CN101441201B - Method for detecting contents of puerarin and rhodiola rosea glycosides in Paotianhongyangshejiu - Google Patents
Method for detecting contents of puerarin and rhodiola rosea glycosides in Paotianhongyangshejiu Download PDFInfo
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- CN101441201B CN101441201B CN2007101708347A CN200710170834A CN101441201B CN 101441201 B CN101441201 B CN 101441201B CN 2007101708347 A CN2007101708347 A CN 2007101708347A CN 200710170834 A CN200710170834 A CN 200710170834A CN 101441201 B CN101441201 B CN 101441201B
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Abstract
The invention provides a method for detecting the content of puerarin and gadol glycoside in 'paotianhong' healthcare wine. The content of the kudzuvine root in 1g of wine is not less than 3.5 mu g in terms of puerarin, and the content of the gadol in 1g of wine is not less than 21 mu g in terms of gadol glycoside. According to the mass standard provided by the invention, the inherent quality of the 'paotianhong' healthcare wine can be well controlled, and the product quality is stable, thereby the healthcare products can better exert their functions.
Description
Technical field:
The present invention relates to Chinese medicine, health products, be specifically related to the detection method of content of Puerarin and rhodioside in the big gun Paotianhongyangshejiu.
Background technology
The red health wine in big gun sky is by radix pseudostellariae, rhodiola root, turtle shell, the bark of eucommia, the root of Dahurain angelica, cassia seed, the fruit of Chinese wolfberry, lotus seeds, the root of kudzu vine, the sophora bud, sealwort ten is the health products of Chinese medicine composition simply, have to improve the health, and improve the function of immunity, are applied to hypoimmunity and fatiguability crowd usually.In the proper mass standard of big gun Paotianhongyangshejiu, do not set up the assay item of reflection composition inherent quality, therefore, can not scientifically control production run, thereby the difficult aborning quality of guaranteeing product, the function that has influenced it is used, limit market efficiency, also the consumer has been brought adverse factors.
Summary of the invention
Technical matters to be solved by this invention is to overcome above-mentioned weak point, and research and design can be controlled big gun sky red health wine method for quality.
At ten simply in the Chinese medicine of the red health wine in big gun sky, the root of kudzu vine and rhodiola root are main efficacy components.The root of kudzu vine has relieving muscles to expel heat, promotes the production of body fluid promoting eruption, the effect of living positive antidiarrheal.Rhodiola root has qi and activate blood circulation, the effect of promoting blood circulation and relievining asthma.Therefore, the present invention selects the root of kudzu vine and rhodiola root two flavor medicines to carry out the foundation of quality standard, so that the check of big gun sky red health vinosity amount to be provided.
1, the development test of puerarin content detection method
Contain the survey method with reference to pharmacopeia one one " root of kudzu vine ", again Puerarin in the big gun Paotianhongyangshejiu root of kudzu vine is set up high performance liquid chromatogram and contain the survey method.
1.1 instrument and reagent
Agilent 1100 high performance liquid chromatographs, Puerarin reference substance (110752-200209, Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides, for assay); Sample: big gun Paotianhongyangshejiu (Shanghai Leiyun Pharmaceutical Industry Co., Ltd. provides, lot number 060801,060802,060803); Methyl alcohol is chromatographically pure, and it is pure that reagent is analysis.
1.2, the test of chromatographic condition and system suitability
1.2.1 detection wavelength
Puerarin has maximum absorption wavelength at 250nm, 306nm place.In 250nm maximum absorption wavelength peak area maximum.Select 250nm as detecting wavelength in conjunction with documents and materials.
1.2.2 chromatographic condition and specificity test
Chromatographic column: Agilent ZORBAX XDB-C18 (4.6 * 150mm; 5.0 μ m), 25 ℃ of column temperatures, moving phase is with methyl alcohol: water (25: 75), flow velocity are 1ml/min.
