WO2017148426A1 - Method for determining fingerprint of chinese medicine composition - Google Patents

Abstract

Provided is a method for determining a high-performance liquid chromatographic fingerprint of a Chinese medicine composition. The Chinese medicine composition consists of forsythia, Japanese honeysuckle, indigowoad root, bitter almond, mentha-camphor, houttuynia cordata, rhubarb, patchouli, male fern rhizome, kirilow rhodiola root and rhizome, ephedra, licorice, and gypsum. The fingerprint determination method detects 15 main chromatographic peaks, and identifies nine of the main chromatographic peaks as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoforsythiaside A, forsythiaside A, forsythin, quercitrin, 4,5-di-O-caffeoylquinic acid, and glycyrrhizic acid ammonium salt, respectively.

Description

Method for determining fingerprint of traditional Chinese medicine composition Technical field

The invention relates to a method for determining a fingerprint of a traditional Chinese medicine composition.

Background technique

The fingerprint of traditional Chinese medicine refers to the chromatogram or spectrum of some Chinese herbal medicines or traditional Chinese medicine preparations which have been subjected to proper analysis and can be marked with chemical characteristics. The fingerprint of traditional Chinese medicine is a comprehensive and quantifiable method for identification. It is based on the study of the chemical composition system of traditional Chinese medicine, and is mainly used to evaluate the authenticity, superiority and stability of the quality of Chinese herbal medicines and semi-finished products of traditional Chinese medicine preparations. "Integrality" and "fuzziness" are distinctive features. The use of fingerprints as a quality control method for extracts of traditional Chinese medicines (natural medicines) and their preparations has become an international consensus, and various fingerprint control technology systems that conform to the characteristics of traditional Chinese medicines (natural medicines) are being researched and established. The US Food and Drug Administration (FDA) allows chromatographic fingerprinting in herbal health product declarations; the World Health Organization (WHO) also provides guidelines for herbal evaluation in 1996 that provide chromatographic fingerprints if the active ingredients of the herbs are not clear. To prove the consistency of product quality; the European Community also said in the herbal quality guidelines that it is not enough to determine the stability of an active ingredient alone, because the herbs and their preparations are active as a whole. Chromatographic fingerprints, especially the sharp fingerprints of thin-layer chromatography, are useful. The application of foreign fingerprints aims to solve the problem of the quality of botanical drug detection and the difference in batch quality between products with complex composition and unclear active ingredients.

High-performance liquid chromatography (HPLC) fingerprint with high resolution, which can separate complex chemical components to form different chromatograms. The height and peak area of these peaks represent various Different chemical compositions and their contents. It can be seen that the fingerprint development of traditional Chinese medicine is further developed than the DNA fingerprinting: not only the characteristic manifestation (the number and relative position of various chemical components - retention time) can be used for qualitative identification, but also the concept of quantity. . The height and peak area of the peak indicate the content of a certain chemical component, and the ratio of the peak height (or peak area) of each peak reflects the relative content of various chemical components; the introduction of the concept of quantity, the combination of qualitative and quantitative It can give greater effect to the fingerprint of traditional Chinese medicine; the fingerprint of traditional Chinese medicine can not only identify the “uniqueness” of individuals and certain species, but also link the characteristics of “quantity” with other systems. Therefore, the fingerprint of traditional Chinese medicine is not only a quality control mode and technology of traditional Chinese medicine, but also can be developed into a research system and research mode that uses various fingerprints to carry out traditional Chinese medicine theory (complex system) and new drug development. The fingerprint should be fingerprinted, ie: (1) strong. It means that the fingerprints developed should be unique to the traditional Chinese medicine and distinguish it from other traditional Chinese medicines. The chemical information reflected is highly selective; (2) the stability is good. That is, the fingerprint of traditional Chinese medicine should be the commonality from multiple batches of a certain Chinese medicine, and the common peak or characteristic peak in the map should be relatively stable; (3) good reproducibility. The fingerprints developed should be able to reproduce the fingerprint features (such as the number, size, location, etc. of the common peaks) under specified conditions, and the error should be within the allowable range. Only in this way can the fingerprints developed have practical value to effectively control the quality of the drugs. The UPLC fingerprint method is used to control the quality of the drug. It is sensitive, rapid, simple and accurate. The chromatographic conditions of the drug preparation can be used to determine the quality of the drug.

The fingerprint of traditional Chinese medicine can be used in various stages of research and production process of traditional Chinese medicine preparations. It can be divided into the fingerprints of traditional Chinese medicines (raw materials), the fingerprints of traditional Chinese medicines (including decoction pieces and compatible particles) and the fingerprints of traditional Chinese medicine preparations. . If it is finer, it can also include fingerprints for intermediates in the process production process. It is worth noting that the establishment of chemical fingerprints of Chinese herbal medicines must take into account the influence of many factors. The variety of traditional Chinese medicines, the historically formed foreign bodies of the same name, the synonyms of the same name, and the fluctuation of the same species in the environment of the place of origin and the components contained in different effective parts have brought many problems to the quality control. Therefore, for a fingerprint of a certain Chinese herbal medicine, a large amount of data accumulation is required to establish a reasonable similarity standard. Traditional Chinese medicines and their preparations are multi-component complex systems. Therefore, the quality should be evaluated according to their suitability. It can provide a wealth of identification information, but the current methods of microscopic identification, physical and chemical identification and content determination are not enough. To solve this problem, the establishment of traditional Chinese medicine fingerprints will be able to more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicines and their preparations, and then describe and evaluate the quality of medicines as a whole. This is also in line with the overall theory of Chinese medicine. On this basis, if the spectral effect study is further carried out, the quality of traditional Chinese medicine and its efficacy can be truly combined, which helps to elucidate the mechanism of action of traditional Chinese medicine. In short, the research and establishment of the fingerprint of traditional Chinese medicine is of great significance for improving the quality of traditional Chinese medicine and promoting the modernization of traditional Chinese medicine.

The patent application number is 201210450660.0, and the invention name is: a preparation process of an antiviral traditional Chinese medicine composition and a method for measuring a fingerprint, and a method for determining a fingerprint of an extract of forsythia, rhubarb, ephedra, and houttuynia by ultrasonic countercurrent extraction is disclosed. Twenty-seven shared peaks were identified, and four of the common peaks were identified, namely, forsythiaside A, forsythin, rosin-4-O-glc, and rhein. The application does not disclose the technical contents of the present invention. The invention requires A method for determining a high performance liquid chromatographic fingerprint of a traditional Chinese medicine composition, which comprises: forsythia, honeysuckle, radix isatidis, bitter almond, menthol, houttuynia, rhubarb, patchouli, cotton Ma Guanzhong, Rhodiola, Ephedra, Licorice, Gypsum, the fingerprinting method, calibrated 15 major chromatographic peaks, identified 9 of them, respectively, new chlorogenic acid, chlorogenic acid, crypto-acid, different Forsythiaside A, forsythiaside A, forsythin, quercetin, 4,5-di-O-caffeoylquinic acid, forsythin, glycyrrhizic acid, and this is not disclosed in the prior art. Technical content.

