Characteristic chromatogram construction method and application of traditional Chinese medicine composition

Abstract

The invention provides a characteristic chromatogram construction method and application of a traditional Chinese medicine composition, and the characteristic chromatogram construction method of the traditional Chinese medicine composition comprises the following steps: obtaining a characteristic chromatogram of a test sample by adopting a high performance liquid chromatography; chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column is a TC-C18 chromatographic column; the detection wavelength is 190 to 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 procedure is adopted for elution; the traditional Chinese medicine composition is a traditional Chinese medicine compound composition comprising semen cuscutae, herba taxilli and radix dipsaci. According to the method disclosed by the invention, effective separation of various chemical components is realized, relatively more chromatographic peak information is obtained, and comprehensive evaluation and comprehensive control of overall quality of internal characteristic components of the traditional Chinese medicine compound composition including semen cuscutae, herba taxilli and radix dipsaci such as the longevity pill are realized.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine detection, and in particular to a method for constructing a characteristic spectrum of a traditional Chinese medicine composition and an application thereof. Background Art

[0002] Shoutai Pills, a classic Chinese medicine prescription, originated from the "Medical Records of Chinese and Western Medicine" written by Zhang Xichun, a physician in the Qing Dynasty. It is a representative prescription for tonifying the kidney and calming the fetus. The original prescription is composed of four herbs: Cuscuta (4 liang, stir-fried and stewed), Viscum album (2 liang), Dipsacus asper (2 liang), and real donkey-hide gelatin (2 liang). The main herb in the prescription is Cuscuta chinensis, which replenishes the kidney and replenishes the essence, and nourishes the fetus; the auxiliary herb, Viscum album, is sweet and mild in nature, nourishes the liver and kidney, and calms the fetus; Dipsacus asper consolidates the Chong and Ren meridians, strengthens the fetal qi, and nourishes the liver and kidney deficiencies; and donkey-hide gelatin nourishes the yin and blood, consolidates the fetus and supports the foundation. The four herbs are matched to play the role of tonifying the kidney and calming the fetus.

[0003] Traditional Chinese medicine and its preparations have many and complex ingredients. Their multi-target mode of action brings advantages to disease treatment, but also brings challenges to its ingredient analysis and quality control. As an important means to characterize the quality of traditional Chinese medicine preparations and ensure the clinical efficacy of traditional Chinese medicine preparations, HPLC characteristic spectrum technology has been recognized as one of the good methods to control the quality of traditional Chinese medicine. It can more comprehensively express the characteristics of the types and numbers of ingredients contained in traditional Chinese medicine and its preparations, and reflect the integrity and intrinsic quality of traditional Chinese medicine compound.

[0004] At present, although there are relevant studies on the Shoutai Pills, most of them only conduct quality analysis on the ingredients of a single medicinal ingredient in the prescription, which cannot fully and accurately reflect the quality status of the characteristic ingredients of each raw material in the Shoutai Pills. Summary of the invention

[0005] Therefore, the purpose of the present invention is to solve the problem that the detection method of Shoutai Pills disclosed in the prior art cannot comprehensively and accurately reflect the quality status of the characteristic components of each raw material in Shoutai Pills, and then to provide a fast, simple, accurate and reliable method for constructing a characteristic spectrum of a Chinese medicine composition, so as to achieve a 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, using high performance liquid chromatography to obtain a characteristic spectrum of a test sample;

[0007] The chromatographic conditions of the high performance liquid chromatography are:

[0008] Chromatographic column: TC-C18 chromatographic column; detection wavelength: 190-240nm; acetonitrile as mobile phase A, phosphoric acid solution as mobile phase B, elution according to the following gradient elution program:

[0009]

[0010] The Chinese medicine composition is a Chinese medicine compound composition comprising Cuscuta australis, Morus alba and Dipsacus asper. Preferably, the Chinese medicine composition is Shoutai Pills.

[0011] Furthermore, the Shoutai pills are prepared from fried Cuscuta seeds, Morus alba, Dipsacus asper and donkey-hide gelatin, wherein the mass ratio of the raw materials fried Cuscuta seeds, Morus alba, Dipsacus asper and donkey-hide gelatin is 2:1:1:1.