The preparation of reference substance: it is an amount of to get the Puerarin reference substance, and accurate the title decides, and adds methyl alcohol and makes the reference substance solution that every 1ml contains 30 μ g.
The preparation of test sample: get this product, the accurate 50ml that draws, water bath method is used 40% dissolve with ethanol solution, is transferred to the 10ml measuring bottle, adds 40% ethanolic solution to scale, and 0.45 μ m filter membrane filters, and gets subsequent filtrate, promptly.
Lack the blank sample preparation of the root of kudzu vine: get the blank sample that lacks the root of kudzu vine, the preparation method prepares with method by test sample, promptly gets blank product solution.
Tool and the consistent chromatographic peak of Puerarin reference substance retention time in the test sample chromatogram, and separate well with the chromatographic peak of other composition.Blank sample does not have chromatographic peak at Puerarin retention time place, does not have interference (seeing accompanying drawing one).
1.3 linear relationship is investigated
Get the Puerarin barbaloin reference substance solution difference sample introduction of variable concentrations, with the peak area is ordinate Y, sample size is horizontal ordinate X, do return typical curve, the result shows that Puerarin is in sample size 0.0490～0.9800 μ g scope, sample size and peak area are good linear relationship, the results are shown in following table.
Table 1 Puerarin typical curve
| Sample size (μ g) | 0.0490 | 0.0980 | 0.2940 | 0.4900 | 0.9800 |
| Peak area | 192.2589 | 389.4302 | 1151.7426 | 1920.8788 | 3799.8210 |
| Regression equation | y＝3873.5x+10.389 r＝0.9999 | ||||
1.4 reference substance solution parallel experiment
Precision is measured 0.098mg/ml Puerarin reference substance solution 3 μ l, injects liquid chromatograph, the sample introduction analysis, and replication 6 times, the record peak area the results are shown in following table.
Table 2 precision test (n=6)
| Peak area | 1 | 2 | 3 | 4 | 5 | 6 | RSD |
| Puerarin | 1153.28 36 | 1150.20 17 | 1151.16 94 | 1151.57 17 | 1151.97 49 | 1156.15 86 | 0.20％ |
1.5 stability test
Get same need testing solution and preparing back 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours injection liquid chromatographs respectively, the sample introduction measurement result, its peak area there is no significant change, illustrates that need testing solution is more stable, the results are shown in following table.
Table 3 stability test
1.6 replica test
Get same batch sample (lot number: 060801), inject liquid chromatograph, measure, the results are shown in Table 5 according to 6 parts of need testing solutions of test sample preparation method preparation.
Table 4 replica test
| Puerarin content (mg/g) | |
| 1 | 0.004887 |
| 2 | 0.004687 |
| 3 | 0.004837 |
| 4 | 0.004838 |
| 5 | 0.004783 |
| 6 | 0.004893 |
| On average | 0.004821 |
| RSD | 1.60％(n＝6) |
The result shows this method repeatability better
1.7 accuracy test
Get 9 parts in the sample (lot number is 060801) of known content, each accurate 25ml that draws adds a certain amount of reference substance, press the preparation of test sample preparation method, injects liquid chromatograph, and mensuration the results are shown in Table 6.
Table 5 Puerarin accuracy test
| Tested number | Sample weighting amount | The amount of recording | Sample size | Addition | The recovery | Mean value (%) | RSD |
| 1 | 25.0000 | 0.2265 | 0.1205 | 0.1078 | 98.33％ | 97.71％ | 0.98％ |
| 2 | 25.0000 | 0.2242 | 0.1205 | 0.1078 | 96.21％ | ||
| 3 | 25.0000 | 0.2254 | 0.1205 | 0.1078 | 97.27％ | ||
| 4 | 25.0000 | 0.2540 | 0.1205 | 0.1372 | 97.29％ | ||
| 5 | 25.0000 | 0.2531 | 0.1205 | 0.1372 | 96.60％ | ||
| 6 | 25.0000 | 0.2553 | 0.1205 | 0.1372 | 98.20％ | ||
| 7 | 25.0000 | 0.2750 | 0.1205 | 0.1568 | 98.55％ | ||
| 8 | 25.0000 | 0.2761 | 0.1205 | 0.1568 | 99.20％ | ||
| 9 | 25.0000 | 0.2737 | 0.1205 | 0.1568 | 97.70％ |
The result shows that this method recovery is good, extracts more complete.
