Chromatographic fingerprint spectrum of Qingxiang tea as well as establishment method and application of chromatographic fingerprint spectrum

Abstract

The invention relates to a Qingyin tea chromatographic fingerprint as well as an establishment method and application thereof, and belongs to the technical field of quality detection of traditional Chinese medicines. The establishment method of the chromatographic fingerprint spectrum comprises the steps of preparation of a test solution, preparation of a reference solution, UPLC (Ultra Performance Liquid Chromatography) detection and the like. The establishment of the chromatographic fingerprint of the Qingyin tea makes up for the deficiency of quality detection of the Qingyin tea in the prior art, so that the quality control of the Qingyin tea is more perfect and scientific.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicines, and relates to a Qingyin tea fingerprint spectrum and an establishment method and application thereof. Background Art

[0002] A traditional Chinese medicine (TCM) chromatographic fingerprint is a pattern that characterizes a TCM or TCM patent medicine after it has been properly processed and analyzed using modern chromatographic techniques. It is a comprehensive, quantifiable identification method based on systematic research into the chemical components of TCM.

[0003] A fingerprint is composed of several characteristic peaks. Their order, size, height, and mutual constraints constitute the overall characteristics, reflecting comprehensive quality analysis information. It emphasizes the integrity of the multiple components of a traditional Chinese medicine in a relatively stable ratio and positional sequence, while also reflecting the inherent ambiguity between individual components based on common characteristics, thus demonstrating the two significant characteristics of chromatographic fingerprints: "integrity" and "ambiguity."

[0004] It can establish a fingerprint pattern with known components as quantitative indicators without fully understanding all the components of Chinese medicinal materials. It can also conduct similarity analysis on the measured fingerprint features using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" of the National Pharmacopoeia Committee to effectively judge the authenticity and evaluate the quality. Summary of the Invention

[0005] The main purpose of the present invention is to provide a chromatographic fingerprint of Qingyin tea and a method for establishing the same, which can simultaneously detect six active ingredients in Qingyin tea.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned purpose:

[0007] A method for establishing a chromatographic fingerprint of Qingyin tea specifically comprises the following steps:

[0008] Step 1, preparation of Qingyin tea test samples: taking different batches of Qingyin tea test paste, adding methanol solution for ultrasonic treatment, filtering, and taking the filtrate as the test solution;

[0009] Step 2, preparation of reference solution: accurately weigh chrysophanol, chlorogenic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, chrysophanol, and luteolin standards, accurately add methanol to dissolve, filter, and collect the filtrate as the reference solution;

[0010] Step 3, UPLC detection: inject the test solution prepared in step 1 and the reference solution prepared in step 2 into an ultra-high performance liquid chromatograph and record the chromatogram for 60 minutes;

[0011] Step 4, import the chromatogram obtained in step 3 into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system; select the common chromatographic peaks in the chromatograms of different batches of Qingyin tea paste as common peaks, calculate the relative retention time and relative peak area of the common peaks; use the median method to generate a control fingerprint of Qingyin tea.

[0012] Furthermore, the preparation method of the test solution in step 1 is:

[0013] Take stir-fried cassia seed, sterculia lychnophora, green fruit, honeysuckle, ophiopogon japonicus, and tea leaves, add water and boil, and concentrate the decoction under reduced pressure to obtain Qingyin tea extract; weigh the Qingyin tea extract, add 70% methanol solution, weigh, ultrasonically treat, cool and weigh, make up the lost weight, filter, and take the filtrate to obtain the test solution.

[0014] Furthermore, the concentration of the test solution is 0.05 g / mL.

[0015] Furthermore, in step 2, the concentration of the aurantium reference solution is 0.095 mg / ml, the concentration of the chlorogenic acid reference solution is 0.095 mg / ml, the concentration of the 3,5-dicaffeoylquinic acid reference solution is 0.092 mg / ml, the concentration of the 4,5-dicaffeoylquinic acid reference solution is 0.08 mg / ml, the concentration of the chrysophanol reference solution is 0.116 mg / ml, and the concentration of the luteolin reference solution is 0.158 mg / ml.

[0016] Furthermore, the UPLC chromatographic conditions in step 3 are as follows: a Waters CORTECS UPLC T3 column, 1.6 μm, 2.1×150 mm; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution; detection wavelength of 220 nm; column temperature of 30° C.; injection volume of 1 μl; and a flow rate of 0.2 ml per minute.

[0017] Furthermore, the gradient elution procedure is:

[0018]

[0019] Furthermore, six common characteristic peaks were selected from the fingerprint spectrum in step 4, namely: chlorogenic acid, retention time 16.364 min; luteolin, retention time 31.374; 3,5-dicaffeoylquinic acid, retention time 35.338 min; 4,5-dicaffeoylquinic acid, retention time 38.212 min; chrysophanol, retention time 49.994 min; and chrysophanol, retention time 56.192 min.

