阅读说明：本技术 一种夏天无指纹图谱建立方法及多成分化学模式识别方法 (Decumbent corydalis tuber fingerprint map establishing method and multi-component chemical pattern recognition method ) 是由 龚千锋 严丽萍 于欢 李潮 温柔 于 2019-10-31 设计创作，主要内容包括：本发明公开了一种夏天无指纹图谱建立方法S1、取十四个产地的大选、统货、小选夏天无粉末,用抢水洗净,放置在阳光下晒干或阴凉通风处阴干,备用；S2、称取1g夏天无粉末,置于具塞锥形瓶中,精密加入50%甲醇50ml,称定重量,超声提取30min；S3、将S3步骤得到的溶液放冷,再称定重量,用50%甲醇溶液补足减失的重量,摇匀,过0.22um滤膜,取续滤液,制得供试品溶液;S4、分别取取10μL溶液通过UltiMate 3000 DAD检测器采用200～400 nm波段进行扫描,建立指纹图谱,可以检测出14个产区的夏天无在成分上的差异,分出各个产区不同等级的夏天无药材,且可判断与夏天无质量相关性较强的化学成分。(The invention discloses a decumbent corydalis tuber fingerprint map establishing method S1, fourteen large-choice, general-purpose and small-choice decumbent corydalis tuber powders of producing areas are taken, cleaned by rush water and placed in the sun for drying or in the shade in a ventilated place for later use; s2, weighing 1g of rhizoma corydalis Decumbentis powder, placing in a conical flask with a plug, precisely adding 50ml of 50% methanol, weighing, and ultrasonically extracting for 30 min; s3, cooling the solution obtained in the step S3, weighing the solution, complementing the lost weight with 50% methanol solution, shaking the solution evenly, filtering the solution with a 0.22um filter membrane, taking the subsequent filtrate to obtain a sample solution, S4, respectively taking 10 mu L of the solution, scanning the solution by an Ultimate 3000DAD detector by adopting a 200-400 nm waveband, establishing a fingerprint spectrum, detecting the difference of the components of corydalis amabilis in 14 production areas, separating the corydalis amabilis medicinal materials in different grades of each production area, and judging the chemical components with strong correlation with the quality of the corydalis amabilis.)

1. A decumbent corydalis tuber fingerprint map establishing method is characterized by comprising the following steps: comprises the following steps of (a) carrying out,

s1, selecting rhizoma corydalis Decumbentis powder from big, general and small fourteen production areas, cleaning with rush water, and drying in the sun or in the shade in a ventilated place;

s2, weighing 1g of rhizoma corydalis Decumbentis powder, placing in a conical flask with a plug, precisely adding 50ml of 50% methanol, weighing, and ultrasonically extracting for 30 min;

s3, cooling the solution obtained in the step S3, weighing, supplementing the weight loss by 50% methanol solution, shaking up, filtering by a 0.22um filter membrane, and taking the subsequent filtrate to prepare a test solution;

s4, respectively taking 10 mu L of solution, scanning by an UlltiMate 3000DAD detector at a wave band of 200-400 nm, and establishing a fingerprint.

2. The multi-row high-power Hall element processing technology according to claim 1, wherein: the chromatographic column of the UltiMate 3000DAD detector used in the step S4 was Diamonsil-C18, acetonitrile was mobile phase a, and the aqueous solution was mobile phase B, and gradient program elution was used; the flow rate was 1.0 mL. min, and the column temperature was 25 ℃.

3. The multi-row high-power Hall element processing technology according to claim 2, wherein: the scanning wavelength used in the step S4 was 289 nm.

4. The multi-row high-power Hall element processing technology according to claim 1, wherein: fingerprint data of corydalis amabilis of 3 grades of 14 producing areas is introduced into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and common characteristic peaks and peaks of four corresponding components are determined.

5. The multi-row high-power Hall element processing technology according to claim 1, wherein: before using UlltiMate 3000DAD detector, precisely weighing 1g of rhizoma corydalis Decumbentis powder of the same batch, preparing test solution according to the steps of S1-S3, automatically feeding 10ul samples continuously for 6 times, and recording each chromatogram.

6. A method for identifying a multi-component chemical pattern of corydalis amabilis is characterized by comprising the following steps,

s1, collecting 14 batches of quantitative data of 6 characteristic components of decumbent corydalis tuber with 3 grades;

s2, introducing 6 characteristic components of 42 batches of rhizoma corydalis decumbentis samples into SIMCA14.1 software for main component analysis;

s3, analyzing the sample by the 0PLS-DA method, deriving a PCA diagram, and observing the PCA diagram.

7. The method as claimed in claim 6, wherein the method comprises the following steps: the quantitative data in S1 is obtained from the fingerprint obtained in the above step.