1.8 sample determination sample thief (1) 060801; (2) 060802; (3) 060803 press the text method measures content, the results are shown in following table.
Table 6 sample determination result
The regulation of content limit is to survey 75% of puerarin content minimum data, and tentative this product contains Puerarin (C
21H
22O
9) meter, every 1ml must not be less than 3.5 μ g.
2, rhodiola root content detection test
Contain the survey method with reference to pharmacopeia one one " rhodiola root ", again rhodioside in this product rhodiola root is set up high performance liquid chromatogram and contain the survey method.
2.1 instrument and reagent
Agilent 1100 high performance liquid chromatographs, rhodioside reference substance (110818-200404, Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides, for assay); Sample: big gun Paotianhongyangshejiu (Shanghai Leiyun Pharmaceutical Industry Co., Ltd. provides, lot number 060801,060802,060803); Methyl alcohol is chromatographically pure, and it is pure that reagent is analysis.
2.2 chromatographic condition and system suitability test
2.2.1 detection wavelength
Rhodioside has maximum absorption wavelength at the 275nm place.Select 275nm as detecting wavelength in conjunction with documents and materials.
2.2.2 chromatographic condition and specificity test
Chromatographic column: Agilent Extend-C18 (4.6 * 150mm; 5.0 μ m), 25 ℃ of column temperatures, moving phase is with methyl alcohol: water (15: 85), flow velocity are 1ml/min.
The preparation of reference substance: it is an amount of to get the rhodioside reference substance, and accurate the title decides, and adds methyl alcohol and makes the reference substance solution that every 1ml contains 0.2mg.
The preparation of test sample: get this product, the accurate 50ml that draws, water bath method is used 40% dissolve with ethanol solution, is transferred to the 10ml measuring bottle, adds 40% ethanolic solution to scale, and 0.45 μ m filter membrane filters, and gets subsequent filtrate, promptly.
Lack the blank sample preparation of rhodiola root: get the blank sample that lacks the root of kudzu vine, the preparation method prepares with method by test sample, promptly gets blank product solution.
Tool and the consistent chromatographic peak of rhodioside reference substance retention time in the test sample chromatogram, and separate well with the chromatographic peak of other composition.Blank sample does not have chromatographic peak at rhodioside retention time place, does not have interference (seeing accompanying drawing two).
2.3 linear relationship is investigated
Get the rhodioside reference substance solution difference sample introduction of variable concentrations, with the peak area is ordinate Y, sample size is horizontal ordinate X, do return typical curve, the result shows that rhodioside is in sample size 0.3920～9.8000 μ g scope, sample size and peak area are good linear relationship, the results are shown in following table.
Table 1 Puerarin typical curve
| Sample size (μ g) | 0.3920 | 0.9800 | 1.9600 | 4.9000 | 9.8000 |
| Peak area | 111.8150 | 271.5869 | 519.1295 | 1338.6754 | 2658.8313 |
| Regression equation | y＝271.31x+1.5428 r＝0.9999 | ||||
2.4 reference substance solution parallel experiment
Precision is measured 0.99mg/ml rhodioside reference substance solution 2 μ l, injects liquid chromatograph, the sample introduction analysis, and replication 6 times, the record peak area the results are shown in following table.
Table 2 precision test (n=6)
| Peak area | 1 | 2 | 3 | 4 | 5 | 6 | RSD |
| Rhodioside | 520.537 2 | 517.721 7 | 525.739 0 | 526.736 0 | 520.380 4 | 532.847 6 | 1.1 2％ |
2.5 stability test
Get same need testing solution and preparing back 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours injection liquid chromatographs respectively, the sample introduction measurement result, its peak area there is no significant change, illustrates that need testing solution is more stable, the results are shown in following table.