[0012] Furthermore, in the chromatographic conditions of the high performance liquid chromatography:

[0013] Mobile phase B is a 0.04% to 0.1% phosphoric acid aqueous solution, for example, the concentration of the phosphoric acid aqueous solution 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° C., for example, the column temperature is 25° C., 28° C., 30° C., 32° C., 35° C., etc.;

[0015] and / or, a flow rate of 0.8-1.2 ml / min, such as a flow rate 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 210-230 nm, for example, the detection wavelength is 210 nm, 220 nm, 230 nm, etc.;

[0017] And / or, the specification of the chromatographic column is 4.6 mm×250 mm, 5 μm, for example, an Agilent 5TC-C18 chromatographic column is used;

[0018] and / or, the injection volume is 5-10 μl, for example, the injection volume is 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 high performance liquid chromatography detection is as follows: take a test sample of the Chinese medicine composition, add an extraction solvent and mix well, weigh it, perform ultrasonic extraction and / or reflux extraction, cool it and then make up the lost weight, shake it well, separate the solid and liquid, take the clear liquid and filter it, and take the filtrate, which is the test solution.

[0021] Further, 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 centrifugal speed is 5000r / min to 20000r / min, for example, 5000r / min, 7000r / min, 9000r / min, 11000r / min, 13000r / min, 15000r / min, 18000r / min, 20000r / min, etc., and the purpose of solid-liquid separation of the present invention can be met within the above centrifugal speed range.

[0025] The present invention also includes a reference solution and / or a negative control solution lacking each single herbal medicine piece; the preparation method of the negative control solution of Shoutai Pills lacking each single herbal medicine piece 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, loganinic acid, hyperoside, isoquercetin, and radix dipsaponin VI, and add a 10% to 100% volume percent methanol aqueous solution, such as about 50% methanol aqueous solution, to prepare the reference solution with a concentration of each component of 1 to 500 μg / ml. Furthermore, the reference solution is a reference solution having 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, a loganinic acid concentration of about 229 μg / ml, a hyperoside concentration of about 474 μg / ml, an isoquercetin concentration of about 128 μg / ml, and a dipsaponin VI concentration of about 473 μg / ml.

[0027] The characteristic spectrum of the test solution at least includes characteristic peaks corresponding to gallic acid, catechin, chlorogenic acid, neochlorogenic acid, loganin, loganinic acid, hyperoside, isoquercetin and dipsaponin 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 isoquercetin, and peak 11 is dipsaponin VI;

[0029] The relative retention times of the characteristic peaks 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 and Peak 9 41.6-42.0 min, Peak 11 80.7-90.0 min.

[0030] Application of the above-mentioned characteristic spectrum construction method of the traditional Chinese medicine composition in quality detection and / or quality evaluation and / or quality control of the traditional Chinese medicine composition.

[0031] The quality control includes overall quality control and content detection of specific active ingredients, and the specific active ingredients include but are not limited to gallic acid, catechins, chlorogenic acid, neochlorogenic acid, loganin, loganinic acid, hyperoside, isoquercetin and radix dipsaponin VI.

[0032] The reference solutions were tested under the same HPLC conditions as the test solution to obtain the chromatogram of the reference solution, and compared with the characteristic spectrum of Shoutai Pills to identify the chromatographic peaks in the characteristic spectrum of Shoutai Pills. When the detection wavelength is 220 nm, peaks 7, 8 and 9 come from the medicinal material Cuscuta, peaks 1 and 4 come from the medicinal material Morus alba, and peaks 3, 6, 10 and 11 come from the medicinal material Dipsacus asper.

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

[0034] 1. The present invention provides a method for constructing a characteristic spectrum of a Chinese medicine composition, which can effectively detect characteristic components such as gallic acid, catechins, chlorogenic acid, neochlorogenic acid, loganin, loganinic acid, hyperoside, isoquercitrin and dipsaponin VI in a Chinese medicine compound composition including dodder seed, mistletoe and dipsaccasis, thereby achieving effective separation of more chemical components in the Chinese medicine composition; and the separation degree of the chromatographic peaks corresponding to the characteristic components in each raw material is greater than 2.0, which can effectively meet the requirements of quality detection and / or quality evaluation and / or quality control of the Chinese medicine compound composition, and achieve the purpose of comprehensive evaluation of the intrinsic characteristic components of the Chinese medicine compound composition including dodder seed, mistletoe and dipsaccasis such as Shoutai Pills and comprehensive control of the overall quality.

