A method for constructing a characteristic chromatogram of a traditional Chinese medicine composition and application thereof

Abstract

The application provides a characteristic map construction method of a traditional Chinese medicine composition and application thereof, wherein the characteristic map construction method of the traditional Chinese medicine composition comprises obtaining a characteristic map of a test product by using high performance liquid chromatography; chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column is a TC-C18 chromatographic column; a detection wavelength is 190-240 nm; acetonitrile is used as a mobile phase A, a phosphoric acid solution is used as a mobile phase B, and a specific gradient elution program is used for elution; and the traditional Chinese medicine composition is a traditional Chinese medicine compound composition comprising Semen cuscutae, Taxilli Herba and Radix Dipsaci. The application realizes effective separation of chemical components, obtains relatively more chromatographic peak information, and realizes comprehensive evaluation of internal characteristic components and overall control of the overall quality of the traditional Chinese medicine compound composition such as Shoutai pills, Semen cuscutae, Taxilli Herba and Radix Dipsaci.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection, specifically to a method for constructing characteristic spectra of a traditional Chinese medicine composition and its application. Background Technology

[0002] The classic Chinese medicine formula Shoutai Wan originates from "Medical Records of Integrating Chinese and Western Medicine" written by Zhang Xichun, a physician of the Qing Dynasty. It is a representative formula for tonifying the kidneys and stabilizing pregnancy. The original formula consists of four herbs: Cuscuta chinensis (4 liang, stir-fried and stewed), Taxillus chinensis (2 liang), Dipsacus asper (2 liang), and donkey-hide gelatin (2 liang). Cuscuta chinensis, the chief herb, nourishes the kidneys and replenishes essence, gently tonifying the fetus; Taxillus chinensis, the assistant herb, is sweet and neutral in nature, tonifying the liver and kidneys, and stabilizing the fetus; Dipsacus asper strengthens the Chong and Ren meridians, invigorates the fetal qi, and nourishes the liver and kidneys; and donkey-hide gelatin nourishes yin and blood, stabilizing the fetus and supporting its foundation. The four herbs work together to tonify the kidneys and stabilize pregnancy.

[0003] Traditional Chinese medicine (TCM) and its preparations contain numerous and complex components. Their multi-target mechanisms of action offer advantages in disease treatment but also present challenges for component analysis and quality control. High-performance liquid chromatography (HPLC) characterization techniques, as an important means of characterizing the quality of TCM preparations and ensuring their clinical efficacy, are widely recognized as one of the best methods for controlling TCM quality. HPLC can comprehensively express the types and numbers of components contained in TCM and its preparations, reflecting the integrity and intrinsic quality of TCM compound formulas.

[0004] Currently, although there are relevant studies on Shoutai Pills, most of them only analyze the quality of the components of a single herb in the formula, which cannot comprehensively and accurately reflect the quality status of the characteristic components of each raw material in Shoutai Pills. Summary of the Invention

[0005] Therefore, the purpose of this invention is to solve the problem that the existing detection methods for Shoutai Pills cannot comprehensively and accurately reflect the quality status of the characteristic components of each raw material in Shoutai Pills, and to provide a rapid, simple, accurate and reliable method for constructing characteristic spectra of traditional Chinese medicine compositions, so as to achieve comprehensive evaluation of the internal components of Shoutai Pills and comprehensive control of the overall quality.

[0006] A method for constructing a characteristic spectrum of a traditional Chinese medicine composition, wherein the characteristic spectrum of the test sample is obtained by high performance liquid chromatography;

[0007] The chromatographic conditions for the high-performance liquid chromatography method are as follows:

[0008] Chromatographic column: TC-C18 column; Detection wavelength: 190-240 nm; Elution was performed using acetonitrile as mobile phase A and phosphoric acid solution as mobile phase B, following a gradient elution program:

[0009]

[0010] The traditional Chinese medicine composition is a compound composition of traditional Chinese medicine including dodder seed, mulberry mistletoe and teaspoon, preferably, the traditional Chinese medicine composition is Shoutai pill.

[0011] Furthermore, the Shoutai Pill is prepared from stir-fried dodder seed, mulberry mistletoe, teasel root, and donkey-hide gelatin, wherein the mass ratio of the raw materials stir-fried dodder seed, mulberry mistletoe, teasel root, and donkey-hide gelatin is 2:1:1:1.