Summary of the invention

The invention provides a method for determining a fingerprint of a traditional Chinese medicine composition.

A method for determining a fingerprint of a traditional Chinese medicine composition, the raw materials of the traditional Chinese medicine composition include: forsythia, ephedra, rhubarb, houttuynia, honeysuckle, radix isatidis, patchouli, Mianma Guanzhong, Rhodiola, menthol , bitter almond, licorice, gypsum; the fingerprinting method comprises the following steps:

Preparation of the test solution: taking the traditional Chinese medicine composition, and extracting with an organic solvent to obtain a test solution;

Preparation of reference solution: fresh chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoylquinine The acid, quercetin, ammonium glycyrrhizinate reference substance is dissolved in an organic solvent to obtain a reference solution;

Preparation of the reference solution: take appropriate amount of forsythiaside A reference substance, add organic solvent to dissolve, to obtain a reference solution;

Chromatographic conditions: a methanol-acetonitrile-phosphoric acid aqueous solution as a mobile phase, gradient elution, the concentration of the aqueous phosphoric acid solution is 0.1% to 0.5%;

Determination method: separately draw the reference solution and the test solution, inject into the liquid chromatograph, and measure, that is, obtain.

Preferably, the organic solvent comprises 50% to 100% methanol solution, 50% to 100% ethanol solution; further preferably, the organic solvent is 50% methanol.

Preferably, the gradient elution procedure is:

Preferably, the detection wavelength is 200-250 nm.

Preferably, the traditional Chinese medicine composition is prepared from the following raw materials by weight: Forsythia 200-300, Ephedra 60-100, Rhubarb 40-60, Houttuynia 200-300, Honeysuckle 200-300, Banlangen 200-300, Patchouli 60-100, Mianma Guanzhong 200-300, Rhodiola 60-100, menthol 5-9, bitter almond 60-100, licorice 60-100, gypsum 200-300, fingerprinting method is as follows:

Preparation of test solution: Take 0.1-0.5g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, precisely add 40-100% methanol 10-50mL, weigh the weight, sonicate for 20-40 minutes , let cool, then weigh the weight, make up the lost weight with 40-100% methanol, shake, filter, take the filtrate, get it;

Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-coffee The acyl quinic acid, quercetin, ammonium glycyrrhizinate reference substance was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml, respectively. Forsythiaside A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4μg/ml ammonium glycyrrhizinate mixed control solution is obtained;

Preparation of the reference solution: Take the appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make a solution containing 101.2 μg per 1 ml, that is.

Chromatographic conditions: the mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, gradient elution procedure was:

The column is a C ₁₈ column; the detection wavelength is 239 nm, and the flow rate is 0.1-0.5 ml/min;

Determination method: respectively, accurately draw the reference solution and the test solution each 1-10μL, inject into the ultra-high liquid chromatograph, determine, record the chromatogram, that is.

The preferred pharmaceutical ingredients of the traditional Chinese medicine composition of the present invention are: Forsythia 200, Honeysuckle 300, Banlangen 200, Rhubarb 40, Patchouli 60, Mianma Guanzhong 300, Rhodiola 100, Menthol 9, Ephedra 60, bitter almond 100, houttuynia cordica 200, licorice 100, gypsum 200.

The preferred parts of the traditional Chinese medicine composition of the present invention are: Forsythia 300, Honeysuckle 200, Banlangen 300, Rhubarb 60, Patchouli 100, Mianma Guanzhong 200, Rhodiola 60, Menthol 5, Ephedra 100, bitter almond 60, houttuyniasis 300, licorice 60, gypsum 300.

The preferred pharmaceutical ingredients of the traditional Chinese medicine composition of the present invention are: Forsythia 278, Honeysuckle 294, Radix Isatidis 285, Rhubarb 55, Patchouli 95, Mianma Guanzhong 290, Rhodiola 87, Menthol 8.5, Ephedra 88, bitter almond 80, houttuynia 284, licorice 95, gypsum 277.

The preferred pharmaceutical ingredients of the traditional Chinese medicine composition of the present invention are: Forsythia 255, Honeysuckle 255, Radix Isatidis 255, Rhubarb 51, Patchouli 85, Mianma Guanzhong 255, Rhodiola 85, Menthol 7.5, Ephedra 85, bitter almond 85, houttuynia 255, licorice 85, gypsum 255.

The preparation method of the traditional Chinese medicine composition preparation of the invention is:

(1) Weigh Chinese medicines according to the weight ratio of raw materials, net selection, and break as appropriate;

(2) Patchouli scented, add 10 times the amount of water to extract volatile oil, oil extraction time 8 hours, collect volatile oil, spare; after the extract is filtered, the residue is discarded, the filtrate is reserved;

(3) Forsythia, Ephedra, Houttuynia cordata, Rhubarb, extracted with 12 times 70% ethanol for 3 times, each time for 2.5 hours, the extract is combined with filtration, ethanol is recovered, and the filtrate is reserved;

(4) Honeysuckle, gypsum, Banlangen, Mianma Guanzhong, Licorice, Rhodiola, add 12 times the amount of water to boil, add bitter almonds, decoct 2 times, each time for 1 hour, extract and filter, the result The filtrate is combined with the filtrate of step (2) patchouli oil extraction, and concentrated to obtain a clear paste having a relative density of 1.10-15.15 at 60 ° C, added with ethanol, adjusted to an alcohol concentration of 70%, refrigerated, filtered, and recovered. Ethanol to no alcohol, get a clear paste;

(5) Combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), and concentrating to obtain a clear paste having a relative density of 1.15 - 1.20 at 60 ° C, and drying to obtain a dry paste powder, which is used;

(6) adding the dry paste powder obtained in the step (5) to a suitable pharmaceutically acceptable auxiliary material for granulation;

(7) The menthol and the volatile oil obtained in the step (2) are dissolved in ethanol, and the granules obtained in the step (6) are sprayed, sealed, mixed, tableted or capsuled, or bagged.

