阅读说明：本技术 紫花地丁指纹图谱的检测方法 (Method for detecting fingerprint spectrum of Chinese violet ) 是由 郑艳萍 刁和芳 成俊 赵开军 于 2020-10-31 设计创作，主要内容包括：本发明公开了紫花地丁指纹图谱的检测方法,紫花地丁指纹图谱建立包括以下步骤：步骤1、紫花地丁供试品溶液的制备；步骤2、参照物的制备的制备：步骤3、分别精密吸取供试品溶液注入液相色谱仪,记录色谱图；步骤4,将步骤3中获得的紫花地丁供试品溶液的指纹图谱导出,并导入中药色谱指纹图谱相似度评价系统2004A；选择不同批次紫花地丁的色谱图中均存在的色谱峰作为共有峰；用平均值计算法生成紫花地丁的对照指纹图谱,并以2号峰为参照峰,计算各共有峰的相对保留时间和相对峰面积。本发明所提供的紫花地丁指纹图谱,能全面,客观地表征紫花地丁的质量。本发明提供的指纹图谱的检测方法具有方法简便、稳定、精密度高、重现性好等优点。(The invention discloses a method for detecting a viola yedoensis fingerprint, which comprises the following steps of: step 1, preparing a test solution of Chinese violet; step 2, preparation of reference substance: step 3, precisely absorbing the test solution to be injected into a liquid chromatograph, and recording a chromatogram; step 4, exporting the fingerprint of the viola philippica test sample solution obtained in the step 3, and importing the viola philippica test sample solution into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of Chinese violet in different batches as common peaks; and generating a control fingerprint of the viola yedoensis by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak by using the No. 2 peak as a reference peak. The fingerprint spectrum of the Chinese violet provided by the invention can comprehensively and objectively characterize the quality of the Chinese violet. The fingerprint detection method provided by the invention has the advantages of simplicity, convenience, stability, high precision, good reproducibility and the like.)

1. A method for detecting a fingerprint of Chinese violet is characterized by comprising the following steps:

step 1, preparing a test solution of Chinese violet:

precisely weighing herba Violae medicinal powder of different batches, placing in a conical flask with a stopper, adding methanol, weighing, ultrasonic extracting, cooling to room temperature, weighing, supplementing the weight loss with methanol, shaking, standing, collecting supernatant, filtering with 0.45 μm microporous membrane, and collecting filtrate;

step 2, preparation of a reference substance: selecting an internal standard substance as a reference substance, wherein the reference peak is a No. 2 peak;

step 3, precisely absorbing the test solution, injecting the test solution into a high performance liquid chromatograph, and recording a chromatogram;

step 4, exporting the fingerprint of the viola philippica test sample solution obtained in the step 3, and importing the viola philippica test sample solution into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of Chinese violet in different batches as common peaks; and generating a control fingerprint of the viola yedoensis by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak by using the No. 2 peak as a reference peak.

2. The method for detecting the fingerprint of the Chinese violet according to claim 1, wherein the step 1: precisely weighing 1.0g of herba Violae medicinal powder, placing into 250ml conical flask with plug, adding 80ml of 80% methanol, weighing, ultrasonic treating for 30min, cooling to room temperature, weighing, supplementing 80% methanol to the reduced weight, shaking, standing, filtering the supernatant with 0.45 μm microporous membrane, and collecting the filtrate to obtain the sample solution.

3. The method for detecting fingerprint of viola yedoensis makino according to claim 1, wherein the internal standard substance in the step 2 is aesculetin.

4. The method for detecting the fingerprint of the viola yedoensis makino according to claim 1, wherein in the step 3, the liquid chromatography conditions are as follows: a chromatographic column: YMC-Pack ODS-A column, 250X 4.6mm in specification, 5 μm; mobile phase: acetonitrile is phase A, 0.1% phosphoric acid water solution is phase B, and the flow rate is as follows: 0.8mL/min, column temperature 30 ℃, gradient elution, detection wavelength of 330nm gradient elution program as follows:

。

5. the method for detecting the fingerprint of viola yedoensis makino according to claim 1, wherein the fingerprint contains 6 peaks.