[0012] Furthermore, the chromatographic conditions for the high-performance liquid chromatography method are as follows:

[0013] Mobile phase B is a 0.04% to 0.1% aqueous solution of phosphoric acid, for example, the concentration of the aqueous solution of phosphoric acid is 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, etc.

[0014] And / or, the column temperature of the chromatographic column is 25-35℃, such as 25℃, 28℃, 30℃, 32℃, 35℃, etc.;

[0015] And / or, flow rate 0.8-1.2 ml / min, for example, flow rates of 0.8 ml / min, 0.9 ml / min, 1.0 ml / min, 1.1 ml / min, 1.2 ml / min, etc.;

[0016] And / or, the detection wavelength is 210-230nm, for example, the detection wavelength is 210nm, 220nm, 230nm, etc.;

[0017] And / or, the chromatographic column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm, for example, an Agilent 5TC-C18 column;

[0018] And / or, the injection volume is 5-10 μl, for example, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, etc.;

[0019] And / or, the theoretical plate number of the chromatographic peak corresponding to the hyperoside is not less than 10,000.

[0020] The preparation method of the test solution for detection by high performance liquid chromatography is as follows: Take the test sample of the traditional Chinese medicine composition, add the extraction solvent and mix evenly, weigh it, extract by ultrasonication and / or reflux extraction, cool it and then make up the weight loss, shake it evenly, separate the solid and liquid, filter the clear liquid, and take the filtrate, which is the test solution.

[0021] Furthermore, the ultrasonic extraction and / or reflux extraction time is 30-60 min, for example, 30 min, 40 min, 50 min, 60 min, etc.;

[0022] And / or, the mass / volume (g / ml) ratio of the test sample to the extraction solvent is 1:10 to 1:50, for example, 1:10, 1:11, 1:12, 1:12.5, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, etc.

[0023] And / or, the extraction solvent is water;

[0024] And / or, the solid-liquid separation method is centrifugation. Preferably, the centrifugation speed is 5000 r / min to 20000 r / min, for example, 5000 r / min, 7000 r / min, 9000 r / min, 11000 r / min, 13000 r / min, 15000 r / min, 18000 r / min, 20000 r / min, etc. The solid-liquid separation purpose of the present invention can be met within the above-mentioned centrifugation speed range.

[0025] The present invention also includes a reference solution and / or a negative control solution lacking each of the single herbal pieces; the preparation method of the negative control solution of Shoutai Pill lacking each of the single herbal pieces is the same as the preparation method of the test solution.

[0026] Preparation of reference solution: Weigh appropriate amounts of gallic acid, catechin, chlorogenic acid, neochlorogenic acid, loganin, loganic acid, hyperoside, isoquercitrin, and dipsaponin VI, and add a methanol aqueous solution with a volume percentage concentration of 10% to 100%, for example, about 50% methanol aqueous solution, to prepare the reference solution with a concentration of 1 to 500 μg / ml for each component. Furthermore, the reference solution is a reference solution with a gallic acid concentration of about 44 μg / ml, a catechin concentration of about 37 μg / ml, a chlorogenic acid concentration of about 52 μg / ml, a neochlorogenic acid concentration of about 38 μg / ml, a loganin concentration of about 292 μg / ml, loganic acid concentration of about 229 μg / ml, hyperoside concentration of about 474 μg / ml, isoquercitrin concentration of about 128 μg / ml, and a dipsacusin VI concentration of about 473 μg / ml.

[0027] The characteristic chromatogram of the test solution includes at least the characteristic peaks corresponding to gallic acid, catechin, chlorogenic acid, neochlorogenic acid, loganin, loganic acid, hyperoside, isoquercitrin, and dipsacin VI.

[0028] When the detection wavelength is 220nm, peak 1 is gallic acid, peak 2 is neochlorogenic acid, peak 3 is loganic acid, peak 4 is catechin, peak 5 is chlorogenic acid, peak 6 is loganin, peak 8 is hyperoside, peak 9 is isoquercitrin, and peak 11 is Dipsacus saponin VI.

[0029] The relative retention times of each characteristic peak are as follows: Peak 1 7.7–7.9 min, Peak 2 18.0–18.2 min, Peak 3 19.9–20.1 min, Peak 4 22.6–22.9 min, Peak 5 23.2–23.4 min, Peak 6 27.2–27.5 min, Peak 8 and Peak 9 41.6–42.0 min, and Peak 11 80.7–90.0 min.

[0030] The above-mentioned method for constructing characteristic spectra of traditional Chinese medicine compositions is applied in the quality detection and / or quality evaluation and / or quality control of traditional Chinese medicine compositions.

