amount of accurately to take by weighing ginsenoside Re's reference substance, adds the reference substance solution that dissolve with methanol is made 1.00mg/mL.
The preparation of B, standard solution
Place 60 ℃ of drying boxes to heat 4 hours to constant weight the genseng standard items in the same place of production, crushing screening is collected the following powder sample of 80 orders;
Accurately weighing 2.0g ginseng pulverate sample adds the liquid-solid extraction apparatus of temperature automatically controlled solvent refluxing, adds 100mL methanol extraction 5h.The extract rotary evaporation reclaims solvent, and concentrate is poured in the 10mL volumetric flask;
With washed with methanol rotary evaporator inwall, each 2ml cleans 3 times altogether, and cleaning fluid is also poured in the volumetric flask and is diluted to scale, and 0.45 μ m filter membrane filters, and gets subsequent filtrate as standard solution;
C, finger-print are set up and are analyzed
Liquid phase chromatogram condition:
Chromatographic column: Inertsil ODS-3,250mm * 4.6mm, 5 μ m;
Detect wavelength: 203nm;
Moving phase: acetonitrile-0.05% phosphate aqueous solution gradient elution;
Flow velocity: 1mL/min;
Elution program: 0～7min, acetonitrile 0%, 7～10min, acetonitrile fade to 10%, 10～30min, acetonitrile fades to 30%, 30～40min, acetonitrile fade to 35%, 40～45min, acetonitrile fade to that 50%, 45～60min, acetonitrile fade to 70%, 60～90min, acetonitrile fades to 90%;
Sample size: 20 μ L;
Column temperature: 30 ℃;
Accurate absorption standard solution and reference substance solution are an amount of, inject liquid chromatograph, the chromatogram of record 90min, according to the correlation parameter that the finger-print of many batch samples provides, all components all goes out the peak at 90min, choose degree of separation and peak shape all preferably 15min～90min as the standard items finger-print, determine that wherein 11 peaks are the common characteristic peak, wherein, add supreme people's court according to retention time and peak height and determine that No. 3 peaks are the ginsenoside Re, with it as interior reference peak;
D, get commercially available samples of Ginseng and use the condition identical to handle and measure respectively with standard items, draw the liquid-phase chromatograph finger print atlas of test sample, both finger-prints are compared or import and analyze as qualitative foundation with chromatographic fingerprints of Chinese materia medica similarity evaluation system, if both similarities can be thought non-identical product less than 90%, similarity is promptly thought identical product greater than 90%.Methodological investigation
(1) mensuration of method precision
Get same Changbai Mountain garden ginsent need testing solution, by above-mentioned chromatographic condition, continuous sample introduction 5 times calculates relative retention time and the relative peak area value of each total peak with respect to interior reference peak.
The test of table 1 method precision
The result shows that the RSD of relative peak area is between 0.20%～1.6%, and the RSD of relative retention time shows that method precision is good between 0.05%～0.27%.
(2) the reproducible mensuration of method
Get five parts in Changbai Mountain, same place of production garden ginsent sample, prepare need testing solution as stated above, by above-mentioned chromatographic condition, sample introduction calculates relative retention time and the relative peak area value of each total peak with respect to interior reference peak respectively.
The reproducible test result of table 2 method
The result shows that the RSD of relative peak area is between 0.20%～2.1%, and the RSD of relative retention time is between 0.03%～0.16%, and the illustration method reappearance is good.
(3) mensuration of sample stability
Get same Changbai Mountain garden ginsent need testing solution, by above-mentioned chromatographic condition, 0,4,12,24, the 48h sample introduction calculates the relative retention time and relative peak face amount of each total peak with respect to interior reference peak.
The test of table 3 sample stability
The result shows that the RSD of relative peak area is between 0.54%～2.8%, and the RSD of relative retention time is between 0.10%～0.22%, and interpret sample is stable in 48 hours at least.
Ginseng crude drug's finger-print preprocess method that the present invention adopts liquid chromatography to set up is simple, and peak number is many and degree of separation is better, and this explanation ginseng crude drug is owing to the different main chemical compositions content difference that cause of geographical environment of growing and weather are very big.The finger-print of the HPLC of genseng can be difference original producton location and the high-end health products genseng in non-original producton location reference frame is provided, and offers reference for the foundation of other Chinese crude drug finger-prints.Investigation through methodology shows: its precision, stability and reappearance all have application promise in clinical practice.
Description of drawings
Fig. 1 is the HPLC finger-print of Changbai Mountain garden ginsent
Fig. 2 is the HPLC finger-print of the white ginseng in Changbai Mountain
Fig. 3 is the HPLC finger-print of mountain, Changbai Mountain ginseng
Fig. 4 is the HPLC finger-print of Liaoning garden ginsent
Fig. 5 is the HPLC finger-print of Jilin garden ginsent
Fig. 6 purchases the HPLC finger-print of culturing ginseng in Baoding pharmacy
Embodiment
1, sets up the liquid-phase chromatograph finger print atlas of origin ginseng
A, set up the liquid-phase chromatograph finger print atlas of origin ginseng
1, the preparation of reference substance solution
It is an amount of accurately to take by weighing ginsenoside Re's reference substance, adds the reference substance solution that dissolve with methanol is made 1.00mg/mL.