Table 3 stability test
2.6 replica test
Get same batch sample (lot number: 060801), inject liquid chromatograph, measure, the results are shown in Table 5 according to 6 parts of need testing solutions of test sample preparation method preparation.
Table 4 replica test
| Rhodioside content (mg/g) | |
| 1 | 0.027251 |
| 2 | 0.026646 |
| 3 | 0.026576 |
| 4 | 0.027686 |
| 5 | 0.027941 |
| 6 | 0.027108 |
| On average | 0.027479 |
| RSD | 2.01％(n＝6) |
The result shows this method repeatability better
2.7 accuracy test
Get 9 parts in the sample (lot number is 060801) of known content, each accurate 25ml that draws adds a certain amount of reference substance, press the preparation of test sample preparation method, injects liquid chromatograph, and mensuration the results are shown in Table 6.
Table 5 Puerarin accuracy test
| Tested number | Sample weighting amount | The amount of recording | Sample size | Addition | The recovery | Mean value | RSD |
| 1 | ?25.0000 | 1.6291 | 0.6870 | 0.9900 | 95.16％ | 96.93％ | 1.35％ |
| 2 | ?25.0000 | 1.6539 | 0.6870 | 0.9900 | 97.67％ | ||
| 3 | ?25.0000 | 1.6571 | 0.6870 | 0.9900 | 97.99％ | ||
| 4 | ?25.0000 | 1.9477 | 0.6870 | 1.2870 | 97.96％ | ||
| 5 | ?25.0000 | 1.9633 | 0.6870 | 1.2870 | 99.17％ | ||
| 6 | ?25.0000 | 1.9250 | 0.6870 | 1.2870 | 96.20％ | ||
| 7 | ?25.0000 | 2.1158 | 0.6870 | 1.4850 | 96.21％ | ||
| 8 | ?25.0000 | 2.1114 | 0.6870 | 1.4850 | 95.92％ | ||
| 9 | ?25.0000 | 2.1135 | 0.6870 | 1.4850 | 96.06％ |
The result shows that this method recovery is good, extracts more complete.
2.8 sample determination sample thief (1) 060801; (2) 060802; (3) 060803 press the text method measures content, the results are shown in following table.
Table 6 sample determination result
The regulation of content limit is to survey 75% of rhodioside content minimum data, and tentative this product contains rhodioside (C
14H
20O
7) meter, every 1ml must not be less than 21 μ g.
Based on the research of above-mentioned test, the present invention has designed the detection method of content of Puerarin and rhodioside in the big gun Paotianhongyangshejiu.
The invention provides the detection method of puerarin content in a kind of big gun sky red wine, this method comprises the following steps:
(1) [assay] Puerarin is measured according to high performance liquid chromatography (Chinese Pharmacopoeia version appendix in 2005 VI D).
Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica, are moving phase with methanol-water (25: 75).The detection wavelength is 250nm.Column temperature is 25 ℃.Theoretical cam curve is calculated by puerarin peak should be not less than 2000.
(2) it is an amount of that the Puerarin reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds 40% ethanol and makes the Puerarin reference substance solution that every 1ml contains 30 μ g.
(3) this product is got in the preparation of need testing solution, the accurate 50ml that draws, and water bath method is used 40% dissolve with ethanol solution, is transferred to the 10ml measuring bottle, adds 40% ethanolic solution to scale, and 0.45 μ m filter membrane filters, and gets subsequent filtrate, promptly.
(4) accurate respectively reference substance and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.
This product contains the root of kudzu vine in Puerarin (C21H20O9), and per 1 milliliter must not be less than 3.5 μ g.
Another purpose of the present invention has provided the detection method of content of rhodioside in the red health wine in a kind of big gun sky, and this method comprises the following steps:
(1) assay: rhodioside is measured according to high performance liquid chromatography (Chinese Pharmacopoeia version appendix in 2005 VI D).
Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica, are moving phase with methanol-water (15: 85).The detection wavelength is 275nm.Column temperature is 25 ℃.Theoretical cam curve is calculated by the rhodioside peak should be not less than 2000.
It is an amount of that the rhodioside reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds 40% ethanol and makes the rhodioside reference substance solution that every 1ml contains 0.2mg.
This product is got in the preparation of need testing solution, the accurate 50ml that draws, and water bath method is used 40% dissolve with ethanol solution, is transferred to the 10ml measuring bottle, adds 40% ethanolic solution to scale, and 0.45 μ m filter membrane filters, and gets subsequent filtrate, promptly.
Accurate respectively reference substance and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.
This product contains rhodiola root with rhodioside (C
14H
20O
7) meter, per 1 milliliter must not be less than 21 μ g.
According to quality standard provided by the present invention, can well control the inherent quality of " big gun Paotianhongyangshejiu ", three batches of pilot scale setting-outs all meet " big gun Paotianhongyangshejiu " quality standard requirement, constant product quality, can bring into play the health products function better, for human health keeps fit to bring glad tidings.
According to the WS3-B-0203-90 inspection provision, to the sampling detection of this product three batch samples, high spot reviews: proterties, amount of alcohol, quantity of methyl alcohol, content, inspection and health examination project, the result is all up to specification.
Experimental data
(1) proterties
| Lot number | 050901 | 050902 | 050903 |
| Proterties | Qualified | Qualified | Qualified |
(2) amount of alcohol
| Lot number | 050901 | 050902 | 050903 |
| Amount of alcohol g/L | 35 | 39 | 40 |
(3) quantity of methyl alcohol
| Lot number | 050901 | 050902 | 050903 |
| Quantity of methyl alcohol g/L | 0.02 | 0.02 | 0.02 |
(4) content uniformity
| Lot number | 050901 | 050902 | 050903 |
| Content uniformity | Qualified | Qualified | Qualified |
(5) assay
| Lot number | 050901 | 050902 | 050903 |
| Puerarin content (μ g/ml) rhodiola root content (μ g/ml) | 4.8 28 | 4.8 28 | 4.8 28 |
(6) microorganism checking
| Lot number | 050901 | 050902 | 050903 |
| Bacterium (cfu/m) mould, saccharomycete (cfu/m) Escherichia coli, mite | ＜10＜10 do not detect | ＜10＜10 do not detect | ＜10＜10 do not detect |
Embodiment:
Method:
[assay] Puerarin is measured according to high performance liquid chromatography (Chinese Pharmacopoeia version appendix in 2000 VI D).
Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica, are moving phase with methanol-water (25: 75).The detection wavelength is 250nm.Column temperature is 25 ℃.Theoretical cam curve is calculated by puerarin peak should be not less than 2000.
It is an amount of that the Puerarin reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds 40% ethanol and makes the Puerarin reference substance solution that every 1ml contains 30 μ g.
This product is got in the preparation of need testing solution, the accurate 50ml that draws, and water bath method is used 40% dissolve with ethanol solution, is transferred to the 10ml measuring bottle, adds 40% ethanolic solution to scale, and 0.45 μ m filter membrane filters, and gets subsequent filtrate, promptly.
Accurate respectively reference substance and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.
This product contains the root of kudzu vine with Puerarin (C
21H
20O
9) meter, per 1 milliliter must not be less than 3.5 μ g.
Rhodioside is measured according to high performance liquid chromatography (Chinese Pharmacopoeia version appendix in 2000 VI D).
Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica, are moving phase with methanol-water (15: 85).The detection wavelength is 275nm.Column temperature is 25 ℃.Theoretical cam curve is calculated by the rhodioside peak should be not less than 2000.
It is an amount of that the rhodioside reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds 40% ethanol and makes the rhodioside reference substance solution that every 1ml contains 0.2mg.