[0031] The quality control includes overall quality control and content detection of specific active ingredients, including but not limited to gallic acid, catechin, chlorogenic acid, neochlorogenic acid, loganin, loganic acid, hyperoside, isoquercitrin, and dipsacin VI.

[0032] The reference solutions were tested under the same high-performance liquid chromatography (HPLC) conditions as the test solution to obtain chromatograms. These chromatograms were then compared with the characteristic chromatogram of Shoutai Pill to identify the chromatographic peaks in the characteristic chromatogram of Shoutai Pill. At the detection wavelength of 220 nm, peaks 7, 8, and 9 were from Cuscuta chinensis, peaks 1 and 4 were from Taxillus chinensis, and peaks 3, 6, 10, and 11 were from Dipsacus asper.

[0033] The technical solution of this invention has the following advantages:

[0034] 1. This invention provides a method for constructing characteristic chromatograms of traditional Chinese medicine compositions, which can effectively detect characteristic components such as gallic acid, catechin, chlorogenic acid, neochlorogenic acid, loganin, loganic acid, hyperoside, isoquercitrin, and dipsacus viniferin VI in traditional Chinese medicine compound compositions including Cuscuta chinensis, Taxillus chinensis, and Dipsacus asperoides. This method achieves effective separation of more chemical components in traditional Chinese medicine compositions; and the resolution of the chromatographic peaks corresponding to the characteristic components in each raw material is greater than 2.0, which can effectively meet the requirements for quality detection and / or quality evaluation and / or quality control of traditional Chinese medicine compound compositions. This method achieves the purpose of comprehensive evaluation of the intrinsic characteristic components and comprehensive quality control of traditional Chinese medicine compound compositions including Cuscuta chinensis, Taxillus chinensis, and Dipsacus asperoides, such as Shoutai Wan.

[0035] 2. The method provided by this invention confirms 11 characteristic peaks, which can achieve better overall quality control. Moreover, the preparation and detection conditions of the test sample are simple and do not involve complicated sample pretreatment steps. It solves the problems of difficult separation of characteristic peaks and interference from impurity peaks in traditional Chinese medicine compound compositions and / or preparations. It can comprehensively reflect the quality level of traditional Chinese medicine compound compositions and / or preparations, and realize the scientific and accurate evaluation of the quality of traditional Chinese medicine compound compositions.

[0036] 3. The method provided by this invention has the advantages of high stability, high precision, and good repeatability.

[0037] 4. The characteristic spectrum of Shoutai Pill established in this invention overcomes the shortcomings of evaluating the whole from a single component, and realizes a scientific and effective comprehensive evaluation of the classic formula Shoutai Pill. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 This is a characteristic chromatogram of Shoutai Pill in Example 1.

[0040] Figure 2 This is a superimposed HPLC comparison chromatogram of the medicinal ingredients in Example 1.

[0041] Figure 3 This is a superimposed HPLC comparison chromatogram of the characteristic peak localization in Example 1.

[0042] Figure 4 This is an overlay of the HPLC chromatograms from the specificity test and the overall investigation in Example 1.

[0043] Figure 5 This is an overlay of the HPLC chromatograms showing the precision results in Example 1.

[0044] Figure 6 This is a superimposed HPLC chromatogram of the stability results in Example 1.

[0045] Figure 7 This is a superimposed HPLC chromatogram of the repeatability results in Example 1.

[0046] Figure 8 This is a full-wavelength scan of 190-400nm in Example 2.

[0047] Figure 9 The above are HPLC comparison chromatograms at different wavelengths in Example 2.

[0048] Figure 10 The above are HPLC comparison chromatograms of different mobile phase systems in Example 2.

[0049] Figure 11 The above are HPLC comparison chromatograms of different gradients in Example 2.

[0050] Figure 12 This is a comparison of HPLC chromatograms of columns from different manufacturers in Example 2.

[0051] Figure 13 The above are HPLC comparison chromatograms at different column temperatures in Example 2.

[0052] Figure 14 The above are HPLC comparison chromatograms of different flow rates in Example 2.

[0053] Figure 15 The images show the HPLC comparison chromatograms of different extraction methods in Example 3. Detailed Implementation

[0054] The instruments and reagents used in the following embodiments and comparative examples of this invention are as follows:

[0055] Instruments: Agilent 1260 high performance liquid chromatograph (Agilent Technologies, USA); METTLER TOLEDO XS105 0.0001 g electronic balance (METTLER-Toledo Instruments (Shanghai) Co., Ltd.), METTLER TOLEDO AL204 (METTLER-Toledo Instruments (Shanghai) Co., Ltd.).