The method for determining the fingerprint of the present invention is preferably:

Preparation of test solution: Take 0.25g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, precisely add 50mL methanol 25mL, weigh the weight, sonication (power 500W, frequency 40kHz) for 30 minutes, Let cool, then weigh the weight, make up the lost weight with 50% methanol, shake it, filter it, take the filtrate, and get it;

Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg /ml ammonium glycyrrhizinate mixed control solution is obtained;

Preparation of the reference solution: Take the appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make a solution containing 101.2 μg per 1 ml, that is.

Chromatographic conditions: the mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, gradient elution procedure was:

The column was a Waters Acquity UPLC HSS T3 column with a column length of 100 mm, an inner diameter of 2.1 mm, a particle size of 1.8 μm, a detection wavelength of 239 nm, and a flow rate of 0.3 ml/min;

Assay method: accurately draw 2 μL of each of the reference solution and the test solution, inject into an ultra-high liquid chromatograph, measure and record the chromatogram, that is, obtain.

The fingerprint mapping method of the present invention is also preferably:

Preparation of test solution: Take 0.1g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, add 50mL of methanol precisely, weigh the weight, sonicate for 40 minutes, let cool, then weigh the weight, use Make up the lost weight of methanol, shake it, filter it, take the filtrate, and get it;

Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg /ml ammonium glycyrrhizinate mixed control solution is obtained;

Preparation of the reference solution: Take the appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make a solution containing 101.2 μg per 1 ml, that is.

Chromatographic conditions: the mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, gradient elution procedure was:

The column was a Waters Acquity UPLC HSS T3 column; the detection wavelength was 239 nm, and the flow rate was 0.1 ml/min;

Determination method: respectively, accurately draw 1 μL of the reference solution and the test solution, inject into the ultra-high liquid chromatograph, measure and record the chromatogram, that is, obtain.

The fingerprint mapping method of the present invention is also preferably:

Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, accurately add 40mL methanol to 10mL, weigh the weight, sonicate for 20 minutes, let cool, then weigh the weight. , use 40% methanol to make up the lost weight, shake, filter, and take the filtrate to obtain;

Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg /ml ammonium glycyrrhizinate mixed control solution is obtained;

Preparation of the reference solution: Take the appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make a solution containing 101.2 μg per 1 ml, that is.

Chromatographic conditions: the mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, gradient elution procedure was:

The column was a column Waters XBridge ^TM Shield RP18; detection wavelength 239nm, flow rate 0.5ml / min;

Determination method: respectively, accurately draw 1 μL of the reference solution and the test solution, inject into the ultra-high liquid chromatograph, measure and record the chromatogram, that is, obtain.

The fingerprint mapping method of the present invention is also preferably:

Preparation of test solution: Take 0.2g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, accurately add 20mL of methanol 20mL, weigh the weight, sonicate for 40 minutes, let cool, then weigh the weight , use 90% methanol to make up the lost weight, shake, filter, and take the filtrate to obtain;

Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg /ml ammonium glycyrrhizinate mixed control solution is obtained;

Preparation of the reference solution: Take the appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make a solution containing 101.2 μg per 1 ml, that is.

Chromatographic conditions: the mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, gradient elution procedure was:

The column was Phenomenex Kinetx XB-C18; the detection wavelength was 239 nm, and the flow rate was 0.4 ml/min;

Assay method: accurately draw 6 μL of each of the reference solution and the test solution, inject into the ultra-high liquid chromatograph, measure and record the chromatogram, that is, obtain.

Using the sample prepared in Example 1, the method for determining the fingerprint of the traditional Chinese medicine composition of the present invention was evaluated in various aspects, and the evaluation method was as follows:

1. Instruments, reagents and reagents

1.1 Instrument

American Waters UPLC H-CLASS Ultra High Performance Liquid Chromatograph (PDA detector, Empower 3.0 chromatography workstation), circulating multi-purpose vacuum pump (Zhengzhou Changcheng Branch Industry and Trade Co., Ltd.), KQ250DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.) , Switzerland METTLER TOLEDOAL204 electronic analytical balance, Switzerland METTLERTOLEDOAB135-S electronic analytical balance, Shanghai Jingke JA2603B electronic balance.

1.2 reagent

Forsythin (China Food and Drug Control Institute, batch number 110821-201213, purity 95.3%), forsythiaside A (China Food and Drug Control Institute, batch number 111810-201304, purity 94.3%), new chlorogenic acid (Chengdupu Ruifa Technology Development Co., Ltd., batch number: 13112712, purity ≥98%), chlorogenic acid (China Food and Drug Control Institute, batch number: 110753-201314, purity, 96.6%), cryptochlorogenic acid (Shanghai Yuanye Biotechnology) Co., Ltd., batch number: ZF0226BA14, purity ≥98%), isophoric acid ester A (Shanghai Yuanye Biotechnology Co., Ltd., batch number: YA0817HB14, purity ≥98%), 4,5-di-O-caffeoylquinine Acid (China Food and Drug Control Institute, batch number: 111894-201102, purity, 94.1%), saponin (HWI ANALYTIK GMBH pharma solutions, batch number: HWI01112, purity, 91.7%), ammonium glycinate (Council of Europe-EDQM, Batch No.: Batch: 1.1, purity ≥98%), methanol (analytical grade, Tianjin Guangfu Technology Development Co., Ltd., batch number January 13, 2014), phosphoric acid (analytical grade, Tianjin Guangfu Technology Development Co., Ltd., batch number 2014 March 26), acetonitrile (chromatographically pure, Fisher, USA, LOT 106246).

1.3 test drugs

Traditional Chinese medicine composition of the invention (Shijiazhuang Yiling Pharmaceutical Co., Ltd.)

2. Optimization of chromatographic conditions

2.1. Selection of detection wavelength

The chromatograms of the test solutions at different detection wavelengths were examined at 210 nm to 400 nm, and the information amount at 239 nm was rich. Therefore, the detection wavelength was optimally selected to be 239 nm. For details, see the following Figures 1-4.

2.2 Selection of mobile phase system

This study compared three mobile phase systems of methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid and methanol-acetonitrile-0.1% phosphoric acid. The results showed that the resolution of the methanol-acetonitrile-0.1% phosphoric acid system peak was the best. See Figure 5-7 for details. Therefore, the mobile phase system was determined to be methanol-acetonitrile-0.1% phosphoric acid. The gradient elution table is shown in Table 1. The flow rate was 0.3 ml/min.