8. The method as claimed in claim 6, wherein the method comprises the following steps: the 6 characteristic components are palmatine hydrochloride, corydaline, tetrahydrojatrorrhizine, bicuculline, protopine and tetrahydropalmatine.

Technical Field

The invention belongs to the technical field of traditional Chinese medicine classification, and particularly relates to a decumbent corydalis tuber fingerprint map establishing method and a multi-component chemical pattern recognition method.

Background

Rhizoma corydalis Decumbentis is dried tuber or whole plant of perennial herb corydalis Decumbentis of corydalis of Papaveraceae, also named YIJINDAN, YEYANHU, DONGLIANSHENXIAN, HOUTOUYAN and DONGZHU. Mainly produced in Jiangxi, widely distributed in Jiangsu, Anhui, Zhejiang, Fujian, Taiwan and Hunan provinces, and is a common Chinese herbal medicine in China. Rhizoma corydalis Decumbentis has effects of promoting blood circulation, dredging collaterals, activating qi-flowing, and relieving pain. Mainly treats hypertension and apoplexy hemiplegia caused by cerebrovascular vessels, has better effects on rheumatoid arthritis, sciatica, sequela of poliomyelitis and the like, and has alkaloid as a main active ingredient. The known alkaloids in the alkaloid extract of corydalis amabilis are mainly: protopine, bicuculline, corydalis tuber alkaloid, corydalmine, tetrahydropalmatine and palmatine, wherein the 6 alkaloids are the main active ingredients in the alkaloid extract of corydalis tuber. In order to fully and accurately reflect the quality of the total alkaloid extract of decumbent corydalis tuber, it is necessary to measure the contents of the 6 alkaloids.

In the decumbent corydalis tuber of each grade of a plurality of producing areas in China, certain difference also exists in actual components, a map is not available for collecting and recording the separation of a plurality of decumbent corydalis tuber components, and a visual display of effective components of the decumbent corydalis tuber is also absent, so that the research and development of the decumbent corydalis tuber components are limited, and the problem needs to be solved by an updated technology.

Disclosure of Invention

The invention aims to provide a corydalis amabilis fingerprint spectrum establishing method and a multi-component chemical pattern recognition method.

In order to achieve the purpose, the invention provides the following technical scheme: a decumbent corydalis tuber fingerprint map establishing method is characterized by comprising the following steps: comprises the following steps of (a) carrying out,

s1, selecting rhizoma corydalis Decumbentis powder from big, general and small fourteen production areas, cleaning with rush water, and drying in the sun or in the shade in a ventilated place;

s2, weighing 1g of rhizoma corydalis Decumbentis powder, placing in a conical flask with a plug, precisely adding 50ml of 50% methanol, weighing, and ultrasonically extracting for 30 min;

s3, cooling the solution obtained in the step S3, weighing, supplementing the weight loss by 50% methanol solution, shaking up, filtering by a 0.22um filter membrane, and taking the subsequent filtrate to prepare a test solution;

s4, respectively taking 10 mu L of solution, scanning by an UlltiMate 3000DAD detector at a wave band of 200-400 nm, and establishing a fingerprint.

Preferably, the chromatographic column of the UltiMate 3000DAD detector used in the S4 step is Diamonsil-C18, acetonitrile is mobile phase a, and aqueous solution is mobile phase B, and elution is performed by a gradient program; the flow rate was 1.0 mL. min, and the column temperature was 25 ℃.

Preferably, the scanning wavelength used in step S4 is 289 nm.

Preferably, fingerprint data of corydalis amabilis of 3 grades of 14 producing areas are introduced into a traditional Chinese medicine chromatography fingerprint similarity evaluation system, and common characteristic peaks and peaks of four corresponding components are determined.

Preferably, before using Ultimate 3000DAD detector, 1g of rhizoma corydalis Decumbentis powder of the same batch is precisely weighed, and sample solution is prepared according to the steps S1-S3, and is subjected to continuous automatic sample injection for 6 times, and each chromatogram is recorded.

The invention also provides a multi-component chemical pattern recognition method of corydalis amabilis, which comprises the following steps,

s1, collecting 14 batches of quantitative data of 6 characteristic components of decumbent corydalis tuber with 3 grades;

s2, introducing 6 characteristic components of 42 batches of rhizoma corydalis decumbentis samples into SIMCA14.1 software for main component analysis;

s3, analyzing the sample by the 0PLS-DA method, deriving a PCA diagram, and observing the PCA diagram.

Preferably, the quantitative data in S1 is obtained from the fingerprint obtained in the above step.

Preferably, the 6 characteristic components are palmatine hydrochloride, corydaline, tetrahydrojatrorrhizine, bicuculline, protopine and tetrahydropalmatine.

