CN104251892A - Method for detecting amentoflavone in Herba Selaginellae extract - Google Patents
Method for detecting amentoflavone in Herba Selaginellae extract Download PDFInfo
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- CN104251892A CN104251892A CN201310274461.3A CN201310274461A CN104251892A CN 104251892 A CN104251892 A CN 104251892A CN 201310274461 A CN201310274461 A CN 201310274461A CN 104251892 A CN104251892 A CN 104251892A
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- amentoflavone
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Abstract
The invention provides a method for detecting amentoflavone in Herba Selaginellae extract. The content of amentoflavone is rapidly and accurately detected through a reversed-phase high-performance liquid chromatography gradient elution method. The method has good precision, reappearance and reliability, is advanced and simple, and can safely, effectively and comprehensively control the quality of the Herba Selaginellae extract.
Description
Technical field
The invention belongs to Chinese medicine detection technique field, relate to the detection method of amentoflavone in a kind of spikemoss extract.
Background technology
Selaginella tamariscina is the dry herb of volume encyclopaedia plant Selaginella tamariscina Selaginella tamariscina (Beauv) Spring or Selaginella pulvinata Selaginella pulvinaat (Hook.et Grev.) Maxim.Containing multiple flavone compound in Selaginella tamariscina, comprise sotetsuflavone (sotetsuflavone), amentoflavone (amentoflavone), hinokiflavone (hinokiflavone), isocryptomerin (isocryptomerin), cryptomeria biflavone (cryptomerin) B, apiolin (apigenin) etc., wherein amentoflavone has another name called Amentoflavone, all exist in a variety of Chinese medicine is as Radix Glycyrrhizae, ginkgo leaf, hypericum perforatum, and in Selaginella tamariscina the content of amentoflavone far away higher than other Chinese herbal medicines.Bis-flavonoid has antibiotic property, can promote blood circulation, controls dermatitis, control hair growth and the functions such as conditioning of invigorating blood circulation.
Amentoflavone detects according to the detection method of Selaginella tamariscina in 2010 editions " state-promulgated pharmacopoeia ", with methyl alcohol and 0.1% phosphoric acid solution be mobile phase A, B carries out gradient elution.Actual mechanical process amentoflavone retention time is late, and analysis time, the longer peak symmetry that causes was not ideal enough.Simultaneously in disclosed patent, be all the description about the preparation and application of amentoflavone in Selaginella tamariscina, progress and pharmacologically active etc., also there is no related to the present invention or similar report.
Summary of the invention
The object of the present invention is to provide a kind of method measuring amentoflavone content in spikemoss extract, this method is relatively simple, the precision that tool is good, reappearance and stability, be applicable to the content measuring amentoflavone, can be used for spikemoss extract quality control.
The invention provides the efficient liquid phase detection method of amentoflavone in a kind of spikemoss extract, the method comprises the following steps:
(1) preparation of reference substance solution: it is appropriate that precision takes amentoflavone reference substance, adds proper amount of methanol dissolving and makes reference substance solution.
(2) preparation of need testing solution: take spikemoss extract appropriate, add proper amount of methanol, ultrasonic dissolution 5 ~ 20, lets cool, and to obtain final product.
(3) chromatographic condition and system flexibility: room temperature, relative humidity 25 ~ 40%; Take octadecylsilane chemically bonded silica as filling agent, chromatographic column (4.6mm × 250mm, 5 μm); Take methanol solution as mobile phase A, with 0.1% trifluoroacetic acid (TFA) aqueous solution for Mobile phase B, the regulation according to the form below carries out gradient elution, flow velocity: 0.8 ~ 1.2ml/min, column temperature 25 ~ 40 DEG C, determined wavelength 330 ~ 337nm.
(4) determination method: accurate absorption reference substance solution, each 20 μ l of need testing solution, injection liquid chromatography, record chromatogram.
In above-mentioned steps (1), reference substance solution can be mixed with the solution of 0.5 ~ 1mg/ml, and preferably every 1ml is containing 0.5mg amentoflavone product solution in contrast.
Institute gets spikemoss extract and can be mixed with the solution of every 1ml containing 0.5 ~ 2mg in step (2), and the preferably concentration of 1mg/ml, ultrasonic time is preferably 10 minutes.
Room temperature described in step (3) is preferably 20 DEG C, and relative humidity is preferably 35%; Flow rate of mobile phase scope is 0.8 ~ 1.2ml/min, is preferably 1ml/min; Column temperature is preferably 30 DEG C from 25 ~ 40 DEG C, and determined wavelength is preferably 335nm.
Mobile phase gradient is preferably as Gradient:
This method, by reverse high performance liquid chromatography, measures the content of amentoflavone in spikemoss extract.Show through test, amentoflavone good separating effect, linear relationship, reappearance, precision, stability, the recovery is all better.It is more economical, simple, quick that this method compares additive method, more conveniently carries out quality control to spikemoss extract accurately.