[0056] Test reagents: Gallic acid (batch number 110831-201906, content calculated as 91.5%, China National Institutes for Food and Drug Control), catechin (batch number 110877-201604, content calculated as 99.2%, China National Institutes for Food and Drug Control), chlorogenic acid (batch number 16428, content calculated as 95.3%, Shanghai Shidander Standard Technical Service Co., Ltd.), neochlorogenic acid (batch number 14604, content calculated as 98.7%, Shanghai Shidander Standard Technical Service Co., Ltd.), loganin (batch number 111640-201808, content calculated as 99.0%, China National Institutes for Food and Drug Control), loganic acid (batch number MUST-19) 081615 (content calculated at 99.3%, Chengdu Mansite Biotechnology Co., Ltd.), hyperoside (batch number MUST-19061210, content calculated at 98.49%, Chengdu Mansite Biotechnology Co., Ltd.), isoquercitrin (batch number MUST-19051005, content calculated at 99.74%, Chengdu Mansite Biotechnology Co., Ltd.), dicoflavone VI (batch number 111685-201908, content calculated at 94.3%, China National Institutes for Food and Drug Control); acetonitrile (chromatographic grade, Hassen Chemical, Germany); phosphoric acid (superior grade, Shanghai Titan Technology Co., Ltd.); purified water (Hangzhou Wahaha Group Co., Ltd.).

[0057] Example 1

[0058] A method for constructing a characteristic spectrum of Shoutai Pill is disclosed, which uses high-performance liquid chromatography (HPLC) to construct the characteristic spectrum of Shoutai Pill. The detection process is as follows:

[0059] 1. Sample preparation

[0060] Preparation of the test solution: Weigh an appropriate amount of Shoutai pills, grind them into a fine powder, take about 2g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of purified water, weigh it, reflux and extract for 60 minutes, cool it, weigh it again, replenish the lost weight with purified water, shake it well, centrifuge it at 10000r / min, take the supernatant and filter it, take the filtrate, and that is the test solution.

[0061] Preparation of reference solutions: Accurately weigh gallic acid reference standards, catechin reference standards, chlorogenic acid reference standards, neochlorogenic acid reference standards, loganin reference standards, loganic acid reference standards, hyperoside reference standards, isoquercitrin reference standards, and dipsacusin VI reference standards. Add 50% methanol to prepare a solution containing 44 μg gallic acid, 37 μg catechin, 52 μg chlorogenic acid, 38 μg neochlorogenic acid, 292 μg loganin, 229 μg loganic acid, 474 μg hyperoside, 128 μg isoquercitrin, and 473 μg dipsacusin VI per ml.

[0062] Preparation of negative control solutions for each missing single herb: Take an appropriate amount of the negative control of Shoutai Pill for each missing single herb, grind it into a fine powder, take about 2g, weigh it accurately, and prepare the negative control solutions for each missing single herb in the same way as the preparation of the test solution, and set them aside.

[0063] 2. High Performance Liquid Chromatography

[0064] An Agilent 5TC-C18 column (250 cm long, 4.6 mm inner diameter, 5 μm particle size) was used as the chromatographic column; acetonitrile was used as mobile phase A, and 0.06% phosphoric acid aqueous solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the detection wavelength was 220 nm; the column temperature was 35 °C; and the flow rate was 1.0 mL / min. The theoretical plate number, calculated based on the characteristic peak of hyperoside at point 8, should not be less than 10,000.

[0065] Table 1

[0066]

[0067] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the ultra-high performance liquid chromatograph and measure to obtain the characteristic chromatograms of the test solution and the reference solution.

[0068] Characteristic peaks were marked on the characteristic spectrum of the test solution, and 11 characteristic peaks were marked, such as... Figure 1As shown, the relative retention times of each characteristic peak are: peak 1 7.7–7.9 min, peak 2 18.0–18.2 min, peak 3 19.9–20.1 min, peak 4 22.6–22.9 min, peak 5 23.2–23.4 min, peak 6 27.2–27.5 min, peak 8, peak 9 41.6–42.0 min, and peak 11 80.7–90.0 min; the resolution of all 11 characteristic peaks is greater than 2.0.