[0020] The present invention has the following beneficial effects:

[0021] 1. The method for establishing the chromatographic fingerprint of Qingyin tea provided by the present invention has the characteristics of simple operation, stability, high precision, good reproducibility, and certain specificity.

[0022] 2. There are 45 common peaks in the standard chromatographic fingerprint established by the present invention, which relatively completely retains the chemical components of Qingyin tea. Among them, 6 common peaks are selected as common characteristic peaks, which can effectively monitor the quality of Qingyin tea.

[0023] 3. Qingyin Tea is an in-house preparation of the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine. The establishment of its chromatographic fingerprint has improved the quality evaluation system of Qingyin Tea and provided a basis for the comprehensive and effective control of Qingyin Tea. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : UPLC chromatograms of different test samples in Example 1, wherein the extraction solvent A is methanol, the extraction solvent B is 70% methanol, the extraction solvent C is 50% methanol, the extraction solvent D is 30% methanol, and the extraction solvent E is water;

[0025] Figure 2 : Partially enlarged views of the chromatograms of different gradient elutions in Example 2, wherein A is the gradient optimized condition I, B is the gradient optimized condition II, and C is the gradient optimized condition III;

[0026] Figure 3 : Chromatogram of the test solution of Qingyin tea extract, where 1 is chlorogenic acid, 2 is luteolin, 3 is 3,5-dicaffeoylquinic acid, 4 is 4,5-dicaffeoylquinic acid, 5 is aurantin, and 6 is chrysophanol;

[0027] Figure 4 : Chromatogram of reference solution-1, where A is chlorogenic acid, B is luteolin, and C is 3,5-dicaffeoylquinic acid;

[0028] Figure 5 : Chromatogram of reference solution-2, wherein A is 4,5-dicaffeoylquinic acid, B is aurantiumin, and C is chrysophanol;

[0029] Figure 6 : Chromatogram of the comparative fingerprint of Qingyin tea. DETAILED DESCRIPTION

[0030] The present invention is further illustrated below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention made by those skilled in the art all fall within the scope of protection of the claims of this application.

[0031] Example 1: Investigation of extraction solvents

[0032] 1.1 Preparation of Qingyin Tea Extract:

[0033] Take Cassia seed (fried), Sterculia lychnophora, green fruit, honeysuckle, Ophiopogon japonicus, and tea leaves, add water and boil twice, combine the decoctions, and concentrate the decoctions under reduced pressure to obtain an extract.

[0034] 1.2 Preparation of test solution:

[0035] Prepare Qingyin tea paste according to the method described in "1.1" above. Weigh about 0.5 g of the paste and place it in a stoppered conical flask, a total of 5 parts, accurately add 10 mL each of methanol, 70% methanol, 50% methanol, 30% methanol, and water, weigh the weight, and ultrasonicate for 30 minutes. Crush with a glass rod while ultrasonicating, rinse with a small amount of solvent, take out, cool, and weigh again. Make up the lost weight with methanol, 70% methanol, 50% methanol, 30% methanol, and water, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution.

[0036] The above-mentioned test samples were detected using a Waters CORTECS UPLC T3 (1.6 μm, 2.1×150 mm) as a chromatographic column; acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, according to the chromatographic conditions shown in Table 1; detection wavelength of 220 nm; column temperature of 30°C; injection volume of 1 μl; and flow rate of 0.2 ml per minute.

[0037] Table 1 Gradient elution program

[0038]

[0039] See attached for the results Figure 4 ,The results showed that when 70% methanol was used as the ,extraction solvent, the number of chromatographic peaks was larger and the ,peak shape was better, therefore, 70% methanol was selected as the ,extraction solvent.

[0040] Example 2 Mobile phase gradient optimization

[0041] The test solution prepared with the 70% methanol solution in step 1.2 was injected into a high performance liquid chromatograph using a Waters CORTECS UPLC T3 (1.6 μm, 2.1×150 mm) column; acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient optimization performed as shown in Tables 2-4 respectively; detection wavelength of 220 nm; column temperature of 30°C; injection volume of 1 μl; and flow rate of 0.2 ml per minute. Results are shown in the attached Figure 2 .

[0042] Table 2 Gradient optimization conditions I

[0043]

[0044]

[0045] Table 3 Gradient optimization conditions II

[0046]

[0047] Table 4 Gradient optimization conditions III

[0048]

[0049] Self-attached Figure 2 As can be seen from the figure, after multiple optimizations, gradient optimization condition III provides better separation at the red frame line, so gradient optimization condition III is selected. That is, the chromatographic conditions are determined as follows:

[0050] A Waters CORTECS UPLC T3 (1.6 μm, 2.1×150 mm) was used as the chromatographic column; acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B, with gradient elution according to Table 5 below; the detection wavelength was 220 nm; the column temperature was 30°C; the injection volume was 1 μl; the flow rate was 0.2 ml per minute, and the acquisition time was set to 60 minutes.