[0035] 2. The method provided by the present invention confirms 11 characteristic peaks, which can achieve a better overall quality control effect; and the test sample preparation and detection conditions are simple, and no complicated sample pretreatment steps are involved, which solves the problem of difficult effective separation of characteristic peaks and interference of impurity peaks in Chinese medicine compound compositions and / or preparations, and can comprehensively reflect the quality level of Chinese medicine compound compositions and / or preparations, and realizes a scientific and accurate evaluation of the quality of Chinese medicine compound.

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

[0037] 4. The characteristic spectrum of Shoutai Pills established by the present invention overcomes the defects of overall evaluation of a single component and realizes a scientific and effective comprehensive evaluation of the classic prescription Shoutai Pills. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 It is the characteristic spectrum of Shoutai Pills in Example 1.

[0040] Figure 2 This is an overlay of HPLC comparison spectra of the medicinal flavor attribution in Example 1.

[0041] Figure 3 This is an overlay of HPLC comparison spectra for positioning characteristic peaks in Example 1.

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

[0043] Figure 5 It is an overlay of the HPLC spectra of the precision results in Example 1.

[0044] Figure 6 This is an overlay of the HPLC spectra of the stability results in Example 1.

[0045] Figure 7 This is an overlay of the HPLC spectra of the repeatability results in Example 1.

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

[0047] Fig. 9 The HPLC comparison charts at different wavelengths in Example 2 are shown.

[0048] Fig.10 This is a comparison chart of HPLC with different mobile phase systems in Example 2.

[0049] Fig.11 The HPLC comparison chart of different gradients in Example 2 is shown.

[0050] Fig.12 This is a comparison chart of HPLC of chromatographic columns from different manufacturers in Example 2.

[0051] Fig.13 The HPLC comparison chart of different column temperatures in Example 2.

[0052] Fig.14 This is a comparison chart of HPLC at different flow rates in Example 2.

[0053] Fig.15 The HPLC comparison chart of different extraction methods in Example 3. DETAILED DESCRIPTION

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

[0055] Instruments: Agilent 1260 high performance liquid chromatograph (Agilent Corporation, USA); METTLER TOLEDO XS105 1 / 100000 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.), METTLER TOLEDO AL204 (Mettler-Toledo Instrument (Shanghai) Co., Ltd.).

[0056] Test drugs: Gallic acid (batch number 110831-201906, content 91.5%, China Food and Drug Inspection Institute), catechin (batch number 110877-201604, content 99.2%, China Food and Drug Inspection Institute), chlorogenic acid (batch number 16428, content 95.3%, Shanghai Shidande Standard Technology Service Co., Ltd.), neochlorogenic acid (batch number 14604, content 98.7%, Shanghai Shidande Standard Technology Service Co., Ltd.), loganin (batch number 111640-201808, content 99.0%, China Food and Drug Inspection Institute), loganinic acid (batch number MUST-19 081615, content is 99.3%, Chengdu Mansite Biotechnology Co., Ltd.), hyperoside (batch number MUST-19061210, content is 98.49%, Chengdu Mansite Biotechnology Co., Ltd.), isoquercetin (batch number MUST-19051005, content is 99.74%, Chengdu Mansite Biotechnology Co., Ltd.), chuanxiong saponin VI (batch number 111685-201908, content is 94.3%, China Food and Drug Inspection Institutes); acetonitrile (chromatographic grade, Hasen Chemie, 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 pills is disclosed. The characteristic spectrum of Shoutai pills is constructed by high performance liquid chromatography. The detection process is as follows:

[0059] 1. Sample preparation

[0060] Preparation of test solution: Weigh an appropriate amount of Shoutai Pills, grind them into powder, take about 2g, weigh accurately, put it in a stoppered conical flask, accurately add 25ml of pure water, weigh the weight, reflux extraction for 60 minutes, cool, weigh again, make up the lost weight with pure water, shake well, centrifuge at 10000r / min, filter the supernatant, and take the filtrate to obtain the test solution.

