CN111007171A - Construction method and identification method of stemona tuberosa fingerprint - Google Patents
Construction method and identification method of stemona tuberosa fingerprint Download PDFInfo
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- CN111007171A CN111007171A CN201911288618.1A CN201911288618A CN111007171A CN 111007171 A CN111007171 A CN 111007171A CN 201911288618 A CN201911288618 A CN 201911288618A CN 111007171 A CN111007171 A CN 111007171A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
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- 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
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Abstract
The invention relates to the field of medicines, and particularly discloses a method for constructing a sessile stemona root fingerprint. The construction method of the stemona tuberosa fingerprint comprises the following steps: preparing reference substance solution and test solution by using 30% methanol, and adopting Halo under the chromatographic conditionR5C18 chromatographic column (column length 250mm, inner diameter 4.6mm, particle size 5 μm), mobile phase 3-40: 97-60 parts of mixed solution; the flow rate is 0.5-1.5 mL/min; the detection wavelength is 254-325 nm; the column temperature is 30-40 ℃, and the number of theoretical plates is not less than 50000 calculated according to chlorogenic acid peaks; precisely absorbing 5-20 mu L of reference solution and sample solution respectively, injecting into a liquid chromatograph, and recording chromatographic peak for 4-30 min to obtain radix Stemonae fingerprint. The fingerprint and the construction method thereof provided by the invention can avoid the conditions of filling, reducing the material feeding, changing the processing, production process and the like which are not strict, thereby cutting off the possibility of producing inferior medicines。
Description
Technical Field
The invention relates to the field of medicines, in particular to a construction method and an identification method of a sessile stemona root fingerprint.
Background
Radix Stemonae, radix Stemonae, and radix Stemonae are collected and used in the first part of Chinese pharmacopoeia (2015 edition). In the pharmacopoeia, the subtle differences in the appearance and the fiber characteristics of the three medicinal parts of the stemona are described only under the characters and identification terms, and the requirements on the professional degree of users in actual use are extremely high. When some products need to use a certain radix stemonae accurately, the accurate judgment is difficult.
Therefore, the research and development of a method for specifically identifying the stemona sessilifolia are urgently needed, and a clear, non-interference fingerprint spectrum capable of identifying a plurality of important active ingredients and proportions in the stemona sessilifolia is constructed to be used as an objective basis and foundation for quality monitoring and evaluation of the product.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a construction method and an identification method of a sessile stemona root fingerprint.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
in a first aspect, the invention provides a method for constructing a sessile stemona root fingerprint, which comprises the following steps:
(1) preparation of reference solutions: precisely weighing appropriate amount of chlorogenic acid reference substance, and adding 30% methanol to obtain solution containing chlorogenic acid 0.025mg per 1 mL;
(2) preparation of a test solution: taking a stemona tuberosa sample, adding water for decocting, keeping slight boiling, concentrating the volume to 20-30 mL, cooling to room temperature, filtering, extracting for multiple times by shaking with water saturated n-butyl alcohol, combining the extracting solutions, evaporating to dryness, adding a proper amount of 30% methanol for dissolving, quantitatively transferring to a 5mL measuring flask, adding 30% methanol for constant volume, and shaking uniformly to obtain the stemona tuberosa extract;
(3) chromatographic conditions are as follows: by HaloR5C18 chromatographic column (column length 250mm, inner diameter 4.6mm, particle diameter 5 μm), octadecylsilane chemically bonded silica as filler;
the mobile phase is 3-40% of acetonitrile-0.4% phosphoric acid solution: 97-60 parts of mixed solution; the flow rate is 0.5-1.5 mL/min; the detection wavelength is 254-325 nm; the column temperature is 30-40 ℃, and the number of theoretical plates is not less than 50000 calculated according to chlorogenic acid peaks;
(4) and (3) determination: precisely absorbing 5-20 mu L of reference solution and sample solution respectively, injecting into a liquid chromatograph, and recording chromatographic peak for 4-30 min to obtain radix Stemonae fingerprint.