[0051] Table 5 UPLC gradient elution program

[0052]

[0053]

[0054] Example 3 Identification of chromatographic peaks

[0055] 3.1 Preparation of reference solution

[0056] Accurately weigh 0.95 mg of cassia bark extract, place it in a 10 ml volumetric flask, add methanol to dissolve it and dilute to the mark, shake well, and the product is obtained.

[0057] Accurately weigh 0.95 mg of chlorogenic acid, place it in a 10 ml volumetric flask, add methanol to dissolve it and dilute to the mark, shake well, and the solution is ready.

[0058] Accurately weigh 0.92 mg of 3,5-dicaffeoylquinic acid, place it in a 10 ml volumetric flask, add methanol to dissolve it and dilute to the mark, shake well, and the product is obtained.

[0059] Accurately weigh 0.80 mg of 4,5-dicaffeoylquinic acid, place it in a 10 ml volumetric flask, add methanol to dissolve it and dilute to the mark, shake well, and the product is obtained.

[0060] Accurately weigh 1.16 mg of chrysophanol, place it in a 10 ml volumetric flask, add methanol to dissolve it and dilute to the mark, shake well, and the product is obtained.

[0061] Accurately weigh 1.58 mg of luteolin, place it in a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0062] 3.2 Preparation of test solution

[0063] Take an appropriate amount of Qingyin tea extract (see "1.1" preparation method), mix well, take about 0.5g, place in a stoppered conical flask, accurately add 10ml of 70% methanol, weigh the weight, and ultrasonicate for 30 minutes. Crush with a glass rod while ultrasonicating, rinse with a small amount of solvent, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter through a 0.22μm filter membrane, and take the filtrate.

[0064] 3.3 Chromatographic peak identification

[0065] The reference solution and the test solution prepared above were analyzed according to the chromatographic conditions confirmed in Example 2.

[0066] By comparing the retention times, the chromatographic peak identification results are as follows.

[0067] Table 6 Chromatographic peak identification results

[0068]

[0069]

[0070] 3.4 Methodological validation

[0071] 3.4.1 Specificity

[0072] Take blank solvent (70% methanol) and the test solution prepared in "3.2" and analyze them according to the chromatographic conditions determined in Example 2.

[0073] The results showed that the blank solvent had no interference in the chromatographic peak position of Qingyin tea fingerprint.

[0074] 3.4.2 Reference Peak Selection

[0075] According to the results of the chromatographic peak identification of "3.3", chlorogenic acid was selected as the reference peak, and the concentration of chlorogenic acid was 1 mg / ml.

[0076] 3.4.3 Precision

[0077] The test solution prepared in "3.2" was injected continuously 6 times according to the chromatographic conditions determined in Example 2. Taking chlorogenic acid as the reference peak, the relative retention time and relative peak area of each chromatographic peak were calculated. The results are shown in the following table.

[0078] Table 7 Precision-Relative Retention Time Results

[0079]

[0080]

[0081] Table 8 Precision-Relative Peak Area Results

[0082]

[0083]

[0084] The results showed that the RSDs of the relative retention times of all chromatographic peaks were less than 1%, and except for peak 20, the RSDs of the relative peak areas of all other chromatographic peaks were less than 6%, indicating that the method had good precision.

[0085] 3.4.4 Repeatability

[0086] Take an appropriate amount of the Qingyin tea extract in "1.1" and mix well. Prepare 6 test solutions according to the method in "3.2". Determine and analyze according to the chromatographic conditions confirmed in Example 2. Using chlorogenic acid as the reference peak, calculate the relative retention time and relative peak area of each chromatographic peak. The results are shown in the following table.

[0087] Table 9 Repeatability-Relative Retention Time Results

[0088]

[0089]

[0090] Table 10 Repeatability-Relative Peak Area Results

[0091]

[0092]

[0093] The results showed that the RSDs of the relative retention times of all chromatographic peaks were less than 1%, and the RSDs of the relative peak areas of the remaining chromatographic peaks were less than 6%, indicating that the method had good repeatability.

[0094] 3.4.5 Stability

[0095] Take the "3.2" test solution and measure and analyze it at regular intervals according to the chromatographic conditions confirmed in Example 2. Using chlorogenic acid as the reference peak, calculate the relative retention time and relative peak area of each chromatographic peak at each time point. The results are shown in the following table.