Technical Field

The invention relates to a detection method of traditional Chinese medicines, in particular to a detection method of a fingerprint spectrum of Chinese violet.

Background

Viola yedoensis Makino is bitter, pungent and cold in taste and is nontoxic; it enters heart and liver meridians, and is cold in nature and slightly bitter in taste, and can clear heat and remove toxicity, cool blood and relieve swelling. Can be used for treating jaundice, dysentery, mastitis, conjunctival congestion, swelling and pain, and pharyngitis; it is indicated for traumatic injury, carbuncle, swelling, snake bite, etc. by external application.

The active ingredients of the Chinese violet are complex and mainly contain active ingredients such as flavonoid, coumarins, organic acids, saccharides and the like. In the prior art, the high performance liquid chromatography is usually adopted to determine individual effective components of the Chinese violet, the analysis time is long, and the quality of the Chinese violet cannot be comprehensively, objectively and accurately evaluated.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to overcome the defects of the prior art, and provides the fingerprint spectrum detection method for the Chinese violet, which can objectively, comprehensively and accurately evaluate the quality of the Chinese violet and has important significance for controlling the quality of the Chinese violet and ensuring the clinical curative effect.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that:

a method for detecting a fingerprint of Chinese violet comprises the following steps:

step 1, preparing a test solution of Chinese violet:

precisely weighing herba Violae medicinal powder of different batches, placing in a conical flask with a stopper, adding methanol, weighing, ultrasonic extracting, cooling to room temperature, weighing, supplementing the weight loss with methanol, shaking, standing, collecting supernatant, filtering with 0.45 μm microporous membrane, and collecting filtrate;

step 2, preparation of a reference substance: selecting an internal standard substance as a reference substance, wherein the reference peak is a No. 2 peak;

step 3, precisely absorbing the test solution, injecting the test solution into a high performance liquid chromatograph, and recording a chromatogram;

step 4, exporting the fingerprint of the viola philippica test sample solution obtained in the step 3, and importing the viola philippica test sample solution into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of Chinese violet in different batches as common peaks; and generating a control fingerprint of the viola yedoensis by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak by using the No. 2 peak as a reference peak.

As a preferred scheme, the method for detecting the fingerprint of the viola yedoensis makino comprises the following steps of 1: precisely weighing 1.0g of herba Violae medicinal powder, placing into 250ml conical flask with plug, adding 80ml of 80% methanol, weighing, ultrasonic treating for 30min, cooling to room temperature, weighing, supplementing 80% methanol to the reduced weight, shaking, standing, filtering the supernatant with 0.45 μm microporous membrane, and collecting the filtrate to obtain the sample solution.

In the preferable scheme, in the method for detecting the fingerprint spectrum of the viola yedoensis makino, the internal standard substance in the step 2 is aesculetin.

As a preferred scheme, in the above method for detecting the fingerprint of viola yedoensis makino, in step 3, the liquid chromatography conditions are as follows: a chromatographic column: YMC-Pack ODS-A column, 250X 4.6mm in specification, 5 μm; mobile phase: acetonitrile is phase A, 0.1% phosphoric acid water solution is phase B, and the flow rate is as follows: 0.8mL/min, column temperature 30 ℃, gradient elution, detection wavelength of 330nm gradient elution program as follows:

。

as a preferred scheme, in the detection method of the fingerprint spectrum of the Chinese violet, 6 peaks are totally contained in the fingerprint spectrum.

Optimization experiment of fingerprint spectrum detection conditions:

1. screening of test solution preparation method

According to the invention, through comparing different extraction methods (an ultrasonic extraction method, reflux, percolation and the like) and different extraction solvents (methanol, water, 70% ethanol aqueous solution, 80% ethanol aqueous solution, 90% ethanol, absolute ethanol) and carrying out a Chinese violet extraction experiment, the result shows that the Chinese violet spectrogram obtained by ultrasonic extraction and reflux extraction has small difference, the ultrasonic extraction efficiency is high, the types of active ingredients are many, and therefore, the ultrasonic extraction method is adopted; in addition, in the investigation of the extraction solvent, the invention finds that the 80% methanol extract chromatogram has the most information content and the highest component content; therefore, 80% methanol was selected for extraction.