Preferably, the construction method comprises the following steps:
(1) preparation of reference solutions: precisely weighing appropriate amount of chlorogenic acid reference substance, and adding 30% methanol to obtain solution containing chlorogenic acid 0.025mg per 1 mL;
(2) preparation of a test solution: taking 1g of stemona tuberosa sample, adding water for decoction, keeping slight boiling, concentrating the volume to about 20-30 mL, cooling to room temperature, filtering, extracting for 3 times by shaking with water saturated n-butyl alcohol, 20mL each time, combining the extract, evaporating to dryness, adding a proper amount of 30% methanol for dissolution, quantitatively transferring to a 5mL measuring flask, adding 30% methanol for constant volume, and shaking uniformly to obtain the stemona tuberosa extract;
(3) chromatographic conditions are as follows: by HaloR5C18 chromatographic column (column length 250mm, inner diameter 4.6mm, particle diameter 5 μm), octadecylsilane chemically bonded silica as filler;
the mobile phase is 3-40% of acetonitrile-0.4% phosphoric acid solution: 97-60 parts of mixed solution; the flow rate is 1 mL/min; the detection wavelength is 254-325 nm; the column temperature is 35 ℃, and the number of theoretical plates is not less than 50000 calculated according to the peak of chlorogenic acid;
(4) and (3) determination: precisely absorbing 10 mu L of reference solution and sample solution respectively, injecting into a liquid chromatograph, and recording chromatographic peak for 4-30 min to obtain radix Stemonae fingerprint.
Further, in the construction method, acetonitrile is used as a mobile phase A, 0.4% phosphoric acid solution is used as a mobile phase B, and gradient elution is carried out according to the specification in the following table; equilibration time 10 minutes; the flow rate was 1.0mL per minute; the detection wavelength is a gradient wavelength; the column temperature is 35 ℃;
the gradient elution and the detection of the gradient wavelength are carried out according to the specification, so that the obtained chromatogram peaks have clear, non-overlapping and non-interference display, and the application of the chromatogram peaks as a fingerprint chromatogram in the identification aspect is facilitated.
Further, in the step (2), the mixture is filtered through filter paper.
In a second aspect, the present invention provides a sessile stemona root standard fingerprint, which is obtained by using the standard fingerprint obtained by the institute of food and drug inspection, lot number: 121221 radix Stemonae sample prepared by using 201003 radix Stemonae as sample, generating average fingerprint by software according to the fingerprint prepared by the above construction method, and obtaining radix Stemonae standard fingerprint, as shown in FIG. 1. The standard fingerprint spectrum of the stemona tuberosa comprises 11 characteristic peaks.
In a third aspect, the invention provides an identification method of radix stemonae, a sample to be detected is taken as a test sample, the fingerprint of the sample to be detected is obtained by adopting the steps (1) to (4) of the construction method, the fingerprint of the sample to be detected and the standard fingerprint of the radix stemonae are analyzed by utilizing traditional Chinese medicine chromatographic fingerprint similarity evaluation software, and the radix stemonae is identified as the radix stemonae if the similarity is more than 0.80.
The raw materials or reagents involved in the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified.
The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.
The invention has the beneficial effects that:
the preparation of the reference substance solution, the preparation of the test solution, the chromatographic conditions and the like are optimized, so that the radix stemonae sessilifoliae fingerprint spectrum with clear characteristic peaks and no interference can be constructed by adopting the method for constructing the radix stemonae sessilifoliae fingerprint spectrum. The standard fingerprint spectrum manufactured by the construction method can simultaneously display 11 clear characteristic peaks which are not overlapped with each other and have no interference.
The standard fingerprint spectrum and the identification method provided by the invention can be used for effectively distinguishing the stemona tuberosa, the stemona sessilifolia and the stemona vitifolia on the market, and the method is adopted for inspection before the medicinal materials are fed, so that the medicinal material variety can be ensured, and the medicine quality can be ensured.
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FIG. 1 shows a standard fingerprint of radix Stemonae.
FIGS. 2 and 3 show the results of the identification of the sample to be tested in example 3 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 construction of fingerprint
1. Preparation of reference solutions: precisely weighing appropriate amount of chlorogenic acid reference substance, and adding 30% methanol to obtain solution containing chlorogenic acid 0.025mg per 1 mL;
2. preparation of a test solution: taking 1g of stemona tuberosa sample, adding water for decoction, keeping slight boiling, concentrating the volume to about 20-30 mL, cooling to room temperature, filtering, extracting for 3 times by shaking with water saturated n-butyl alcohol, 20mL each time, combining the extract, evaporating to dryness, adding a proper amount of 30% methanol for dissolution, quantitatively transferring to a 5mL measuring flask, adding 30% methanol for constant volume, and shaking uniformly to obtain the stemona tuberosa extract;
3. chromatographic conditions are as follows: by HaloR5C18 chromatographic column (column length 250mm, inner diameter 4.6mm, particle diameter 5 μm, manufacturer Wotter), octadecylsilane chemically bonded silica as filler;
acetonitrile is taken as a mobile phase A, 0.4 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; equilibration time 10 minutes; the flow rate was 1.0mL per minute; the detection wavelength is a gradient wavelength; the column temperature is 35 ℃; the number of theoretical plates is not less than 50000 calculated according to chlorogenic acid peak;
4. and (3) determination: precisely absorbing 5-20 mu L of reference solution and sample solution respectively, injecting into a liquid chromatograph, and recording chromatographic peak for 4-30 min to obtain radix Stemonae fingerprint.