Table 1 Mobile phase gradient elution table

3. Preparation of test solution and reference solution

3.1 Investigation of extraction solvent

The extraction results of methanol, 90% methanol, 70% methanol and 50% methanol were compared. The results showed that the chromatographic peaks of the fingerprints obtained by 50% methanol extraction were the most. See Figure 8-11 for details. Therefore, 50% methanol was selected as the extraction. Solvent.

3.2 Investigation of extraction methods

The ultrasonic and reflow methods were compared. The number and intensity of the chromatographic peaks obtained by the ultrasonic and reflux extraction methods were not significantly different. For details, see Figure 12-13. Since the ultrasonic extraction method is simple, the ultrasound is selected. extract.

3.3 Ultrasonic time investigation

Ultrasound was compared for 20 minutes, 30 minutes, and 40 minutes. The results showed that the amount of peak information in the fingerprints obtained by ultrasound for 30 minutes and 40 minutes was large, and there was no significant difference. For details, see Figure 14-16. 30 minutes.

3.4 Determination of the preparation method of the test solution: Take the appropriate amount of the content of the traditional Chinese medicine composition of the invention, grind finely, take 0.25g, accurately weigh, place in a conical flask, precisely add 50mL methanol 25mL, weigh the weight, Ultrasonic treatment (power 500W, frequency 40kHz) for 30 minutes, let cool, then weigh the weight, use 50% methanol to make up the lost weight, shake, filter, and take the filtrate to obtain.

3.5 Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeyl Quinine, quercetin, ammonium glycyrrhizinate reference substance, respectively, made of 18.4μg / ml new chlorogenic acid, 27.2μg / ml chlorogenic acid, 21.2μg / ml cryptochlorogenic acid, 27.2μg / ml Phytosterol A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 Μg/ml ammonium glycyrrhizinate mixed control solution is obtained.

3.6 Preparation of reference solution: Take appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make 101.2μg solution per 1ml, that is.

Spectral characteristics and identification of the main chromatographic peaks Under the determined chromatographic conditions, the reference solution and the test solution were injected separately, and 16 main chromatographic peaks were determined in the chromatogram of the test solution. According to the retention time and spectral characteristics of the chromatographic peak, it is determined that the peak of No. 2 in the fingerprint is new chlorogenic acid, the peak of No. 3 is chlorogenic acid, the peak of No. 5 is cryptochlorogenic acid, and the peak of No. 8 is isoprenoid A. Peak 11 is forsythiaside A, peak 13 is quercetin, peak 14 is 4,5-di-O-caffeoylquinic acid, peak 15 is forsythin, and peak 16 is glycyrrhizic acid. The chromatograms and spectra of the above reference and test samples are shown in Figures 17-28.

Precision

The test solution was taken and continuously injected 6 times according to the determined chromatographic conditions. The RSD of each major peak area was less than 5%, indicating that the injection precision was good, and the results are shown in Table 2.

Table 2 precision inspection results

Repeatability

Taking 6 parts of the content of the traditional Chinese medicine composition of the present invention, prepared according to the determined "preparation method of the test solution", and injecting into a liquid chromatograph to determine that the RSD of each major peak area is less than 5%, indicating the method repeatability. Preferably, the results are shown in Table 3.

Table 3 results of repetitive investigation

stability

The test solution was taken at 0h, 2h, 4h, 8h, 12h, 24h, and the RSD of each major peak area was less than 10%, indicating that the test solution was stable within 24h. The results are shown in Table 4. .

Table 4 stability investigation results

Fingerprint Mapping and Similarity Analysis

8.1 Fingerprint establishment: Take 10 batches of the sample of the traditional Chinese medicine composition of the invention, prepare the test solution according to the requirements of 3.4, and carry out the sample analysis under the above chromatographic conditions to obtain the fingerprints of 10 batches of the traditional Chinese medicine composition of the invention, and adopt The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A version developed by the National Pharmacopoeia Committee analyzed the fingerprints of 10 batches of samples. The median method was used to correct and automatically match the chromatographic peaks of each fingerprint to calculate the similarity. A comparison fingerprint was generated, as shown in Figure 29-30. The similarity results are shown in Table 5. There are 16 chromatographic peaks in the fingerprint, and 9 common peaks were identified by standard: new chlorogenic acid (peak 2), green Acid (peak 3), cryptochlorogenic acid (peak 5), forsythine A (peak 8), forsythiaside A (peak 11), quercetin (peak 13), 4 , 5-di-O-caffeoylquinic acid (peak 14), forsythin (peak 15), glycyrrhizic acid (peak 16). Among them, the 11th chromatographic peak forsythiaside A with stable peak and large peak area is the reference peak:

Table 5 Fingerprint similarity of capsules in 10 batches of traditional Chinese medicine composition

The invention establishes a liquid phase fingerprinting method of the traditional Chinese medicine composition of the invention, which has 16 chromatographic peaks, and 9 common peaks are identified by standard products: new chlorogenic acid (peak No. 2) and chlorogenic acid (peak No. 3). , cryptochlorogenic acid (peak 5), forsythine A (peak 8), forsythiaside A (peak 11), quercetin (peak 13), 4,5-di-O - caffeoyl quinic acid (peak 14), forsythin (peak 15), glycyrrhizic acid (peak 16). Among them, the 11th chromatographic peak forsythiaside A with a stable peak and a large peak area was used as a reference peak, and the similarity of the fingerprints of the 10 batches of the traditional Chinese medicine composition of the present invention was high. It can be seen from the above experimental results that the method has good precision and stability, and repeatability, and provides a new method for improving the quality control of the traditional Chinese medicine composition.

DRAWINGS

Figure 1: Chromatogram of the test solution at a detection wavelength of 320 nm

Figure 2: Chromatogram of the test solution at a detection wavelength of 290 nm

Figure 3: Chromatogram of the test solution at a detection wavelength of 254 nm

Figure 4: Chromatogram of the test solution at a detection wavelength of 230 nm

Figure 5: Chromatogram of methanol-0.1% phosphoric acid test solution

Figure 6: Chromatogram of acetonitrile-0.1% phosphoric acid test solution

Figure 7: Chromatogram of methanol-acetonitrile-0.1% phosphoric acid test solution

Figure 8: Chromatogram of 50% methanol extraction test solution

Figure 9: Chromatogram of 70% methanol extraction test solution

Figure 10: Chromatogram of 90% methanol extraction test solution

Figure 11: Chromatogram of methanol extraction test solution

Figure 12: Chromatogram of the sample solution for 30 minutes of ultrasound

Figure 13: Chromatogram of the test solution for 1 hour of reflux

Figure 14: Chromatogram of the test solution for 20 minutes of ultrasound

Figure 15: Chromatogram of the sample solution for 30 minutes of ultrasound

Figure 16: Chromatogram of the solution for 40 minutes of ultrasound

Figure 17: Chromatogram of the reference solution

Figure 18: Chromatogram of the forsythiaside A reference solution

Figure 19: Chromatogram of the test solution

Figure 20: Comparison of the spectrum of the new chlorogenic acid in the reference solution and the test solution, the dotted line is the spectrum of the sample, and the solid line is the spectrum of the reference.