This product is got in the preparation of need testing solution, the accurate 50ml that draws, and water bath method is used 40% dissolve with ethanol solution, is transferred to the 10ml measuring bottle, adds 40% ethanolic solution to scale, and 0.45 μ m filter membrane filters, and gets subsequent filtrate, promptly.
Accurate respectively reference substance and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.
This product contains rhodiola root with rhodioside (C
14H
20O
7) meter, per 1 milliliter must not be less than 21 μ g.
Get many batch samples and carry out assay, data are as follows:
| Lot number | 070801 | 070802 | 070803 | 070901 | 070902 | 070903 | 070904 | 070905 |
| Puerarin (μ g/ml) | 4.8 | 4.7 | 4.7 | 4.7 | 4.7 | 4.7 | 4.8 | 4.7 |
| Rhodioside (μ g/ml) | 27 | 28 | 28 | 28 | 27 | 27 | 28 | 28 |
Claims (2)
1. the detection method of puerarin content in the big gun Paotianhongyangshejiu is characterized in that this method comprises the following steps:
(1) assay: Puerarin
High effective liquid chromatography for measuring:
Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica, are moving phase with methanol-water 25: 75v/v; The detection wavelength is 250nm; Column temperature is 25 ℃; Theoretical cam curve is calculated by puerarin peak should be not less than 2000;
(2) it is an amount of that the Puerarin reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds 40% ethanol and makes the Puerarin reference substance solution that every 1ml contains 30 μ g;
(3) this product is got in the preparation of need testing solution, the accurate 50ml that draws, and water bath method is used 40% dissolve with ethanol solution, is transferred to the 10ml measuring bottle, adds 40% ethanolic solution to scale, and 0.45 μ m filter membrane filters, and gets subsequent filtrate, promptly;
(4) accurate respectively reference substance and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.
2. the detection method of content of rhodioside in the big gun Paotianhongyangshejiu is characterized in that this method comprises the following steps:
(1) assay: rhodioside
High effective liquid chromatography for measuring:
Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica; With methanol-water 15: 85v/v is moving phase; The detection wavelength is 275nm; Column temperature is 25 ℃; Theoretical cam curve is calculated by the rhodioside peak should be not less than 2000;
(2) it is an amount of that the rhodioside reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds 40% ethanol and makes the rhodioside reference substance solution that every 1ml contains 0.2mg;
(3) this product is got in the preparation of need testing solution, the accurate 50ml that draws, and water bath method is used 40% dissolve with ethanol solution, is transferred to the 10ml measuring bottle, adds 40% ethanolic solution to scale, and 0.45 μ m filter membrane filters, and gets subsequent filtrate, promptly;
Accurate respectively reference substance and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101708347A CN101441201B (en) | 2007-11-23 | 2007-11-23 | Method for detecting contents of puerarin and rhodiola rosea glycosides in Paotianhongyangshejiu |
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|---|---|---|---|
| CN2007101708347A CN101441201B (en) | 2007-11-23 | 2007-11-23 | Method for detecting contents of puerarin and rhodiola rosea glycosides in Paotianhongyangshejiu |
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| CN101441201B true CN101441201B (en) | 2011-10-05 |
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| 王锦红 |
| 王锦红;孙传梅;冉瑞模;赵定风;龙正文.高效液相色谱法测定长寿长乐补酒中补骨脂素和异补骨脂素含量.《中国药业》.2005,第15卷(第11期),42. * |
| 胡海云 |
| 胡海英 |
| 胡海英;杨丽嘉;胡海云;李继成.不同产地红景天药材中红景天苷含量的测定.《河南医学研究》.2007,第16卷(第3期),203-205. * |
| 赵定风 |
| 郑楚光.葛酒中葛根素及其挥发性成分分析.《光谱实验室》.2004,第21卷(第2期),396-398. |
| 龙正文.高效液相色谱法测定长寿长乐补酒中补骨脂素和异补骨脂素含量.《中国药业》.2005,第15卷(第11期),42. |
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