Example 2 construction of Standard fingerprints
The method comprises the following steps of A, testing research institute of Chinese food and drug, batch number: 121221 an 201003 radix Stemonae sample is used as a sample, and a fingerprint representing the average state is generated by software according to the fingerprint prepared by the above construction method, i.e. the radix Stemonae standard fingerprint (as shown in FIG. 1).
The standard fingerprint spectrum of the stemona tuberosa comprises 11 characteristic peaks.
Example 3 verification of Standard fingerprints
Fingerprint spectra of 9 batches of radix stemonae products selected from the market and folium eriobotryae which can be easily used for replacing radix stemonae sessilifoliae are constructed according to the method in the embodiment 1, and the fingerprint spectra of the obtained samples are analyzed by utilizing traditional Chinese medicine chromatography fingerprint spectrum similarity evaluation software with the standard fingerprint spectra obtained in the embodiment 2.
| Numbering | Sample (I) | Producing area | Origin of |
|
| 1 | Stemona root | Guangxi | User mailing | |
| 2 | Stemona root | Guangxi province | Dongbei Dajie 13780426848 as a kind of Anguo medicine in Baoding city of North province in Wanyao Hebei | |
| 3 | Stemona root | Yunnan province | Junjun of Zhongzhou city Zhongyi Tang Chinese medicinal material sales Limited company Junjun Pan 01 | |
| 4 | Stemona root | Guangxi province | Anto drug Source commerce and trade Limited | |
| 5 | Stemona root | Guangxi province | User mailing | |
| 6 | Stemona root | Guangxi province | User mailing | |
| 7 | Stemona root | Yunnan | User mailing | |
| 8 | Stemona root | Yunnan province | User mailing | |
| 9 | Stemona root | North of a | User mailing | |
| 10 | Folium Eriobotryae | Guangxi province | User mailing |
The software screenshot is shown in FIG. 2, and the data in the table shows that the similarity between the samples with the numbers 1-7 and 9 and the standard spectrum is 0.907-0.990, and the samples are identified as the stemona tuberosa.
The similarity between the samples with the numbers of 8 and 10 and the standard map is 0.842 and 0.737, and the two medicinal materials are not stemona sessilifolia according to the identification method of the invention.
And detecting by thin-layer chromatography, wherein samples numbered 1-7 and 9 are identified as radix Stemonae, numbered 8, and folium Eriobotryae, numbered 10. It can be seen that the method of the present invention has an authentication accuracy of 100%.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (7)
1. A method for constructing a radix stemonae sessilifoliae fingerprint spectrum is characterized by comprising the following steps:
(1) preparation of reference solutions: precisely weighing appropriate amount of chlorogenic acid reference substance, and adding 30% methanol to obtain solution containing chlorogenic acid 0.025mg per 1 mL;
(2) preparation of a test solution: taking a stemona tuberosa sample, adding water for decocting, keeping slight boiling, concentrating the volume to 20-30 mL, cooling to room temperature, filtering, extracting for multiple times by shaking with water saturated n-butyl alcohol, combining the extracting solutions, evaporating to dryness, adding a proper amount of 30% methanol for dissolving, quantitatively transferring to a 5mL measuring flask, adding 30% methanol for constant volume, and shaking uniformly to obtain the stemona tuberosa extract;
(3) chromatographic conditions are as follows: by HaloR5C18 chromatographic column, octadecylsilane chemically bonded silica is filler;
the mobile phase is 3-40% of acetonitrile-0.4% phosphoric acid solution: 97-60 parts of mixed solution; the flow rate is 0.5-1.5 mL/min; the detection wavelength is 254-325 nm; the column temperature is 30-40 ℃, and the number of theoretical plates is not less than 50000 calculated according to chlorogenic acid peaks;
(4) and (3) determination: precisely absorbing 5-20 mu L of reference solution and sample solution respectively, injecting into a liquid chromatograph, and recording chromatographic peak for 4-30 min to obtain radix Stemonae fingerprint.