Figure 21: Comparison of the chlorogenic acid spectrum of the reference solution and the test solution, the dotted line is the spectrum of the sample, and the solid line is the spectrum of the reference.

Figure 22: Comparison of the spectrum of the cryptochlorogenic acid in the reference solution and the test solution, the dotted line is the spectrum of the sample, and the solid line is the spectrum of the reference.

Figure 23: Comparison of the reference solution of the reference solution with the testosterone A in the test solution, the dotted line is the spectrum of the test sample, and the solid line is the spectrum of the reference product.

Figure 24: Comparison of the reference solution of forsythiaside A in the reference solution and the test solution, the dotted line is the spectrum of the test sample, and the solid line is the spectrum of the reference product.

Figure 25: Comparison of the quercetin spectrum of the reference solution and the test solution, the dotted line is the spectrum of the sample, and the solid line is the spectrum of the reference.

Figure 26: Comparison of the spectrum of 4,5-di-O-caffeoylquinic acid in the reference solution and the test solution. The dotted line is the spectrum of the sample, and the solid line is the spectrum of the reference.

Figure 27: Comparison of the forsythiaside spectra of the reference solution and the test solution, the dotted line is the spectrum of the test sample, and the solid line is the reference spectrum of the reference product.

Figure 28: Comparison of the spectrum of glycyrrhizic acid in the reference solution and the test solution, the dotted line is the spectrum of the sample, and the solid line is the spectrum of the reference.

Figure 29: Chromatographic fingerprint of 10 batches of traditional Chinese medicine composition of the present invention

Figure 30: Control fingerprint of traditional Chinese medicine composition of the present invention

Figure 31: Fingerprint of Example 1, wherein the peak 1 in Figure A is new chlorogenic acid, 2 is chlorogenic acid, 3 is cryptochlorogenic acid, 4 is berberine A, and 5 is forsythiaside A. 6 is quercetin, 7 is 4,5-di-O-caffeoylquinic acid, 8 is forsythin, 9 is glycyrrhizic acid; B is a chromatogram of forsythiaside A reference solution; C is Fingerprint chromatogram of test solution

Figure 32: Fingerprint of Example 2, wherein the peak 1 in Figure A is new chlorogenic acid, 2 is chlorogenic acid, 3 is cryptochlorogenic acid, 4 is berberine A, and 5 is forsythiaside A. 6 is quercetin, 7 is 4,5-di-O-caffeoylquinic acid, 8 is forsythin, 9 is glycyrrhizic acid; B is a chromatogram of forsythiaside A reference solution; C is Fingerprint chromatogram of test solution

Figure 33: Fingerprint of Example 3, wherein the peak 1 in Figure A is neochlorogenic acid, 2 is chlorogenic acid, 3 is cryptochlorogenic acid, 4 is isopenic acid ester A, and 5 is forsythiaside A. 6 is quercetin, 7 is 4,5-di-O-caffeoylquinic acid, 8 is forsythin, 9 is glycyrrhizic acid; B is a chromatogram of forsythiaside A reference solution; C is Fingerprint chromatogram of test solution

Figure 34: Fingerprint of Example 4, wherein the peak 1 in Figure A is new chlorogenic acid, 2 is chlorogenic acid, 3 is cryptochlorogenic acid, 4 is berberine A, and 5 is forsythiaside A. 6 is quercetin, 7 is 4,5-di-O-caffeoylquinic acid, 8 is forsythin, 9 is glycyrrhizic acid; B is a chromatogram of forsythiaside A reference solution; C is Fingerprint chromatogram of test solution

detailed description

Example 1

Weighed according to the ratio: Forsythia 200g, Honeysuckle 300g, Radix Isatidis 200g, Rhubarb 40g, Patchouli 60g, Mianma Guanzhong 300g, Rhodiola 100g, Menthol 9g, Ephedra 60g, Bitter Almond 100g, Houttuynia 200g, Licorice 100g, gypsum 200g, extracted according to the following process:

(1) Weigh Chinese medicines according to the weight ratio of raw materials, net selection, and break as appropriate;

(2) Patchouli scented, add 10 times the amount of water to extract volatile oil, oil extraction time 8 hours, collect volatile oil, spare; after the extract is filtered, the residue is discarded, the filtrate is reserved;

(3) Forsythia, Ephedra, Houttuynia cordata and Rhubarb, extracted with 12 times 70% ethanol for 3 times, each time for 2.5 hours, the extract is combined with filtration, and the ethanol is recovered and filtered. Liquid reserve

(4) Honeysuckle, gypsum, Banlangen, Mianma Guanzhong, Licorice, Rhodiola, add 12 times the amount of water to boil, add bitter almonds, decoct 2 times, each time for 1 hour, extract and filter, the result The filtrate is combined with the filtrate of step (2) patchouli oil extraction, and concentrated to obtain a clear paste having a relative density of 1.10-15.15 at 60 ° C, added with ethanol, adjusted to an alcohol concentration of 70%, refrigerated, filtered, and recovered. Ethanol to no alcohol, get a clear paste;

(5) Combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), and concentrating to obtain a clear paste having a relative density of 1.15 - 1.20 at 60 ° C, and drying to obtain a dry paste powder, which is used;

(6) adding the dry paste powder obtained in the step (5) to a suitable pharmaceutically acceptable auxiliary material for granulation;

(7) The menthol and the volatile oil obtained in the step (2) are dissolved in ethanol, and the granules obtained in the step (6) are sprayed, and capsules are obtained.

Fingerprint measurement method:

Preparation of test solution: Take 0.25g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, precisely add 50mL methanol 25mL, weigh the weight, ultrasonic power 500W, frequency 40kHz, ultrasonic 30 minutes, put Cold, weigh the weight again, make up the lost weight with 50% methanol, shake it, filter it, take the filtrate, and get it;

Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg /ml ammonium glycyrrhizinate mixed control solution is obtained;

Preparation of the reference solution: Take the appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make a solution containing 101.2 μg per 1 ml, that is.