Embodiment
Detecting instrument in following embodiment: Shimadzu high performance liquid chromatograph, comprises LC10ATvp plus binary pump, SPD10Avp plus UV-detector, LC-solution chromatographic work station.Methyl alcohol is chromatographically pure, and water is ultrapure water, and it is pure that all the other reagent are analysis.Amentoflavone reference substance is that therefrom state's drug assay research institute buys, and spikemoss extract is provided by Jiangsu Tiansheng Pharmaceutical Co., Ltd..
Following embodiment is to be described the technical characteristic of the claims in the present invention, for further illustrating practicality of the present invention, should not be construed as limitation of the scope of the invention.
Embodiment one:
1 chromatographic condition and system flexibility
Chromatographic column: C18 chromatographic column (Agilent TC-C18 (2) 4.6 × 250mm, 5 μm); Take methanol solution as mobile phase A, with 0.1%TFA aqueous solution for Mobile phase B, the regulation according to the form below carries out gradient elution, flow velocity: 1ml/min, column temperature 30 DEG C, determined wavelength 335nm.
2 experimental techniques and result
The preparation of 2.1 reference substance solution
Precision takes amentoflavone reference substance 5.26mg, puts in 10ml measuring bottle, adds proper amount of methanol dissolved dilution constant volume, in contrast product solution.
The preparation of 2.2 need testing solutions
Precision takes spikemoss extract 50.14mg, puts in 50ml measuring bottle, adds a certain amount of methyl alcohol and dissolves, after ultrasonic 10 minutes, let cool, then by methanol constant volume to scale, to obtain final product.
2.2 measurement result
Accurate absorption reference substance solution and need testing solution 20 μ l respectively, injection liquid chromatography, measures.
There is identical peak in sample in the identical retention time of reference substance, and calculate amentoflavone content in spikemoss extract is 34.4% simultaneously.
Embodiment two:
1 chromatographic condition and system flexibility
Chromatographic column: C18 chromatographic column (Agilent TC-C18 (2) 4.6 × 250mm, 5 μm); Take methanol solution as mobile phase A, with 0.1%TFA aqueous solution for Mobile phase B, the regulation according to the form below carries out gradient elution, flow velocity: 1.2ml/min, column temperature 35 DEG C, determined wavelength 330nm.
2 experimental techniques and result
The preparation of 2.1 reference substance solution
Precision takes amentoflavone reference substance 10.08mg, puts in 10ml measuring bottle, adds after proper amount of methanol is dissolved and is settled to scale, to obtain final product.
The preparation of 2.2 need testing solutions
Precision takes spikemoss extract sample 25.78mg, puts in 50ml measuring bottle, adds a certain amount of methyl alcohol and dissolves, after ultrasonic 20 minutes, let cool, then by methanol constant volume to scale, to obtain final product.
2.3 assay
Accurate absorption reference substance solution and need testing solution 20 μ l respectively, injection liquid chromatography, measures.
There is identical peak in sample in the identical retention time of reference substance, and calculating amentoflavone content is 33.8%.
Embodiment three:
1 chromatographic condition and system flexibility
Chromatographic column: C18 chromatographic column (Agilent TC-C18 (2) 4.6 × 250mm, 5 μm); Take methanol solution as mobile phase A, with 0.1%TFA aqueous solution for Mobile phase B, the regulation according to the form below carries out gradient elution, flow velocity: 0.8ml/min, column temperature 25 DEG C, determined wavelength 337nm.
2 experimental techniques and result
The preparation of 2.1 reference substance solution
Precision takes amentoflavone 7.25mg, puts in 10ml measuring bottle, adds proper amount of methanol and dissolves and constant volume, in contrast product solution.
The preparation of 2.2 need testing solutions
Precision takes spikemoss extract 30.21mg, puts in 50ml measuring bottle, adds a certain amount of methyl alcohol and dissolves, after ultrasonic 15 minutes, let cool, then by methanol constant volume to scale, to obtain final product.
2.3 assay
Accurate absorption reference substance solution and need testing solution 20 μ l respectively, injection liquid chromatography, measures.
There is identical peak in sample in the identical retention time of reference substance, and calculating amentoflavone content is 34.7%.
Claims (1)
1. the detection method of amentoflavone in spikemoss extract, it is characterized in that, the method is specifically carried out according to following steps:
(1) preparation of reference substance solution:
Precision takes amentoflavone reference substance, adds proper amount of methanol and dissolves the reference substance solution made every 1ml and contain 0.5mg amentoflavone.
(2) preparation of need testing solution:
It is appropriate that precision takes spikemoss extract, adds methyl alcohol ultrasonic dissolution 10 minutes, let cool, then make the solution of 1mg/ml by methanol constant volume, obtain need testing solution.