[0061] Preparation of reference solution: Take gallic acid reference, catechin reference, chlorogenic acid reference, neochlorogenic acid reference, loganin reference, loganinic acid reference, hyperoside reference, isoquercetin reference, and dipsaponin VI reference respectively, weigh accurately, add 50% methanol to make a solution containing 44 μg of gallic acid, 37 μg of catechin, 52 μg of chlorogenic acid, 38 μg of neochlorogenic acid, 292 μg of loganin, 229 μg of loganinic acid, 474 μg of hyperoside, 128 μg of isoquercetin, and 473 μg of dipsaponin VI per 1 ml.

[0062] Preparation of negative control solutions for the lack of each single ingredient: Take appropriate amount of Shoutai Pills negative control for the lack of each single ingredient, grind it into powder, take about 2g, weigh it accurately, and prepare negative control solutions for the lack of each single ingredient in the same way as "Preparation of test solution", and set aside.

[0063] 2. High performance liquid chromatography

[0064] Agilent 5TC-C18 (column length 250cm, column inner diameter 4.6mm, particle size 5μm) was used as the chromatographic column; acetonitrile was used as the mobile phase A, 0.06% phosphoric acid aqueous solution was used as the mobile phase B, and gradient elution was performed according to the provisions in Table 1 below; the detection wavelength was 220nm; the column temperature was 35°C; the flow rate was 1.0ml / min. The theoretical plate number calculated based on the characteristic peak of hyperoside No. 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 respectively, inject into the ultra-high performance liquid chromatography, and measure to obtain the characteristic spectra 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 time of each characteristic peak is: 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, peak 11 80.7-90.0 min; the separation degrees of the above 11 characteristic peaks are all greater than 2.0.

[0069] The reference comparison results of the reference solution are as follows Figure 2 As shown, Figure 2 A to J in the table are respectively Shoutai Wan test solution, Chuanxuduan saponin VI, isoquercetin, hyperoside, loganinic acid, loganin, neochlorogenic acid, chlorogenic acid, catechin, and gallic acid. Figure 2 By comparison, 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 isoquercetin, and peak 11 is Dipsaconoside VI.

[0070] The HPLC spectrum of the negative control solution is as follows Figure 3 As shown, based on the chromatographic results of the negative control solution, the compound name and medicinal taste of each characteristic component were obtained. The specific results are shown in Table 2 below.

[0071] Table 2

[0072]

[0073] Among the above-mentioned compound components, gallic acid, chlorogenic acid, and neochlorogenic acid are organic acids, hyperoside, isoquercitrin, and loganin are flavonoids, catechins are flavanol compounds, and chuanxiong saponin VI is a saponin. The results of the comparison of Table 3 above show that the method of the present invention can effectively identify the above-mentioned various compound components, especially gallic acid, catechins, loganin, loganinic acid, isoquercitrin, and neochlorogenic acid, proving that the method for constructing the characteristic spectrum disclosed in the present invention can more comprehensively reflect the integrity and characteristic of the internal chemical composition of Shoutai Pills. At the same time, the three medicines of Cuscuta, Morus alba, and Dipsacus in the Shoutai Pills can be identified in the characteristic spectrum constructed by the characteristic spectrum construction method of the present invention, indicating that the characteristic spectrum disclosed in the present invention can be used for the quality control of the Shoutai Pills.

[0074] 3. Methodological investigation

[0075] 3.1. Specificity test and overall investigation

[0076] Test solution: Take an appropriate amount of Shoutai pills, grind them into powder, take about 2g, weigh accurately, put it in a stoppered conical flask, add 25ml of pure water accurately, weigh the weight, reflux for 60 minutes, cool, weigh again, make up the lost weight with pure water, shake well, centrifuge at 10000r / min, filter the supernatant, and take the filtrate to obtain the solution;

[0077] Reference solution: Take an appropriate amount of hyperoside reference substance, weigh accurately, and add 50% methanol to make a solution containing 474 μg per 1 ml.

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

[0079] The test solution, reference solution and blank solvent were respectively injected into the liquid chromatograph, and the same chromatographic conditions as in "2. High Performance Liquid Chromatography" were used for determination, and the chromatogram was recorded. At the same time, the integrity of the test sample was examined, and the results are shown in Figure 4 . Figure 4 In the figure, A: reference solvent blank, B: test solvent blank, C: reference solution, D: test solution, E: integrity test.