Chromatographic conditions: the mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, gradient elution procedure was:

The column was a Waters Acquity UPLC HSS T3 column with a column length of 100 mm, an inner diameter of 2.1 mm, a particle size of 1.8 μm, a detection wavelength of 239 nm, and a flow rate of 0.3 ml/min;

Assay method: accurately draw 2 μL of each of the reference solution and the test solution, inject into an ultra-high liquid chromatograph, measure and record the chromatogram, that is, obtain.

Conclusion: The fingerprint is shown in Figure 31, and the results are satisfactory for controlling the quality of the Chinese medicine composition.

Example 2

Weighed in proportion: Forsythia 300g, Honeysuckle 200g, Radix Isatidis 300g, Rhubarb 60g, Patchouli 100g, Mianma Guanzhong 200g, Rhodiola 60g, Menthol 5g, Ephedra 100g, Bitter Almond 60g, Houttuynia 300g, Glycyrrhiza 60g, gypsum 300g, extracted according to the following process:

(1) Weigh Chinese medicines according to the weight ratio of raw materials, net selection, and break as appropriate;

(2) Patchouli scented, add 10 times the amount of water to extract volatile oil, oil extraction time 8 hours, collect volatile oil, spare; after the extract is filtered, the residue is discarded, the filtrate is reserved;

(3) Forsythia, Ephedra, Houttuynia cordata, Rhubarb, extracted with 12 times 70% ethanol for 3 times, each time for 2.5 hours, the extract is combined with filtration, ethanol is recovered, and the filtrate is reserved;

(4) Honeysuckle, gypsum, Banlangen, Mianma Guanzhong, Licorice, Rhodiola, add 12 times the amount of water to boil, add bitter almonds, decoct 2 times, each time for 1 hour, extract and filter, the result The filtrate is combined with the filtrate of step (2) patchouli oil extraction, and concentrated to obtain a clear paste having a relative density of 1.10-15.15 at 60 ° C, added with ethanol, adjusted to an alcohol concentration of 70%, refrigerated, filtered, and recovered. Ethanol to no alcohol, get a clear paste;

(5) Combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), and concentrating to obtain a clear paste having a relative density of 1.15 - 1.20 at 60 ° C, and drying to obtain a dry paste powder, which is used;

(6) adding the dry paste powder obtained in the step (5) to a suitable pharmaceutically acceptable auxiliary material for granulation;

(7) The menthol and the volatile oil obtained in the step (2) are dissolved in ethanol, and the granules obtained in the step (6) are sprayed and tableted.

Fingerprint measurement method:

Preparation of test solution: Take 0.1g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, accurately add 50mL of methanol, 50mL, weigh the weight, sonicate for 40 minutes, let cool, then weigh the weight. , use 100% methanol to make up the lost weight, shake, filter, and take the filtrate to obtain;

Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg /ml ammonium glycyrrhizinate mixed control solution is obtained;

Preparation of the reference solution: Take the appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make a solution containing 101.2 μg per 1 ml, that is.

Chromatographic conditions: the mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, gradient elution procedure was:

The column was a Waters Acquity UPLC HSS T3 (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm) column; detection wavelength 239 nm, flow rate 0.1 ml / min;

Determination method: respectively, accurately draw 1 μL of the reference solution and the test solution, inject into the ultra-high liquid chromatograph, measure and record the chromatogram, that is, obtain.

Conclusion: The fingerprint is shown in Figure 32. The results are satisfactory and can be used to control the quality of the Chinese herbal composition.

Example 3

The raw material formula is: 278g forsythia, 294g for honeysuckle, 285g for radix isatidis, 55g for rhubarb, 95g for patchouli, 290g for cotton horse, and 87g for Rhodiola Menthol 8.5g, ephedra 88g, bitter almond 80g, houttuynia 284g, licorice 95g, gypsum 277g, extracted according to the following process:

(1) Weigh Chinese medicines according to the weight ratio of raw materials, net selection, and break as appropriate;

(2) Patchouli scented, add 10 times the amount of water to extract volatile oil, oil extraction time 8 hours, collect volatile oil, spare; after the extract is filtered, the residue is discarded, the filtrate is reserved;

(3) Forsythia, Ephedra, Houttuynia cordata, Rhubarb, extracted with 12 times 70% ethanol for 3 times, each time for 2.5 hours, the extract is combined with filtration, ethanol is recovered, and the filtrate is reserved;

(4) Honeysuckle, gypsum, Banlangen, Mianma Guanzhong, Licorice, Rhodiola, add 12 times the amount of water to boil, add bitter almonds, decoct 2 times, each time for 1 hour, extract and filter, the result The filtrate is combined with the filtrate of step (2) patchouli oil extraction, and concentrated to obtain a clear paste having a relative density of 1.10-15.15 at 60 ° C, added with ethanol, adjusted to an alcohol concentration of 70%, refrigerated, filtered, and recovered. Ethanol to no alcohol, get a clear paste;

(5) Combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), and concentrating to obtain a clear paste having a relative density of 1.15 - 1.20 at 60 ° C, and drying to obtain a dry paste powder, which is used;

(6) adding the dry paste powder obtained in the step (5) to a suitable pharmaceutically acceptable auxiliary material for granulation;

(7) The menthol and the volatile oil obtained in the step (2) are dissolved in ethanol, and the granules obtained in the step (6) are sprayed, and the bag is obtained.

Fingerprint measurement method:

Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, accurately add 40mL methanol to 10mL, weigh the weight, sonicate for 20 minutes, let cool, then weigh the weight. , use 40% methanol to make up the lost weight, shake, filter, and take the filtrate to obtain;

Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg /ml ammonium glycyrrhizinate mixed control solution is obtained;

Preparation of the reference solution: Take the appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make a solution containing 101.2 μg per 1 ml, that is.

Chromatographic conditions: the mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, gradient elution procedure was:

The column was ^{Waters XBridge TM Shield RP18 (5μm,} 4.6mm × 250mm) column; detection wavelength 239nm, flow rate 0.5ml / min;

Determination method: respectively, accurately draw 1 μL of the reference solution and the test solution, inject into the ultra-high liquid chromatograph, measure and record the chromatogram, that is, obtain.

Results: The fingerprint is shown in Figure 33 and the results were satisfactory to control the quality of the Chinese herbal composition.