[0069] The reference comparison results of the reference solution are as follows: Figure 2 As shown, Figure 2 Substances A through J are, in order, the test solution of Shoutai Pill, Dipsacus saponin VI, isoquercitrin, hyperoside, loganic acid, loganin, neochlorogenic acid, chlorogenic acid, catechin, and gallic acid. Figure 2 The comparison shows that peak 1 is gallic acid, peak 2 is neochlorogenic acid, peak 3 is loganic acid, peak 4 is catechin, peak 5 is chlorogenic acid, peak 6 is loganin, peak 8 is hyperoside, peak 9 is isoquercitrin, and peak 11 is Dipsacus saponin VI.

[0070] The HPLC chromatogram of the negative control solution is as follows: Figure 3 As shown in Table 2 below, based on the chromatogram results of the negative control solution, the compound names and medicinal properties of each characteristic component were obtained.

[0071] Table 2

[0072]

[0073] Of the aforementioned compounds, gallic acid, chlorogenic acid, and neochlorogenic acid are organic acids; hyperoside, isoquercitrin, and loganin are flavonoids; catechin is a flavanol; and dipsacus viniferin VI is a saponin. The results of the comparison in Table 3 show that the method of this invention can effectively identify the various compounds, especially gallic acid, catechin, loganin, loganic acid, isoquercitrin, and neochlorogenic acid. This demonstrates that the characteristic spectrum construction method disclosed in this invention can comprehensively reflect the integrity and characteristics of the intrinsic chemical composition of Shoutai Pill. Furthermore, the three herbs in Shoutai Pill—Cuscuta chinensis, Taxillus chinensis, and Dipsacus asper—can all be identified through the characteristic spectrum constructed using the characteristic spectrum construction method of this invention, indicating that the characteristic spectrum disclosed in this invention can be used for the quality control of Shoutai Pill.

[0074] 3. Methodological Examination

[0075] 3.1 Specificity Test and Holistic Examination

[0076] Test solution: Take an appropriate amount of Shoutai pills, grind them into a fine powder, take about 2g, weigh accurately, place in a stoppered conical flask, accurately add 25ml of purified water, weigh, reflux for 60 minutes, cool, weigh again, replenish the lost weight with purified water, shake well, centrifuge at 10000r / min, take the supernatant and filter, take the filtrate, and the test solution is obtained.

[0077] Reference solution: Weigh an appropriate amount of hyperoside reference standard accurately, add 50% methanol to prepare a solution containing 474 μg per ml.

[0078] Blank solvent: purified water for the test sample and 50% methanol for the reference solvent.

[0079] Inject the test solution, reference solution, and blank solvent into the liquid chromatograph, respectively, and perform the determination under the same chromatographic conditions as described in "2. High Performance Liquid Chromatography," recording the chromatograms. Simultaneously, the integrity of the test sample was examined, and the results are shown in [Figure 1]. Figure 4 . Figure 4 In the table, A: reference solvent blank, B: test sample solvent blank, C: reference solution, D: test sample solution, and E: integrity test.

[0080] from Figure 4 The results show that the reference peak corresponds to the corresponding position of the test solution, and the blank solvent has no interference, indicating that the method has good specificity. The chromatographic peaks were basically collected when the acquisition time was 95 minutes, and the chromatographic conditions have reached the maximum information acquisition. The acquisition time of 95 minutes is reasonable.

[0081] 3.2 Instrument precision test

[0082] The test solution was prepared using the same method as in "1. Sample Preparation". Under the same chromatographic conditions as in "2. High Performance Liquid Chromatography", it was injected six times consecutively, with 10 μl injected each time. The retention time of the hyperoside peak (peak 8) was used as a reference to calculate the RSD% value of the relative retention time of each characteristic peak. Specific results are shown in Table 3. Figure 5 .

[0083] Table 3. Relative retention time results of instrument precision test (n=6)

[0084]

[0085]

[0086] The results showed that the relative retention times were basically consistent, and the relative retention time RSD% of each characteristic peak was less than 2.0%, indicating good instrument precision.

[0087] 3.3 Stability Test

[0088] The test solution was prepared using the same method as in "1. Sample Preparation". Under the same chromatographic conditions as in "2. High Performance Liquid Chromatography", 10 μl of sample was injected once at 0, 6, 12, 24, 30, 36, 42, and 48 hours each time. The retention time of the hyperoside peak (peak 8) was used as a reference to calculate the RSD% value of the relative retention time of each characteristic peak. The specific results are shown in Table 4. Figure 6 , Figure 6 The numbers from bottom to top are 0h, 6h, 12h, 24h, 30h, 36h, 42h, and 48h.