[0096] Table 11 Stability-Relative Retention Time Results

[0097]

[0098]

[0099] Table 12 Stability-Relative Peak Area Results

[0100]

[0101]

[0102] The results showed that the RSDs of the relative retention times of all chromatographic peaks were less than 1%, and except for peak 20, the RSDs of the relative peak areas of the remaining chromatographic peaks were less than 7%, indicating that the method had good precision.

[0103] Example 4 Establishment of Chromatographic Fingerprint

[0104] Eighteen batches of Qingyin tea extract were prepared according to the method in "1.1" and test solutions were prepared according to the method in "3.2". The chromatographic conditions confirmed in Example 2 were used for measurement. The data was imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition), with a time window set to 0.1 min. The median method was used to generate a reference spectrum. The reference spectrum is shown in the figure below. Using the reference spectrum as a reference, the similarity of the 18 batches of Qingyin tea extract was calculated. The results are shown in the table below. The similarity of each batch was above 0.99.

[0105] Table 13 Similarity results of 18 batches of extracts

[0106]

[0107] A liquid chromatography fingerprint detection method for Qingyin tea was established using liquid chromatography. The method was feasible after verification and provided a basis for the comprehensive evaluation of the quality of Qingyin tea.

Claims

1. A method for establishing a chromatographic fingerprint of Qingyin tea, characterized in that: The following steps are involved: Step 1, preparation of Qingyin tea test samples: taking different batches of Qingyin tea test paste, adding methanol solution for ultrasonic treatment, filtering, and taking the filtrate as the test solution; Step 2, preparation of reference solution: accurately weigh chrysophanol, chlorogenic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, chrysophanol, and luteolin standards, accurately add methanol to dissolve, filter, and collect the filtrate as the reference solution; Step 3, UPLC detection: inject the test solution prepared in step 1 and the reference solution prepared in step 2 into an ultra-high performance liquid chromatograph and record the chromatogram for 60 minutes; Step 4, import the chromatogram obtained in step 3 into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system; select the common chromatographic peaks in the chromatograms of different batches of Qingyin tea paste as common peaks, select appropriate chromatographic peaks as reference peaks, calculate the relative retention time and relative peak area after comparison between each common peak and the reference peak; use the median method to generate a control fingerprint of Qingyin tea.

2. The establishment method according to claim 1, wherein: The preparation method of the test solution in step 1 is: Take stir-fried cassia seed, sterculia lychnophora, green fruit, honeysuckle, ophiopogon japonicus, and tea leaves, add water and boil, and concentrate the decoction under reduced pressure to obtain Qingyin tea extract; weigh the Qingyin tea extract, add 70% methanol solution, weigh, ultrasonically treat, cool and weigh, make up the lost weight, filter, and take the filtrate to obtain the test solution.

3. The establishment method according to claim 2, characterized in that: The concentration of the test solution is 0.05 g / mL.

4. The establishment method according to claim 1, wherein: In step 2, the concentration of the aurantium reference solution is 0.095 mg / ml, the concentration of the chlorogenic acid reference solution is 0.095 mg / ml, the concentration of the 3,5-dicaffeoylquinic acid reference solution is 0.092 mg / ml, the concentration of the 4,5-dicaffeoylquinic acid reference solution is 0.08 mg / ml, the concentration of the chrysophanol reference solution is 0.116 mg / ml, and the concentration of the luteolin reference solution is 0.158 mg / ml.

5. The establishment method according to claim 1, wherein: The UPLC chromatographic conditions in step 3 are as follows: a Waters CORTECS UPLC T3 column, 1.6 μm, 2.1×150 mm; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution; detection wavelength, 220 nm; column temperature, 30° C.; injection volume, 1 μl; and flow rate, 0.2 ml per minute.

6. The establishment method according to claim 5, characterized in that: The gradient elution program is: 0-8 min, 4%-8% mobile phase A, 95%-92% mobile phase B; 8-17 min, 8%-10% mobile phase A, 92%-90% mobile phase B; 17-20 min, 10%-15% mobile phase A, 90%-85% mobile phase B; 20-30 min, 15%-20% mobile phase A, 85%-80% mobile phase B; 30-40 min, 20%-25% mobile phase A, 80%-75% mobile phase B; 40-60 min, 25%-100% mobile phase A, 75%-0% mobile phase B; 60-70 min, 100% mobile phase A, 0% mobile phase B.

7. The establishment method according to claim 1, wherein: Six common characteristic peaks are selected from the fingerprint spectrum of step 4, namely: chlorogenic acid, retention time 16.364 min; luteolin, retention time 31.374; 3,5-dicaffeoylquinic acid, retention time 35.338 min; 4,5-dicaffeoylquinic acid, retention time 38.212 min; chrysophanol, retention time 49.994 min; and chrysophanol, retention time 56.192 min.

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