(3) chromatographic condition and system suitability test: take octadecylsilane chemically bonded silica as filling agent, chromatographic column (4.6mm × 250mm, 5 μm); Eluent gradient is
At 0.01 minute, flow velocity 1.0ml/min, methyl alcohol and 0.1%TFA aqueous solution volume ratio are 75: 25;
At 0.01-10 minute, flow velocity 1.0ml/min, methyl alcohol and 0.1%TFA aqueous solution volume ratio are 66: 34;
At 10-13 minute, flow velocity 1.0ml/min, methyl alcohol and 0.1%TFA aqueous solution volume ratio are 85: 15;
At 13-20 minute, flow velocity 1.0ml/min, methyl alcohol and 0.1%TFA aqueous solution volume ratio are 90: 10;
Room temperature 20 DEG C, column temperature 30 DEG C; Determined wavelength 335nm, sample size 20 μ l.
(4) determination method:
The reference substance solution obtained according to step (1) and (2) and need testing solution are injected high performance liquid chromatograph under the chromatographic test strip part of step (3) detect, record chromatogram, then calculates the content of amentoflavone.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104749275A (en) * | 2015-03-16 | 2015-07-01 | 兰州大学 | Method for detecting flavone component in roundhead wormwood by using high performance liquid chromatography |
| CN105699577A (en) * | 2016-04-22 | 2016-06-22 | 广西壮族自治区梧州食品药品检验所 | Method for analyzing amentoflavone in selaginella tamariscina |
| CN105954377A (en) * | 2016-04-22 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Extraction method for amentoflavone in Selaginella tamariscina |
| CN106872611A (en) * | 2017-04-17 | 2017-06-20 | 广西壮族自治区梧州食品药品检验所 | A kind of method that ASE HPLC methods determine amentoflavone content in Selaginella tamariscina |
| CN108627596A (en) * | 2018-04-26 | 2018-10-09 | 湖北祥瑞丰红豆杉科技股份有限公司 | The detection method and preparation method thereof of a variety of chromocor compound contents in a kind of Chinese yew branch |
| CN109320486A (en) * | 2017-08-01 | 2019-02-12 | 复旦大学 | A kind of method of amentoflavone in infrared extraction Selaginella tamariscina |
| CN111337605A (en) * | 2020-05-19 | 2020-06-26 | 中国农业科学院蜜蜂研究所 | Method for evaluating authenticity of lotus bee pollen |
| CN113466385A (en) * | 2021-08-13 | 2021-10-01 | 五源本草(山东)健康科技有限公司 | Selaginella pulvinata total flavone extraction preparation and HPLC fingerprint detection method |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104749275A (en) * | 2015-03-16 | 2015-07-01 | 兰州大学 | Method for detecting flavone component in roundhead wormwood by using high performance liquid chromatography |
| CN105699577A (en) * | 2016-04-22 | 2016-06-22 | 广西壮族自治区梧州食品药品检验所 | Method for analyzing amentoflavone in selaginella tamariscina |
| CN105954377A (en) * | 2016-04-22 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Extraction method for amentoflavone in Selaginella tamariscina |
| CN106872611A (en) * | 2017-04-17 | 2017-06-20 | 广西壮族自治区梧州食品药品检验所 | A kind of method that ASE HPLC methods determine amentoflavone content in Selaginella tamariscina |
| CN109320486A (en) * | 2017-08-01 | 2019-02-12 | 复旦大学 | A kind of method of amentoflavone in infrared extraction Selaginella tamariscina |
| CN108627596A (en) * | 2018-04-26 | 2018-10-09 | 湖北祥瑞丰红豆杉科技股份有限公司 | The detection method and preparation method thereof of a variety of chromocor compound contents in a kind of Chinese yew branch |
| CN108627596B (en) * | 2018-04-26 | 2021-05-11 | 湖北祥瑞丰红豆杉科技股份有限公司 | Method for detecting content of multiple flavone compounds in taxus chinensis branches and preparation method thereof |
| CN111337605A (en) * | 2020-05-19 | 2020-06-26 | 中国农业科学院蜜蜂研究所 | Method for evaluating authenticity of lotus bee pollen |
| CN113466385A (en) * | 2021-08-13 | 2021-10-01 | 五源本草(山东)健康科技有限公司 | Selaginella pulvinata total flavone extraction preparation and HPLC fingerprint detection method |
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Address after: 212415 Jurong city of Jiangsu province Baohua Town Development Zone No. 10 Patentee after: Jiangsu Tiansheng Pharmaceutical Co., Ltd. Address before: 212415 Zhenjiang city of Jiangsu province Jurong Baohua Development Zone No. 10 Jiangsu Tiansheng Pharmaceutical Co. Ltd. Patentee before: Jiangsu Tiansheng Pharmaceutical Co., Ltd. |
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