2. Screening experiments under chromatographic conditions

In the invention, chromatograms at 254nm, 280nm, 300nm and 330nm positions are screened, and when the detection wavelength is 330nm, the information content contained in the 80% methanol extract chromatogram is most comprehensive and the base line is stable, so that 330nm is selected as the detection wavelength;

in addition, the invention screens the flow rate (1mL/min, 0.8mL/min, 0.7mL/min, 0.6mL/min, 0.5mL/min), so the separation can not be carried out at high flow rate, the separation effect is better at low flow rate, and finally the substances with similar polarity are separated under the gradient condition of the flow rate of 0.8 mL/min.

The invention compares the elution effects of 5 different elution systems of acetonitrile-0.1% phosphoric acid water, acetonitrile and 0.05% phosphoric acid water, methanol-water, acetonitrile-water and acetonitrile-0.1% formic acid under different gradients. As a result, the acetonitrile-0.1% phosphoric acid water is used as a mobile phase, the components in the Chinese violet can be well separated, so that the acetonitrile and the water are finally selected as the mobile phase. Because the ingredients of the Chinese violet contain a plurality of flavones with close polarity, organic acids and other coumarin ingredients with extremely similar polarity, the isocratic elution can not achieve good separation degree, different gradient elutions are screened through a large number of experiments, and the finally obtained optimal gradient elution program is as follows: 0mim, 90% B, 0 to 20min, 90% to 80% B, 20 to 40min, 80% to 75% B, 40 to 52min, 75% to 70% B, 52 to 52.01min, 70% to 90% B, 52.01 to 60min, 90% to 90% B.

Has the advantages that:

1. according to the structural property characteristics of active ingredients contained in the Chinese violet, the best mobile phase composition is screened out through a large number of experiments, and analysis conditions such as gradient elution procedures, flow rate, detection wavelength, chromatographic column and column temperature are verified through a plurality of experiments.

2. The method for detecting the fingerprint of the viola yedoensis makino provided by the invention has the advantages of simplicity, convenience, good stability, high precision, good reproducibility and the like.

Drawings

FIG. 1 shows the fingerprint of 16 batches of Chinese violet samples.

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples, in which specific conditions are not specified, according to conventional conditions or conditions recommended by manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The instruments and reagents used in the examples were as follows:

experimental equipment

1.1 instruments

A dual-wavelength scanning high-performance liquid chromatography system of Shimadzu corporation in Japan comprises a full-automatic online degassing system, a full-automatic sample injection system SIL-20A, an ultraviolet detector SPD-20A, an automatic temperature control column temperature box CTD-20AC, KH-500E type ultrasonic cleaner (Kunshan Seama ultrasonic instruments Co., Ltd.), and an ML104/02 electronic analytical balance (Mettler Toledo).

1.2 drugs and reagents

The sources of the Chinese violet samples are shown in the table 1; methanol (analytically pure); acetonitrile (chromatographically pure); water (Wahaha purified water).

TABLE 1 Viola yedoensis sample information sheet

Sample number	Batch number	Manufacturer of the product
			S1	191201	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
S2	191202	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
			S3	191203	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
S4	191204	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
			S5	191205	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
S6	200101	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
			S7	200102	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
S8	200201	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
			S9	200202	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
S10	200401	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
			S11	200402	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
S12	200502	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
			S13	200502	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
S14	200601	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
			S15	200602	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.
S16	200603	NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd.