[0089] Table 4. Results of relative retention times in stability tests

[0090]

[0091]

[0092] The results showed that the relative retention times were basically consistent, and the relative retention time RSD% of each characteristic peak was less than 2.0%, indicating that the test solution had good stability within 48 hours.

[0093] 3.4 Repeatability Test

[0094] Six test solutions were prepared using the same method as in "1. Sample Preparation". Under the same chromatographic conditions as in "2. High Performance Liquid Chromatography", 10 μl was injected each time. The relative retention time (RSD%) of each characteristic peak was calculated using the retention time of the hyperoside peak (peak 8) as a reference. Specific results are shown in Table 5. Figure 7 .

[0095] Table 5. Relative retention time results of repeatability tests (n=6)

[0096]

[0097]

[0098] The results show that the relative retention times are basically consistent, and the RSD% of the relative retention times of each characteristic peak is less than 2.0%, indicating that the method has good repeatability.

[0099] Example 2

[0100] This embodiment uses different chromatographic conditions to construct the characteristic chromatograms, while other conditions are the same as in Example 1, as detailed below:

[0101] 1. Selection of detection wavelength

[0102] The DAD detector was used to scan the entire wavelength range of the test sample from 190 to 400 nm. The results are as follows: Figure 8As shown. Simultaneously, the chromatograms at wavelengths of 220, 254, 280, and 320 nm were compared, and the comparison results are as follows. Figure 9 As shown, Figure 9 In the given information, A: wavelength 220nm, B: wavelength 254nm, C: wavelength 280nm, and D: wavelength 320nm.

[0103] pass Figure 8 It is known that there is more chromatographic peak information at detection wavelengths below 240nm. Therefore, in this invention, a detection wavelength in the range of 190-240nm is used for detection.

[0104] pass Figure 9 It can be seen that there are differences in the number of chromatographic peaks, absorption intensity, and resolution at the four wavelengths. Since Cuscuta chinensis is the principal herb, Taxillus chinensis is the assistant herb, and Dipsacus asperoides is the adjuvant herb in Shoutai Pill, according to the formulation principles and relevant regulations, the characteristic components of the principal and assistant herbs are given priority. When the detection wavelength is greater than 254nm (280nm, 320nm), there is less chromatographic peak information, and the absorption intensity of some peaks is weak. When the detection wavelength is 254nm, catechin (peak 4) and Dipsacus asperoides saponin VI (peak 11) have almost no absorption, and peak 7 has a small absorption. When the wavelength changes from 240nm to 220nm, the absorption of catechin (peak 4), Dipsacus asperoides saponin VI (peak 11), peak 1, and peak 7 gradually increase. When the detection wavelength is 220nm, there are many chromatographic peaks with suitable peak heights. Hyperoside (peak 8) and isoquercitrin (peak 9) in Cuscuta chinensis, the principal drug; gallic acid (peak 1) and catechin (peak 4) in Taxillus chinensis, the assistant drug; and loganic acid (peak 3), loganin (peak 6), and Dipsacus saponin VI (peak 11) in Dipsacus asperoides, the adjuvant drug, all showed good absorption.

[0105] Since there are more chromatographic peaks at a wavelength of 220 nm, the absorption intensity of each chromatographic peak is more average, and the separation is good, the detection wavelength in the range of 210-230 nm is preferred for detection, and 220 nm is further selected as the optimal detection wavelength.

[0106] 2. Selection of mobile phase

[0107] Screening was conducted on mobile phase systems of acetonitrile-0.04% phosphoric acid solution, acetonitrile-0.06% phosphoric acid solution, and acetonitrile-0.1% phosphoric acid solution, and the results are as follows: Figure 10 As shown, Figure 10 In the solution, A is acetonitrile-0.04% phosphoric acid solution, B is acetonitrile-0.06% phosphoric acid solution, and C is acetonitrile-0.1% phosphoric acid solution.

[0108] The results showed that when the phosphoric acid concentration was 0.06%–0.1%, the chromatographic peak elution time was suitable, the symmetry was good, the separation was good, and the characteristic peaks were obvious. Therefore, acetonitrile-0.06%–0.1% phosphoric acid solution can be used as the mobile phase system.