[0080] from Figure 4 From the results, we can see that the reference peaks correspond to the corresponding positions of the test solution, and the blank solvent has no interference, indicating that the method has good specificity. The chromatographic peaks have been basically collected when the acquisition time is 95 minutes. The chromatographic conditions have achieved maximum information collection, and it is reasonable to set the acquisition time at 95 minutes.

[0081] 3.2 Instrument precision test

[0082] The test solution was prepared by the same method as in "1. Sample preparation". The sample was injected 6 times in succession under the same chromatographic conditions as in "2. High performance liquid chromatography". 10 μl was injected each time. The retention time of the hyperoside chromatographic peak (peak 8) was used as a reference to calculate the RSD% value of the relative retention time of each characteristic peak. The specific measurement 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, the RSD% of the relative retention time of each characteristic peak was less than 2.0%, and the instrument precision was good.

[0087] 3.3 Stability test

[0088] The test solution was prepared by the same method as in "1. Sample preparation". According to the same chromatographic conditions as in "2. High performance liquid chromatography", the sample was injected once at 0, 6, 12, 24, 30, 36, 42, and 48 h, with 10 μl injected each time. The retention time of the hyperoside chromatographic peak (peak 8) was used as a reference to calculate the RSD% value of the relative retention time of each characteristic peak. The specific measurement results are shown in Table 4. Figure 6 , Figure 6 From bottom to top they are 0h, 6h, 12h, 24h, 30h, 36h, 42h, and 48h.

[0089] Table 4 Relative retention time results of stability test

[0090]

[0091]

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

[0093] 3.4 Repeatability test

[0094] The same method as in "1. Sample preparation" was used to prepare 6 test solutions. According to the same chromatographic conditions as in "2. High performance liquid chromatography", 10 μl was injected each time. The retention time of the hyperoside chromatographic peak (peak 8) was used as a reference to calculate the RSD% value of the relative retention time of each characteristic peak. The specific measurement results are shown in Table 5. Figure 7 .

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

[0096]

[0097]

[0098] The results showed that the relative retention times were basically consistent, and the RSD% of the relative retention time of each characteristic peak was less than 2.0%, indicating that this method had good repeatability.

[0099] Example 2

[0100] This example uses different chromatographic conditions to construct the characteristic spectrum, and other conditions are the same as those in Example 1, as follows:

[0101] 1. Selection of detection wavelength

[0102] The DAD detector was used to scan the full wavelength range of 190-400nm for the test sample. The results were as follows: Figure 8At the same time, the chromatograms at wavelengths of 220, 254, 280, and 320 nm are compared, and the comparison results are shown in Fig. 9 As shown, Fig. 9 Among them, A: wavelength 220nm, B: wavelength 254nm, C: wavelength 280nm, D: wavelength 320nm.

[0103] pass Figure 8 It can be seen that at a detection wavelength below 240 nm, there is more chromatographic peak information, so the present invention adopts a detection wavelength within the range of 190-240 nm for detection.

[0104] pass Fig. 9 It can be seen that there are differences in the number, absorption intensity and separation of chromatographic peaks under the four wavelengths. Since Cuscuta chinensis is the main drug, Morus alba is the auxiliary drug, and Dipsacus asper is the adjuvant drug in Shoutai Pills, according to the principles of formulation and relevant regulations, the characteristic components of the main and auxiliary drugs are given priority. When the detection wavelength is greater than 254nm (280nm, 320nm), the chromatographic peak information is less, and the absorption intensity of individual peaks is weak; when the detection wavelength is 254nm, catechin (peak 4) and Dipsacus asper saponin VI (peak 11) have basically no absorption, and peak 7 has a small absorption. When the wavelength changes from 240nm to 220nm, the absorption of catechin (peak 4), Dipsacus asper saponin VI (peak 11), peak 1 and peak 7 gradually increases. When the detection wavelength is 220nm, the chromatographic peak information is more and the peak height is appropriate. The components such as hyperoside (peak 8) and isoquercetin (peak 9) in the main drug Cuscuta chinensis, gallic acid (peak 1) and catechins (peak 4) in the auxiliary drug Viscum album, and loganinic acid (peak 3), loganin (peak 6) and dipsaponin VI (peak 11) in the adjuvant drug Dipsacus asper all have good absorption.