2, the preparation of standard solution
Place 60 ℃ of drying boxes to heat 4 hours to constant weight as standard items commercially available Changbai Mountain garden ginsent, crushing screening is collected the following powder sample of 80 orders;
Accurately weighing 2.0g Changbai Mountain garden ginsent powder sample adds the liquid-solid extraction apparatus of temperature automatically controlled solvent refluxing, adds 100mL methanol extraction 5h, and the extract rotary evaporation reclaims solvent, and concentrate is poured in the 10mL volumetric flask;
With washed with methanol rotary evaporator inwall, each 2ml cleans 3 times altogether, and cleaning fluid is also poured in the volumetric flask and is diluted to scale, and 0.45 μ m filter membrane filters, and gets subsequent filtrate as standard solution;
3, finger-print is set up and is analyzed
Liquid phase chromatogram condition:
Chromatographic column: Inertsil ODS-3,250mm * 4.6mm, 5 μ m;
Detect wavelength: 203nm;
Moving phase: acetonitrile-0.05% phosphate aqueous solution gradient elution;
Flow velocity: 1mL/min;
Elution program: 0～7min, acetonitrile 0%, 7～10min, acetonitrile fade to 10%, 10～30min, acetonitrile fades to 30%, 30～40min, acetonitrile fade to 35%, 40～45min, acetonitrile fade to that 50%, 45～60min, acetonitrile fade to 70%, 60～90min, acetonitrile fades to 90%;
Sample size: 20 μ L;
Column temperature: 30 ℃;
Accurate absorption standard solution and reference substance solution are an amount of, inject liquid chromatograph, the chromatogram of record 90min, according to the correlation parameter that the finger-print of many batch samples provides, all components all goes out the peak at 90min, choose degree of separation and peak shape all preferably 15min～90min as the standard items finger-print, as shown in Figure 1, determine that wherein 11 peaks are the common characteristic peak, wherein, add supreme people's court according to retention time and peak height and determine that No. 3 peaks are the ginsenoside Re, with it as interior reference peak;
4, get again that commercially available Changbai Mountain is joined in vain, mountain, Changbai Mountain ginseng, Liaoning garden ginsent, Jilin garden ginsent and purchase and culture the ginseng sample in Baoding pharmacy and use the condition identical to handle and measure respectively with standard items, draw the liquid-phase chromatograph finger print atlas of test sample such as Fig. 2-shown in Figure 6, both finger-prints are compared or import and analyze as qualitative foundation with chromatographic fingerprints of Chinese materia medica similarity evaluation system, if both similarities can be thought non-identical product less than 90%, similarity is promptly thought identical product greater than 90%.The result shows the individual difference because of sample, and the content at common characteristic peak has very notable difference.
The different places of production of table 4 and kind genseng finger-print detect
By Fig. 1-6 and table 4 as can be known, the relative retention time reappearance fine (RSD is between 0.11%～0.30%) at the ginseng crude drug's of several places of production and kind HPLC finger-print common characteristic peak, but there is very significant difference (RSD is between 26%～102%) in the relative peak area value, and this ginseng crude drug that above six places of production and kind are described is owing to the different main chemical compositions content difference that cause of geographical environment of growing and weather are very big.
The finger-print preprocess method of the HPLC of the genseng that the present invention set up is simple, and peak number is many and degree of separation is better, can be difference original producton location and the high-end health products genseng in non-original producton location reference frame is provided, and offer reference for the foundation of other Chinese crude drug finger-prints.
Listed examples of the present invention is intended to further illustrate this concrete operations of HPLC fingerprint discrimination method and the application direction that is used for origin ginseng protection, and scope of the present invention is not constituted any restriction.
Claims (2)
1, a kind of HPLC fingerprint discrimination method that is used for origin ginseng protection is characterized in that comprising the steps:
A, set up the liquid-phase chromatograph finger print atlas of origin ginseng
Genseng with same original producton location is standard items, sets up its finger-print by liquid-phase chromatographic analysis;
B, test sample
Get samples of Ginseng to be detected and use the condition identical to detect respectively, draw the spectrogram of test sample with standard items, with both finger-print with direct observational method or finger-print software analysis as qualitative foundation.
2, the HPLC fingerprint discrimination method that is used for origin ginseng protection according to claim 1 is characterized in that comprising the steps:
The preparation of A, reference substance solution
It is an amount of accurately to take by weighing ginsenoside Re's reference substance, adds the reference substance solution that dissolve with methanol is made 1.00mg/mL.