[0109] 3. Selection of mobile phase gradient

[0110] The mobile phase gradient settings are shown in Table 6-8 below. The detection results using this mobile phase gradient are as follows: Figure 11 As shown, Figure 11 In the diagram, A represents gradient 1, B represents gradient 2, and C represents gradient 3.

[0111] Table 6 Characteristic chromatograms of traditional Chinese medicine compound compositions, gradient 1 elution table

[0112]

[0113]

[0114] Table 7 Characteristic spectrum of traditional Chinese medicine compound composition, gradient 2 elution table

[0115]

[0116] Table 8 Characteristic spectrum of traditional Chinese medicine compound composition gradient 3 elution table

[0117]

[0118] pass Figure 11 It can be seen that under gradient elution condition 3, the separation of each characteristic peak is good, the retention time is appropriate, the symmetry is good, and the theoretical plate number is high. Therefore, gradient 3 is finally selected as the best elution gradient.

[0119] 4. Selection of chromatographic column

[0120] Column 1: Agilent 5TC-C18 (4.6×250mm, 5μm);

[0121] Column 2: InertSustain C18 (4.6×250mm, 5μm).

[0122] The separation effect of the Shoutai Pill test solution was verified by using different chromatographic columns, and the results are as follows: Figure 12 As shown, Figure 12 In the diagram, A is an Agilent 5TC-C18 column, and B is an InertSustain C18 column.

[0123] pass Figure 12 It can be seen that when using the Agilent 5TC-C18 column, under the same gradient elution program, each characteristic peak can be effectively separated, the chromatographic peaks are evenly distributed and have good peak shapes. Therefore, the Agilent 5TC-C18 was finally selected as the chromatographic column for the characteristic chromatogram.

[0124] 5. Column Temperature Selection

[0125] Column temperature conditions of 20℃, 25℃, 30℃, and 35℃ were screened and optimized, and the results are as follows: Figure 13 As shown, Figure 13 In the given information, A is 35℃, B is 30℃, C is 25℃, and D is 20℃.

[0126] pass Figure 13 It can be seen that when the column temperature is below 30℃, the separation of peak 4 (catechin) and peak 5 (chlorogenic acid) is poor. Increasing the column temperature is beneficial to improving the separation of peak 4 (catechin) and peak 5 (chlorogenic acid). When the column temperature is increased to 35℃, peak 4 (catechin) and peak 5 (chlorogenic acid) achieve baseline separation. The separation of each characteristic peak is relatively good, the symmetry is good, and the influence of interference factors is small. Therefore, 35℃ is finally selected as the optimal column temperature condition for the characteristic spectrum.

[0127] 6. Selection of flow rate

[0128] Flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were screened and optimized, and the results are as follows: Figure 14 As shown. Figure 14 In the above, A was 0.8 ml / min, B was 1.0 ml / min, and C was 1.2 ml / min; via Figure 14 It can be seen that the requirements of this invention can be met within the flow rate range of 0.8-1.2 ml / min. However, when the flow rate is 1.0 ml / min, the peak shape and separation are relatively better and less affected by interference factors. Therefore, a flow rate of 1.0 ml / min is finally selected as the optimal flow rate condition for the characteristic spectrum.

[0129] Example 3

[0130] This embodiment uses different extraction methods for the test samples to construct the feature maps, while other conditions are the same as in Example 1, as follows:

[0131] The first extraction method: the solvent is water, and the extraction is carried out under reflux for 60 minutes; specifically, weigh an appropriate amount of Shoutai pills, grind them into a fine powder, take about 2g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of purified water, weigh it, reflux for 60 minutes, cool it, weigh it again, replenish the lost weight with purified water, shake it well, centrifuge it at 10000r / min, take the supernatant and filter it, and take the filtrate to obtain the product.

[0132] The second extraction method: The solvent is water. Reflux extraction is performed for 60 minutes, followed by filtration. After evaporating the filtrate to dryness, 70% methanol is added for ultrasonic extraction for 30 minutes. Specifically, an appropriate amount of Shoutai pills is weighed, finely ground, and approximately 2g is accurately weighed. This is placed in a stoppered conical flask, and 25ml of water is accurately added. The weight is then measured, reflux extraction is performed for 60 minutes, followed by filtration. After evaporating the filtrate to dryness, 70% methanol is added for ultrasonic extraction for 30 minutes. The mixture is cooled, and the weight is measured again. Water is used to replenish the lost weight, and the mixture is shaken well. After centrifugation at 10000r / min, the supernatant is collected and filtered. The filtrate is then collected to obtain the final product.