The invention has the technical effects and advantages that: the fingerprint of alkaloid components of corydalis amabilis established by the method has strong characteristics and simple and convenient method, the quality of the fingerprint can be better controlled by combining the determination of 6 alkaloid contents, the fingerprint has guiding significance and reference value for the grade classification of corydalis amabilis, the research and the application of the components of medicinal materials of corydalis amabilis are promoted, and the fingerprint has great benefits for the selective purchase of corydalis amabilis in the market.

Detailed Description

The embodiments described herein are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a method for establishing a decumbent corydalis tuber fingerprint, which comprises the following steps,

s1, selecting rhizoma corydalis Decumbentis powder from big, general and small fourteen production areas, cleaning with rush water, and drying in the sun or in the shade in a ventilated place;

s2, weighing 1g of rhizoma corydalis Decumbentis powder, placing in a conical flask with a plug, precisely adding 50ml of 50% methanol, weighing, and ultrasonically extracting for 30 min;

s3, cooling the solution obtained in the step S3, weighing, supplementing the weight loss by 50% methanol solution, shaking up, filtering by a 0.22um filter membrane, and taking the subsequent filtrate to prepare a test solution;

s4, respectively taking 10 mu L of solution, scanning by an UlltiMate 3000DAD detector at a wave band of 200-400 nm, and establishing a fingerprint. Gradient elution is carried out by taking an e2695 type aqueous solution and an acetonitrile-0.5% triethylamine solution (pH is adjusted by acetic acid =9.99) as a mobile phase, 14 batches of 3 grades of corydalis amabilis medicinal materials are determined by an HPLC method, similarity is calculated by using 'traditional Chinese medicine spectrum fingerprint spectrum similarity evaluation software' 2012A edition, content determination is carried out on 6 alkaloid components, and accordingly corydalis amabilis component values of various grades are obtained and are arranged into a spectrum.

The chromatographic column of the UltiMate 3000DAD detector used in the step S4 was Diamonsil-C18, acetonitrile was mobile phase a, and the aqueous solution was mobile phase B, and gradient program elution was used; the flow rate was 1.0 mL. min, and the column temperature was 25 ℃.

The scanning wavelength used in the step S4 is 289nm, and the separation speed of the decumbent corydalis tuber components is faster and the effect is better under the scanning of the wavelength.

The fingerprint data of corydalis amabilis of 3 grades in 14 producing areas is led into a traditional Chinese medicine chromatography fingerprint similarity evaluation system, common characteristic peaks and peaks of four corresponding components are determined, HPLC fingerprints of alkaloid components of corydalis amabilis of different grades in 14 producing areas are established, the similarity is larger than 0.94, the difference of the corydalis amabilis of different producing areas in terms of components is proved, 12 peaks are calibrated, and 6 characteristic peaks are appointed.

Before an Ultimate 3000DAD detector is used, precisely weighing 1g of corydalis amabilis powder of the same batch, preparing a test solution according to the steps of S1-S3, carrying out continuous automatic sample injection for 6 times with 10ul, recording each spectrogram, carrying out a precision test, calibrating the detection precision of an instrument, precisely weighing 6 parts of corydalis amabilis powder with known content of the same batch, each part being about 1.0g, precisely adding 6 reference substances such as protopine and the like respectively, wherein the adding amount is 80%, 100% and 120% of the content of the test sample respectively, preparing the test solution according to the condition of '2.4', measuring through the step of S4, recording the peak area of each reference substance and calculating the recovery rate, and the result shows that the recovery rates of the protopine, the palmatine hydrochloride, the fumaryl ethyl, the bicuculline, the tetrahydrojaponine and the viologen are respectively: 97.54%, 97.85%, 97.31%, 97.61%, 97.78%, 98.23%; RSD values are 1.21%, 0.76%, 1.26%, 1.93%, 1.52% and 1.24%, respectively, which indicates that the established content determination method has high accuracy. .

The method for identifying the multi-component chemical pattern of corydalis amabilis comprises the following steps,

s1, collecting 14 batches of quantitative data of 6 characteristic components of decumbent corydalis tuber with 3 grades;

s2, introducing 6 characteristic components of 42 batches of rhizoma corydalis decumbentis samples into SIMCA14.1 software for main component analysis;

s3, analyzing the sample by the 0PLS-DA method, deriving a PCA diagram, and observing the PCA diagram. The method comprises the steps of performing pattern recognition on a sample by using Principal Component Analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA), further analyzing the sample by using a 0PLS-DA method in order to better investigate whether a chemical pattern recognition method can completely distinguish different grades, and observing from a PCA diagram, wherein large selection and small selection can be well separated, so that a reliable basis is provided for grade division of corydalis amabilis.

The quantitative data in S1 is obtained from the fingerprint obtained in the above step.

The 6 characteristic components are palmatine hydrochloride, corydaline, tetrahydrojatrorrhizine, bicuculline, protopine and tetrahydropalmatine.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.