2. The construction method according to claim 1, comprising the steps of:
(1) preparation of reference solutions: precisely weighing appropriate amount of chlorogenic acid reference substance, and adding 30% methanol to obtain solution containing chlorogenic acid 0.025mg per 1 mL;
(2) preparation of a test solution: taking 1g of stemona tuberosa sample, adding water for decoction, keeping slight boiling, concentrating the volume to about 20-30 mL, cooling to room temperature, filtering, extracting for 3 times by shaking with water saturated n-butyl alcohol, 20mL each time, combining the extract, evaporating to dryness, adding a proper amount of 30% methanol for dissolution, quantitatively transferring to a 5mL measuring flask, adding 30% methanol for constant volume, and shaking uniformly to obtain the stemona tuberosa extract;
(3) chromatographic conditions are as follows: by HaloR5C18 chromatographic column, octadecylsilane chemically bonded silica is filler;
the mobile phase is 3-40% of acetonitrile-0.4% phosphoric acid solution: 97-60 parts of mixed solution; the flow rate is 1 mL/min; the detection wavelength is 254-325 nm; the column temperature is 35 ℃, and the number of theoretical plates is not less than 50000 calculated according to the peak of chlorogenic acid;
(4) and (3) determination: precisely absorbing 10 mu L of reference solution and sample solution respectively, injecting into a liquid chromatograph, and recording chromatographic peak for 4-30 min to obtain radix Stemonae fingerprint.
3. The method of claim 1 or 2, wherein acetonitrile is used as mobile phase a, 0.4% phosphoric acid solution is used as mobile phase B, and gradient elution is performed as specified in the following table; equilibration time 10 minutes; the flow rate was 1.0mL per minute; the detection wavelength is a gradient wavelength; the column temperature is 35 ℃;
4. the method of claim 1 or 2, wherein the filtration is performed by using filter paper in the step (2).
5. The standard fingerprint spectrum of radix stemonae tuberosa is characterized in that the standard fingerprint spectrum is prepared by the following raw materials in China food and drug inspection research institute, batch number: 121221 an radix Stemonae sample of 201003 an radix Stemonae sample as a sample, preparing a fingerprint according to the construction method of any one of claims 1-4, and generating a spectrum representing the average state by software, namely, the radix Stemonae standard fingerprint.
6. The radix stemonae sessilifoliae standard fingerprint spectrum of claim 5, wherein the radix stemonae sessilifoliae standard fingerprint spectrum comprises 11 characteristic peaks.
7. An identification method of radix stemonae, which is characterized in that a sample to be detected is taken as a test sample, the fingerprint of the sample to be detected is obtained by adopting the steps (1) to (4) of the construction method of any one of claims 1 to 4, the fingerprint of the sample to be detected and the standard fingerprint of radix stemonae as claimed in claim 5 or 6 are analyzed by utilizing traditional Chinese medicine chromatographic fingerprint similarity evaluation software, and the radix stemonae with the similarity of more than 0.80 is identified as the radix stemonae.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112362788A (en) * | 2020-11-30 | 2021-02-12 | 四川新绿色药业科技发展有限公司 | Method for determining content of chlorogenic acid in radix stemonae tuberose medicinal material and application of method |
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| US20170216306A1 (en) * | 2014-07-30 | 2017-08-03 | Jiangsu Kanion Parmaceutical Co. Ltd. | A Use of Alkaloid in Preparing Pharmaceutical Compositions for Preventing or Treating Pulmonary Fibrosis |
| CN109374787A (en) * | 2018-12-21 | 2019-02-22 | 广东方制药有限公司 | The construction method and detection method of radix stemonae tuberosae medicinal material UPLC characteristic spectrum |
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Patent Citations (2)
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| US20170216306A1 (en) * | 2014-07-30 | 2017-08-03 | Jiangsu Kanion Parmaceutical Co. Ltd. | A Use of Alkaloid in Preparing Pharmaceutical Compositions for Preventing or Treating Pulmonary Fibrosis |
| CN109374787A (en) * | 2018-12-21 | 2019-02-22 | 广东方制药有限公司 | The construction method and detection method of radix stemonae tuberosae medicinal material UPLC characteristic spectrum |
Non-Patent Citations (3)
| Title |
|---|
| LAN-LAN FAN ET AL.: "Binary chromatographic fingerprint analysis of Stemonae Radix from three Stemona plants and its applications", 《JOURNAL OF NATURAL MEDICINES》 * |
| 孙冬梅 等: "百部配方颗粒的高效液相色谱指纹图谱研究", 《时珍国医国药》 * |
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Cited By (1)
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| CN112362788A (en) * | 2020-11-30 | 2021-02-12 | 四川新绿色药业科技发展有限公司 | Method for determining content of chlorogenic acid in radix stemonae tuberose medicinal material and application of method |
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