Example 4

The raw material formula is: weighed according to the ratio: Forsythia 255g, Honeysuckle 255g, Radix Isatidis 255g, Rhubarb 51g, Patchouli 85g, Mianma Guanzhong 255g, Rhodiola 85g, Menthol 7.5g, Ephedra 85g, Bitter Almond 85g Houttuynia 255g, licorice 85g, gypsum 255g, extracted according to the following process:

(1) Weigh Chinese medicines according to the weight ratio of raw materials, net selection, and break as appropriate;

(2) Patchouli scented, add 10 times the amount of water to extract volatile oil, oil extraction time 8 hours, collect volatile oil, spare; after the extract is filtered, the residue is discarded, the filtrate is reserved;

(3) Forsythia, Ephedra, Houttuynia cordata, Rhubarb, extracted with 12 times 70% ethanol for 3 times, each time for 2.5 hours, the extract is combined with filtration, ethanol is recovered, and the filtrate is reserved;

(4) Honeysuckle, gypsum, Banlangen, Mianma Guanzhong, Licorice, Rhodiola, add 12 times the amount of water to boil, add bitter almonds, decoct 2 times, each time for 1 hour, extract and filter, the result The filtrate is combined with the filtrate of step (2) patchouli oil extraction, and concentrated to obtain a clear paste having a relative density of 1.10-15.15 at 60 ° C, added with ethanol, adjusted to an alcohol concentration of 70%, refrigerated, filtered, and recovered. Ethanol to no alcohol, get a clear paste;

(5) Combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), and concentrating to obtain a clear paste having a relative density of 1.15 - 1.20 at 60 ° C, and drying to obtain a dry paste powder, which is used;

(6) adding the dry paste powder obtained in the step (5) to a suitable pharmaceutically acceptable auxiliary material for granulation;

(7) The menthol and the volatile oil obtained in the step (2) are dissolved in ethanol, and the granules obtained in the step (6) are sprayed, and capsules are obtained.

Fingerprint measurement method:

Preparation of test solution: Take 0.2g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, accurately add 20mL of methanol 20mL, weigh the weight, sonicate for 40 minutes, let cool, then weigh the weight , use 90% methanol to make up the lost weight, shake, filter, and take the filtrate to obtain;

Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg /ml ammonium glycyrrhizinate mixed control solution is obtained;

Preparation of the reference solution: Take the appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make a solution containing 101.2 μg per 1 ml, that is.

Chromatographic conditions: the mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, gradient elution procedure was:

The column was a Phenomenex Kinetx XB-C18 column; the detection wavelength was 239 nm, and the flow rate was 0.4 ml/min;

Assay method: accurately draw 6 μL of each of the reference solution and the test solution, inject into the ultra-high liquid chromatograph, measure and record the chromatogram, that is, obtain.

Conclusion: The fingerprint is shown in Figure 34 and the results are satisfactory for controlling the quality of the Chinese herbal composition.

Claims (14)

1. A method for determining a fingerprint of a traditional Chinese medicine composition, the raw materials of the traditional Chinese medicine composition include: forsythia, ephedra, rhubarb, houttuynia, honeysuckle, radix isatidis, patchouli, Mianma Guanzhong, Rhodiola, menthol , bitter almond, licorice, gypsum, wherein the fingerprinting method comprises the following steps:

   Preparation of the test solution: taking the traditional Chinese medicine composition, and extracting with an organic solvent to obtain a test solution;

   Preparation of reference solution: fresh chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoylquinine The acid, quercetin, ammonium glycyrrhizinate reference substance is dissolved in an organic solvent to obtain a reference solution;

   Preparation of the reference solution: take appropriate amount of forsythiaside A reference substance, add organic solvent to dissolve, to obtain a reference solution;

   Chromatographic conditions: a methanol-acetonitrile-phosphoric acid aqueous solution as a mobile phase, gradient elution, the concentration of the aqueous phosphoric acid solution is 0.1% to 0.5%;

   Determination method: separately draw the reference solution and the test solution, inject into the liquid chromatograph, and measure, that is, obtain.
2. The fingerprinting method according to claim 1, wherein the organic solvent comprises 50% to 100% of a methanol solution and 50% to 100% of an ethanol solution.
3. The method of determining a fingerprint according to claim 1, wherein said gradient elution program is:

   
4. The fingerprint mapping method according to claim 1, wherein the detection wavelength is 200 to 250 nm.
5. The method for determining a fingerprint according to claim 1, wherein the traditional Chinese medicine composition is made of the following raw materials by weight: Forsythia 200-300, Ephedra 60-100, Rhubarb 40-60, Houttuynia cordata 200 -300, honeysuckle 200-300, Banlangen 200-300, patchouli 60-100, Mianma Guanzhong 200-300, Rhodiola 60-100, menthol 5-9, bitter almond 60-100, licorice 60- 100, gypsum 200-300; the fingerprinting method is as follows:

   Preparation of test solution: Take 0.1-0.5g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, precisely add 40-100% methanol 10-50mL, weigh the weight, sonicate for 20-40 minutes , let cool, then weigh the weight, make up the lost weight with 40-100% methanol, shake, filter, take the filtrate, get it;

   Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg/ Methyl glycyrrhizinate mixed control solution is obtained;

   Preparation of reference solution: Take appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make 101.2μg solution per 1ml, that is;

   The mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, and the gradient elution procedure was:

   

   The column is a C ₁₈ column; the detection wavelength is 239 nm, and the flow rate is 0.1-0.5 ml/min;

   Determination method: respectively, accurately draw the reference solution and the test solution each 1-10μL, inject into the ultra-high liquid chromatograph, determine, record the chromatogram, that is.
6. The method of determining a fingerprint according to claim 5, wherein the preparation of the traditional Chinese medicine composition is made of the following raw materials by weight:

   Forsythia 200, Honeysuckle 300, Banlangen 200, Rhubarb 40, Patchouli 60, Mianma Guanzhong 300, Rhodiola 100, Menthol 9, Ephedra 60, Bitter Almond 100, Houttuynia 200, Licorice 100, Gypsum 200 .
7. The method of determining a fingerprint according to claim 5, wherein the preparation of the traditional Chinese medicine composition is made of the following raw materials by weight:

   Forsythia 300, Honeysuckle 200, Banlangen 300, Rhubarb 60, Patchouli 100, Mianma Guanzhong 200, Rhodiola 60, Menthol 5, Ephedra 100, Bitter Almond 60, Houttuynia 300, Licorice 60, Gypsum 300 .
8. The method of determining a fingerprint according to claim 5, wherein the preparation of the traditional Chinese medicine composition is made of the following raw materials by weight:

   Forsythia 278, Honeysuckle 294, Banlangen 285, Rhubarb 55, Patchouli 95, Mianma Guanzhong 290, Rhodiola 87, Menthol 8.5, Ephedra 88, Bitter Almond 80, Houttuynia 284, Licorice 95, Gypsum 277 .
9. The method of determining a fingerprint according to claim 5, wherein the preparation of the traditional Chinese medicine composition is made of the following raw materials by weight:

   Forsythia 255, Honeysuckle 255, Banlangen 255, Rhubarb 51, Patchouli 85, Mianma Guanzhong 255, Rhodiola 85, Menthol 7.5, Ephedra 85, Bitter Almond 85, Houttuynia 255, Licorice 85, Gypsum 255 .
10. The method for determining a fingerprint according to any one of claims 1 to 9, wherein the preparation method of the traditional Chinese medicine composition preparation is:

    (1) Weigh Chinese medicines according to the weight ratio of raw materials, net selection, and break as appropriate;

    (2) Patchouli scented, add 10 times the amount of water to extract volatile oil, oil extraction time 8 hours, collect volatile oil, spare; after the extract is filtered, the residue is discarded, the filtrate is reserved;

    (3) Forsythia, Ephedra, Houttuynia cordata, Rhubarb, extracted with 12 times 70% ethanol for 3 times, each time for 2.5 hours, the extract is combined with filtration, ethanol is recovered, and the filtrate is reserved;

    (4) Honeysuckle, gypsum, Banlangen, Mianma Guanzhong, Licorice, Rhodiola, add 12 times the amount of water to boil, add bitter almonds, decoct 2 times, each time for 1 hour, extract and filter, the result The filtrate is combined with the filtrate of step (2) patchouli oil extraction, and concentrated to obtain a clear paste having a relative density of 1.10-15.15 at 60 ° C, added with ethanol, adjusted to an alcohol concentration of 70%, refrigerated, filtered, and recovered. Ethanol to no alcohol, get a clear paste;

    (5) Combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), and concentrating to obtain a clear paste having a relative density of 1.15 - 1.20 at 60 ° C, and drying. Get dry powder, spare;

    (6) adding the dry paste powder obtained in the step (5) to a suitable pharmaceutically acceptable auxiliary material for granulation;

    (7) The menthol and the volatile oil obtained in the step (2) are dissolved in ethanol, and the granules obtained in the step (6) are sprayed, sealed, mixed, tableted or capsuled, or bagged.
11. The fingerprint mapping method according to any one of claims 1 to 9, wherein the fingerprinting method is as follows:

    Preparation of test solution: Take 0.25g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, precisely add 50mL methanol 25mL, weigh the weight, ultrasonic power 500W, frequency 40kHz, ultrasonic 30 minutes, put Cold, weigh the weight again, make up the lost weight with 50% methanol, shake it, filter it, take the filtrate, and get it;

    Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg/ Methyl glycyrrhizinate mixed control solution is obtained;

    Preparation of reference solution: Take appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make 101.2μg solution per 1ml, that is;

    The mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, and the gradient elution procedure was:

    

    The column was a Waters Acquity UPLC HSS T3 column with a column length of 100 mm, an inner diameter of 2.1 mm, a particle size of 1.8 μm, a detection wavelength of 239 nm, and a flow rate of 0.3 ml/min;

    Assay method: accurately draw 2 μL of each of the reference solution and the test solution, inject into an ultra-high liquid chromatograph, measure and record the chromatogram, that is, obtain.
12. The fingerprint mapping method according to any one of claims 1 to 9, wherein the fingerprinting method is as follows:

    Preparation of test solution: Take 0.1g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, accurately add 50mL of methanol, 50mL, weigh the weight, sonicate for 40 minutes, let cool, then weigh the weight. , use 100% methanol to make up the lost weight, shake, filter, and take the filtrate to obtain;

    Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg/ Methyl glycyrrhizinate mixed control solution is obtained;

    Preparation of reference solution: Take appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make 101.2μg solution per 1ml, that is;

    The mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, and the gradient elution procedure was:

    

    The column was a Waters Acquity UPLC HSS T3 column; the detection wavelength was 239 nm, and the flow rate was 0.1 ml/min;

    Determination method: respectively, accurately draw 1 μL of the reference solution and the test solution, inject into the ultra-high liquid chromatograph, measure and record the chromatogram, that is, obtain.
13. The fingerprint mapping method according to any one of claims 1 to 9, wherein the fingerprinting method is as follows:

    Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, accurately add 40mL methanol to 10mL, weigh the weight, sonicate for 20 minutes, let cool, then weigh the weight. , use 40% methanol to make up the lost weight, shake, filter, and take the filtrate to obtain;

    Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg/ Methyl glycyrrhizinate mixed control solution is obtained;

    Preparation of reference solution: Take appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make 101.2μg solution per 1ml, that is;

    The mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, and the gradient elution procedure was:

    

    The column was Waters XBridge ^TM Shield RP18; detection wavelength 239nm, flow rate 0.5ml / min;

    Determination method: respectively, accurately draw 1 μL of the reference solution and the test solution, inject into the ultra-high liquid chromatograph, measure and record the chromatogram, that is, obtain.
14. The fingerprint mapping method according to any one of claims 1 to 9, wherein the fingerprinting method is as follows:

    Preparation of test solution: Take 0.2g of the traditional Chinese medicine composition, accurately weigh it, place it in a conical flask, accurately add 20mL of methanol 20mL, weigh the weight, sonicate for 40 minutes, let cool, then weigh the weight , use 90% methanol to make up the lost weight, shake, filter, and take the filtrate to obtain;

    Preparation of reference solution: weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, forsythiaside A, forsythin, 4,5-di-O-caffeoyl The appropriate amount of linic acid, quercetin and ammonium glycyrrhizinate was prepared to contain 18.4 μg/ml neochlorogenic acid, 27.2 μg/ml chlorogenic acid, 21.2 μg/ml cryptochlorogenic acid, and 27.2 μg/ml. Ester A, 21.6 μg/ml forsythiaside A, 16.0 μg/ml forsythin, 14.4 μg/ml 4,5-di-O-caffeoylquinic acid, 9.0 μg/ml quercetin, 24.4 μg/ Methyl glycyrrhizinate mixed control solution is obtained;

    Preparation of reference solution: Take appropriate amount of forsythiaside A reference substance, accurately weighed, add 50% methanol to make 101.2μg solution per 1ml, that is;

    The mobile phase was methanol-acetonitrile-0.1% phosphoric acid, gradient elution, and the gradient elution procedure was:

    

    The column was Phenomenex Kinetx XB-C18; the detection wavelength was 239 nm, and the flow rate was 0.4 ml/min;

    Assay method: accurately draw 6 μL of each of the reference solution and the test solution, inject into the ultra-high liquid chromatograph, measure and record the chromatogram, that is, obtain.

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