The preparation of B, standard solution
Place 60 ℃ of drying boxes to heat 4 hours to constant weight the genseng standard items in the same place of production, crushing screening is collected the following powder sample of 80 orders;
Accurately weighing 2.0g ginseng pulverate sample adds the liquid-solid extraction apparatus of temperature automatically controlled solvent refluxing, adds 100mL methanol extraction 5h, and the extract rotary evaporation reclaims solvent, and concentrate is poured in the 10mL volumetric flask;
With washed with methanol rotary evaporator inwall, each 2ml cleans 3 times altogether, and cleaning fluid is also poured in the volumetric flask and is diluted to scale, and 0.45 μ m filter membrane filters, and gets subsequent filtrate as standard solution;
C, finger-print are set up and are analyzed
Liquid phase chromatogram condition:
Chromatographic column: InertSil ODS-3,250mm * 4.6mm, 5 μ m;
Detect wavelength: 203nm;
Moving phase: acetonitrile-0.05% phosphate aqueous solution gradient elution;
Flow velocity: 1mL/min;
Elution program: 0～7min, acetonitrile 0%, 7～10min, acetonitrile fade to 10%, 10～30min, acetonitrile fades to 30%, 30～40min, acetonitrile fade to 35%, 40～45min, acetonitrile fade to that 50%, 45～60min, acetonitrile fade to 70%, 60～90min, acetonitrile fades to 90%;
Sample size: 20 μ L;
Column temperature: 30 ℃;
Accurate absorption standard solution and reference substance solution are an amount of, inject liquid chromatograph, the chromatogram of record 90min, according to the correlation parameter that the finger-print of many batch samples provides, all components all goes out the peak at 90min, choose degree of separation and peak shape all preferably 15min～90min as the standard items finger-print, determine that wherein 11 peaks are the common characteristic peak, wherein, add supreme people's court according to retention time and peak height and determine that No. 3 peaks are the ginsenoside Re, with it as interior reference peak;
D, get commercially available samples of Ginseng and use the condition identical to handle and measure respectively with standard items, draw the liquid-phase chromatograph finger print atlas of test sample, both finger-prints are compared or import and analyze as qualitative foundation with chromatographic fingerprints of Chinese materia medica similarity evaluation system, if both similarities can be thought non-identical product less than 90%, similarity is promptly thought identical product greater than 90%.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102621264A (en) * | 2012-03-20 | 2012-08-01 | 吉林省中医药科学院 | Method for constructing finger-print chromatogram for ginsenosides-containing crude drugs and preparations |
| CN106546580A (en) * | 2015-09-16 | 2017-03-29 | 北京博肽未名生物技术有限公司 | A kind of polypeptide microarrays chip for differentiating the ginseng place of production |
| CN107737144A (en) * | 2017-10-18 | 2018-02-27 | 中国农业科学院特产研究所 | The extracting method of ginsenoside in a kind of ginseng roots |
| CN109991330A (en) * | 2019-04-04 | 2019-07-09 | 上海上药杏灵科技药业股份有限公司 | A kind of detection method of the finger-print of ginseng under forest |
| CN111220719A (en) * | 2019-11-11 | 2020-06-02 | 云南生物谷药业股份有限公司 | Method for evaluating quality of ginseng medicinal material by using fingerprint spectrum |
| CN113960213A (en) * | 2021-11-09 | 2022-01-21 | 上海海虹实业(集团)巢湖今辰药业有限公司 | Alisma orientale component selection method based on alisma orientale decoction |
Families Citing this family (1)
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| CN107478753A (en) * | 2017-10-18 | 2017-12-15 | 中国农业科学院特产研究所 | A kind of assay method of general ginsenoside |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1670529B (en) * | 2005-03-28 | 2010-04-21 | 中山大学 | Method for constructing Compound Xueshuantong preparation HPLC fingerprint pattern |
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2008
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102621264A (en) * | 2012-03-20 | 2012-08-01 | 吉林省中医药科学院 | Method for constructing finger-print chromatogram for ginsenosides-containing crude drugs and preparations |
| CN106546580A (en) * | 2015-09-16 | 2017-03-29 | 北京博肽未名生物技术有限公司 | A kind of polypeptide microarrays chip for differentiating the ginseng place of production |
| CN106546580B (en) * | 2015-09-16 | 2019-06-28 | 北京博肽未名生物技术有限公司 | It is a kind of for identifying the polypeptide microarrays chip in the ginseng place of production |
| CN107737144A (en) * | 2017-10-18 | 2018-02-27 | 中国农业科学院特产研究所 | The extracting method of ginsenoside in a kind of ginseng roots |
| CN109991330A (en) * | 2019-04-04 | 2019-07-09 | 上海上药杏灵科技药业股份有限公司 | A kind of detection method of the finger-print of ginseng under forest |
| CN111220719A (en) * | 2019-11-11 | 2020-06-02 | 云南生物谷药业股份有限公司 | Method for evaluating quality of ginseng medicinal material by using fingerprint spectrum |
| CN113960213A (en) * | 2021-11-09 | 2022-01-21 | 上海海虹实业(集团)巢湖今辰药业有限公司 | Alisma orientale component selection method based on alisma orientale decoction |
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