[0133] Test results as follows Figure 15 As shown, Figure 15 In the middle, from bottom to top, are the first extraction method and the second extraction method.

[0134] The results show that there is no significant difference between the two extraction methods, and both extraction methods provided by this invention can meet the requirements of this invention.

[0135] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic spectrum of a traditional Chinese medicine composition, characterized in that, The characteristic chromatograms of the test sample were obtained by high performance liquid chromatography. The chromatographic conditions for the high-performance liquid chromatography method are as follows: Chromatographic column: TC-C18 column; Detection wavelength: 190-240 nm; Elution was performed using acetonitrile as mobile phase A and phosphoric acid solution as mobile phase B, following a gradient elution program: The traditional Chinese medicine composition is a compound composition of traditional Chinese medicine including Cuscuta chinensis, Taxillus chinensis, and Dipsacus asper. In the chromatographic conditions of the high performance liquid chromatography method: mobile phase B is a 0.04%~0.1% aqueous solution of phosphoric acid; The column temperature for chromatography is 25-35℃; Flow rate: 0.8-1.2 ml / min; The detection wavelength is 210-230nm; The chromatographic column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. The traditional Chinese medicine composition is Shoutai Pill; The characteristic spectrum includes at least the characteristic peaks corresponding to gallic acid, catechin, chlorogenic acid, neochlorogenic acid, loganin, loganic acid, hyperoside, isoquercitrin, and dipsacin VI.

2. The feature map construction method according to claim 1, characterized in that, The column temperature of the chromatographic column is 30-35℃; And / or, the detection wavelength is 220nm; And / or, the injection volume is 5-10 μl.

3. The feature map construction method according to claim 2, characterized in that, Mobile phase B is a 0.1% aqueous solution of phosphoric acid; And / or, the column temperature is 35°C; And / or, flow rate 1.0 ml / min; And / or, the detection wavelength is 220nm; And / or, the injection volume is 10 μl.

4. The feature map construction method according to claim 1, characterized in that, The preparation method of the test solution for detection by high performance liquid chromatography is as follows: Take the test sample of the traditional Chinese medicine composition, add the extraction solvent and mix evenly, weigh it, extract by ultrasonication and / or reflux extraction, cool it and then make up the weight loss, shake it evenly, separate the solid and liquid, filter the clear liquid, and take the filtrate, which is the test solution.

5. The feature map construction method according to claim 4, characterized in that, The ultrasonic extraction and / or reflux extraction time is 30-60 min; And / or, the extraction solvent is water; And / or, the solid-liquid separation method is centrifugation.

6. The feature map construction method according to claim 5, characterized in that, The ultrasonic extraction and / or reflux extraction time is 60 min; And / or, the mass / volume ratio of the test sample to the extraction solvent is 1:12.5; And / or, the extraction solvent is an aqueous methanol solution with a volume percentage concentration of 10% to 100%; And / or, the centrifugation speed is 10000 r / min.

7. The feature map construction method according to claim 1, characterized in that, When the detection wavelength is 220nm, peak 1 is gallic acid, peak 2 is neochlorogenic acid, peak 3 is loganic acid, peak 4 is catechin, peak 5 is chlorogenic acid, peak 6 is loganin, peak 8 is hyperoside, peak 9 is isoquercitrin, and peak 11 is Dipsacus saponin VI. The relative retention times of each characteristic peak are as follows: Peak 1 7.7~7.9 min, Peak 2 18.0~18.2 min, Peak 3 19.9~20.1 min, Peak 4 22.6~22.9 min, Peak 5 23.2~23.4 min, Peak 6 27.2~27.5 min, Peak 8 and Peak 9 41.6~42.0 min, and Peak 11 80.7~90.0 min.

8. The application of the method for constructing characteristic spectra of a traditional Chinese medicine composition according to any one of claims 1-7 in the quality detection and / or quality evaluation and / or quality control of the traditional Chinese medicine composition.

9. The application according to claim 8, characterized in that, When the traditional Chinese medicine composition is Shoutai Pill, the intrinsic characteristic components of stir-fried Cuscuta chinensis include hyperoside and isoquercitrin in the quality testing and / or quality evaluation and / or quality control of Shoutai Pill; the intrinsic characteristic components of Loranthus parasiticus include gallic acid and catechin; and the intrinsic characteristic components of Dipsacus asperoides include loganic acid, loganin and Dipsacus asperoides saponin VI.

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