[0105] Since there are more chromatographic peaks at a wavelength of 220nm, the absorption intensity of each chromatographic peak is relatively even, and the separation is good, after comprehensive consideration, it is preferred to use a detection wavelength within the range of 210-230nm for detection, and further select a wavelength of 220nm as the optimal detection wavelength.

[0106] 2. Selection of mobile phase

[0107] The mobile phase systems acetonitrile-0.04% phosphoric acid solution, acetonitrile-0.06% phosphoric acid solution, and acetonitrile-0.1% phosphoric acid solution were screened. Fig.10 As shown, Fig.10 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 show that when the phosphoric acid concentration is 0.06% to 0.1%, the chromatographic peak has an appropriate elution time, good symmetry, good separation, and obvious characteristic peaks. Therefore, acetonitrile-0.06% to 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. The detection results using this mobile phase gradient are as follows: Fig.11 As shown, Fig.11 In the figure, A is gradient 1, B is gradient 2, and C is gradient 3.

[0111] Table 6 Characteristic spectrum of Chinese medicine compound composition Gradient 1 elution table

[0112]

[0113]

[0114] Table 7 Gradient 2 elution table of characteristic spectrum of Chinese medicine compound composition

[0115]

[0116] Table 8 Gradient 3 elution table of characteristic spectrum of Chinese medicine compound composition

[0117]

[0118] pass Fig.11 It can be seen that under the gradient elution 3 condition, the separation of the characteristic peaks 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 optimal elution gradient.

[0119] 4. Selection of chromatographic columns

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

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

[0122] Different chromatographic columns were used to verify the separation effect of Shoutai Pills test solution. The results are as follows: Fig.12 As shown, Fig.12 In the figure, A is an Agilent 5TC-C18 column and B is an InertSustain C18 column.

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

[0124] 5. Selection of column temperature

[0125] The column temperature conditions of 20℃, 25℃, 30℃ and 35℃ were screened and optimized respectively. The results are as follows Fig.13 As shown, Fig.13 In the figure, A is 35°C, B is 30°C, C is 25°C, and D is 20°C.

[0126] pass Fig.13 It can be seen that when the column temperature is lower than 30°C, the separation of peak 4 (catechins) and peak 5 (chlorogenic acid) is poor. Increasing the column temperature is beneficial to improving the separation of peak 4 (catechins) and peak 5 (chlorogenic acid). When the column temperature is increased to 35°C, peak 4 (catechins) and peak 5 (chlorogenic acid) achieve baseline separation, and the separation of each characteristic peak is relatively good, with good symmetry and little influence by interference factors. Therefore, 35°C was finally selected as the optimal column temperature condition for the characteristic spectrum.

[0127] 6. Selection of flow rate

[0128] The flow rate conditions of 0.8ml / min, 1.0ml / min and 1.2ml / min were screened and optimized respectively. The results are as follows: Fig.14 shown. Fig.14 Among them, A is 0.8ml / min, B is 1.0ml / min, and C is 1.2ml / min; Fig.14 It can be seen that the requirements of the present invention can be met within the flow rate range of 0.8-1.2 ml / min, but when the flow rate is 1.0 ml / min, the peak shape and separation of each peak are relatively better, and are 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 example uses different extraction methods of the test sample to construct the characteristic spectrum, and other conditions are the same as those in Example 1, specifically as follows:

[0131] The first extraction method: the solvent is water, and reflux extraction is performed for 60 minutes; specifically, an appropriate amount of Shoutai Pills is weighed, ground into powder, about 2 g is taken, accurately weighed, placed in a stoppered conical flask, 25 ml of pure water is accurately added, the weight is determined, reflux extraction is performed for 60 minutes, cooled, and the weight is determined again, and the lost weight is supplemented with pure water, shaken, centrifuged at a speed of 10000 r / min, the supernatant is filtered, and the filtrate is obtained.