。

Embodiment 1 a method for detecting a fingerprint of viola yedoensis makino, comprising the following steps:

step 1, preparing a test solution of Chinese violet:

precisely weighing 1.0g of 16 batches of Chinese violet medicinal material powder in the table 1, placing the powder in a 250ml conical flask with a plug, adding 80ml of 80% methanol, weighing, carrying out ultrasonic treatment for 30min, cooling to room temperature, weighing, supplementing the lost weight with 80% methanol, shaking up, standing, taking supernatant, filtering through a 0.45 mu m microporous filter membrane, and taking subsequent filtrate to obtain a sample solution;

step 2, preparation of a reference substance: selecting an internal standard substance aesculetin as a reference substance, wherein a reference peak is a No. 2 peak;

step 3, precisely absorbing the test solution, injecting the test solution into a high performance liquid chromatograph, and recording a chromatogram;

step 4, exporting the fingerprint of the 16 batches of Chinese violet test sample solution obtained in the step 3, and introducing the fingerprint into a Chinese medicine chromatographic fingerprint similarity evaluation system 2004A; the fingerprint similarity calculation results of 16 batches of Chinese violet samples are shown in table 3, and 6 chromatographic peaks existing in the chromatograms of 16 batches of Chinese violet are selected as common peaks; the control fingerprint of Viola yedoensis Makino is generated by an average calculation method, and the relative retention time and the relative peak area of each common peak are calculated by taking the No. 2 peak as a reference peak and are shown in Table 4.

In the step 3, the liquid chromatography conditions are as follows: a chromatographic column: YMC-Pack ODS-A column, 250X 4.6mm in specification, 5 μm; mobile phase: acetonitrile is phase A, 0.1% phosphoric acid water solution is phase B, and the flow rate is as follows: 0.8mL/min, column temperature 30 ℃, gradient elution, detection wavelength of 330nm gradient elution program as following table 2:

TABLE 2 gradient elution procedure

。

TABLE 316 calculation of the fingerprint similarity of Viola yedoensis Makino samples

，

TABLE 4 common fingerprint Peak relative Retention time and relative Peak area

Example 2 fingerprint detection methodology study

The invention carries out methodology investigation according to the requirements of the fingerprint, including stability, precision and repeatability, RSD of the common peak relative retention time of the result is less than 3%, and RSD of the common peak with peak area more than 5% is less than 3%, which shows that the relative retention time and the consistency of the relative peak area of the chromatographic peak are detected by using the detection method to carry out fingerprint detection, namely, the components are stable within 8 hours after the solution is prepared, and the sample injection precision and the repeatability of the sample are good.

(1) Stability test: precisely weighing 1.0g of herba Violae medicinal powder, placing into 250ml conical flask with plug, adding 80ml of 80% methanol, weighing, ultrasonic treating for 30min, cooling to room temperature, weighing, supplementing 80% methanol to the reduced weight, shaking, standing, collecting supernatant, filtering with 0.45 μm microporous membrane, and collecting filtrate. The solution was measured according to the method at 0, 2, 4, 6, and 8 hours after the preparation, and the results showed that the test solution was stable within 8 hours, and the results are shown in Table 5.

TABLE 5 stability of finger print

(2) And (3) precision experiment: precisely weighing 1.0g of herba Violae medicinal powder, placing into 250ml conical flask with plug, adding 80ml of 80% methanol, weighing, ultrasonic treating for 30min, cooling to room temperature, weighing, supplementing 80% methanol to the reduced weight, shaking, standing, collecting supernatant, filtering with 0.45 μm microporous membrane, and collecting filtrate. The sample introduction was repeated 5 times to determine the relative standard deviation, and the results are shown in Table 6.

TABLE 6 fingerprint precision test results

(3) And (3) repeatability experiment: precisely weighing 1.0g of herba Violae medicinal powder, placing into 250ml conical flask with plug, adding 80ml of 80% methanol, weighing, ultrasonic treating for 30min, cooling to room temperature, weighing, supplementing 80% methanol to the reduced weight, shaking, standing, collecting supernatant, filtering with 0.45 μm microporous membrane, and collecting filtrate. 5 test solutions were prepared and measured to find the relative standard deviation, and the results are shown in Table 7.

TABLE 7 results of the fingerprint repeatability experiments

The experimental results show that the viola yedoensis fingerprint spectrum detection method provided by the invention has the advantages of good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of the viola yedoensis, and has important significance for ensuring the clinical curative effect.

The above embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and the scope of the present invention is defined by the claims. Various modifications and equivalents may be made by those skilled in the art within the spirit and scope of the present invention, and such modifications and equivalents should also be considered as falling within the scope of the present invention.