[0132] The second extraction method: the solvent is water, reflux extraction for 60 minutes, filter, evaporate the filtrate and add 70% methanol for ultrasonic extraction for 30 minutes; specifically, weigh an appropriate amount of Shoutai Pills, grind them into powder, take about 2g, accurately weigh, put them in a stoppered conical flask, accurately add 25ml of water, weigh the weight, reflux extraction for 60 minutes, filter, evaporate the filtrate and add 70% methanol for ultrasonic extraction for 30 minutes, cool, weigh again, make up the lost weight with water, shake well, centrifuge at 10000r / min, take the supernatant and filter, take the filtrate, and get it.

[0133] Test results such as Fig.15 As shown, Fig.15 , from bottom to top they are the first extraction method and the second extraction method.

[0134] The results show that the difference between the two extraction methods is not significant, and the two extraction methods provided by the present invention can meet the requirements of the present invention.

[0135] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing a characteristic spectrum of a traditional Chinese medicine composition, characterized in that: The characteristic spectrum of the test sample was obtained by high performance liquid chromatography; The chromatographic conditions of the high performance liquid chromatography are: Chromatographic column: TC-C18 chromatographic column; detection wavelength: 190-240nm; acetonitrile as mobile phase A, phosphoric acid solution as mobile phase B, elution according to the following gradient elution program: The traditional Chinese medicine composition is a traditional Chinese medicine compound composition comprising dodder seeds, loranthus parasiticus and dipsacus.

2. The method for constructing a characteristic map according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography method are as follows: mobile phase B is a 0.04% to 0.1% phosphoric acid aqueous solution; and / or, the column temperature of the chromatographic column is 25-35° C., preferably 30-35° C.; and / or, flow rate 0.8-1.2 ml / min; and / or, the detection wavelength is 210-230 nm, preferably 220 nm; and / or, the specification of the chromatographic column is 4.6 mm×250 mm, 5 μm; and / or, the injection volume is 5-10 μl; And / or, the Chinese medicine composition is Shoutai Pills.

3. The method for constructing a characteristic map according to claim 2, characterized in that: Mobile phase B was 0.1% phosphoric acid in water; and / or, the column temperature of the chromatographic column is 35°C; and / or, flow rate 1.0 ml / min; and / or, the detection wavelength is 220 nm; And / or, the injection volume is 10 μl.

4. The method for constructing a characteristic map according to claim 1, characterized in that: The preparation method of the test solution for high performance liquid chromatography detection is as follows: take a test sample of the Chinese medicine composition, add an extraction solvent and mix well, weigh it, perform ultrasonic extraction and / or reflux extraction, cool it and then make up the lost weight, shake it well, separate the solid and liquid, take the clear liquid and filter it, and take the filtrate, which is the test solution.

5. The method for constructing a characteristic spectrum according to claim 4, characterized in that: The ultrasonic extraction and / or reflux extraction time is 30-60min; And / or, the extraction solvent is water; And / or, the solid-liquid separation method is centrifugation.

6. The method for constructing a characteristic map 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 a methanol aqueous solution with a volume percentage concentration of 10% to 100%; And / or, the centrifugal rotation speed is 10000r / min.

7. The method for constructing a characteristic spectrum according to any one of claims 1 to 6, characterized in that: The characteristic spectrum at least includes characteristic peaks corresponding to gallic acid, catechin, chlorogenic acid, neochlorogenic acid, loganin, loganinic acid, hyperoside, isoquercetin and dipsaponin VI.

8. The method for constructing a characteristic spectrum according to claim 7, 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 isoquercetin, and peak 11 is dipsaponin VI; The relative retention times of the characteristic peaks 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 and Peak 9 41.6-42.0 min, Peak 11 80.7-90.0 min.

9. Application of the method for constructing a characteristic spectrum of a traditional Chinese medicine composition according to any one of claims 1 to 8 in quality detection and / or quality evaluation and / or quality control of the traditional Chinese medicine composition.

10. The use according to claim 9, characterized in that: When the Chinese medicine composition is Shoutai Pills, in the quality inspection and / or quality evaluation and / or quality control of Shoutai Pills, the intrinsic characteristic ingredients of stir-fried Cuscuta australis include hyperoside and isoquercetin; the intrinsic characteristic ingredients of Viscum album include gallic acid and catechins, and the intrinsic characteristic ingredients of Dipsacus asper include loganinic acid, loganin and Dipsacus asper saponin VI.

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