Preparation method of artemisia capillaris decoction reference sample and construction method of high performance liquid characteristic chromatogram of artemisia capillaris decoction reference sample

Abstract

The invention provides a preparation method of an artemisia capillaris decoction reference sample. The invention also provides a construction method of the high performance liquid characteristic chromatogram of the artemisia capillaris decoction reference sample. According to the invention, the characteristic chromatogram of the artemisia capillaris decoction is constructed by adopting the high performance liquid chromatography for the first time, the blank of quality control of the artemisia capillaris decoction is filled through the method of the characteristic chromatogram, meanwhile, the quality condition of the prescription is comprehensively reflected, the quality control of the artemisia capillaris decoction is more perfect, and a means is added for the quality standard aspect of the artemisia capillaris decoction. According to the method disclosed by the invention, gradient elution is adopted for determination by adopting a high-performance liquid chromatography, and the method has the advantages of simplicity in operation, good separation degree, high precision, good repeatability, good stability and the like. Under specific detection conditions, the identification of the artemisia capillaris decoction characteristic spectrum method can be realized by taking a chlorogenic acid chromatographic peak as a reference substance S peak.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method of a reference sample of Artemisiae Capillariae Decoction and a construction method of a high-performance liquid chromatography characteristic map of the reference sample of Artemisiae Capillariae Decoction. BACKGROUND

[0002] Artemisiae Capillariae Decoction is first recorded in Zhang Zhongjing's Treatise on Febrile Diseases and is composed of three medicines, i.e., Artemisiae Capillariae, Gardeniae and Radix et Rhizoma Rhei. The original record is "Artemisiae Capillariae, 6 liang, Gardeniae, 14 pieces (cut), Radix et Rhizoma Rhei, 2 liang (with the skin removed), the three medicines, 1 dou 2 liang of water, first decoct Artemisiae Capillariae, reduce 6 liang, add the other two medicines, decoct to take 3 liang, take the dregs, and divide into three doses for taking". Artemisiae Capillariae Decoction has the effects of clearing heat and removing dampness and relieving jaundice. The guiding ideology of its treatment is to use dampness-removing and heat-clearing, dampness-removing and purgation. It is a basic and representative prescription for treating damp-heat jaundice and has the effects of removing jaundice and resisting liver injury. The monarch drug Artemisiae Capillariae has the effects of clearing heat and removing dampness and the main chemical components are flavonoids, coumarins, organic acids and the like and has the effects of protecting liver and gallbladder, resisting inflammation, resisting oxidation and resisting tumor. The minister drug Gardeniae can strengthen the effect of Artemisiae Capillariae, remove dampness and discharge heat and contains chemical components such as iridoid glycosides, terpenes, organic acid esters and flavonoids and has the effects of resisting inflammation, resisting virus and resisting tumor. The auxiliary drug Radix et Rhizoma Rhei can clear heat and remove dampness and contains chemical components such as anthraquinones, anthracenes, stilbenes and tannins and has the effects of protecting liver, resisting inflammation and pain, eliminating oxygen free radicals and resisting tumor. Therefore, the chemical components in Artemisiae Capillariae Decoction can be divided into organic acids, anthraquinones, iridoid glycosides, crocin and flavonoids and the like and the components such as chlorogenic acid, capillarin, crocin I and emodin-8-O-beta-D-glucoside contained in Artemisiae Capillariae Decoction can be used as quality markers and are closely related to the main effects of Artemisiae Capillariae Decoction, i.e., clearing heat and removing dampness and relieving jaundice.

[0003] Liang Hui, et al., Comparison of Chemical Components in Artemisiae Capillariae Decoction Prepared by Different Processes, Chinese Traditional Patent Drugs, August 2011, Vol. 33, No. 8, investigated the differences in chemical components of Artemisiae Capillariae Decoction granules prepared by combined decoction, granules prepared by separate decoction and traditional decoction concentrate and provided a basis for the application of the granules prepared by combined decoction, the granules prepared by separate decoction and the traditional decoction concentrate in clinic. The granules prepared by combined decoction and the granules prepared by separate decoction can be used in clinic and the clinical effect of the granules prepared by combined decoction is more consistent with that of the traditional decoction concentrate. The document shows the complexity of the components in Artemisiae Capillariae Decoction.

[0004] Application No. 201310139307.5, Invention Name: A kind of Artemisia capillaris decoction formula granules and its preparation method and detection method, provide a kind of Artemisia capillaris decoction formula granules and its preparation method and detection method, the Artemisia capillaris decoction formula granules are extracted by the water with the mass ratio of 3:1.5:1 of Artemisia capillaris, gardenia and rhubarb, filtering, concentrated spray drying and are prepared;The detection method of the Artemisia capillaris decoction formula granules, the detection method includes the following steps: the Artemisia capillaris decoction formula granules are identified by thin layer chromatography;The fingerprint of the Artemisia capillaris decoction formula granules is determined by high performance liquid chromatography;The content of the substance in the Artemisia capillaris decoction formula granules is determined by high performance liquid chromatography;The extract content of the Artemisia capillaris decoction formula granules is determined by hot immersion method;The heavy metal content in the Artemisia capillaris decoction formula granules is determined by ignition residue inspection method;The organic chlorine pesticide residue content in the Artemisia capillaris decoction formula granules is determined by pesticide residue determination method.The raw material of the Artemisia capillaris decoction formula granules in this patent document is different from the prescription of Zhang Zhongjing, and is intended for the preparation of compound preparation, not the reference sample of Artemisia capillaris decoction.

[0005] Application No. 202210143197.9, Invention Name: A kind of Artemisia capillaris decoction compound preparation fingerprint detection method, discloses a kind of Artemisia capillaris decoction compound preparation fingerprint detection, the detection method includes the following steps: S1: preparation of test solution: mix Artemisia capillaris, gardenia and rhubarb in methanol, ultrasonic, filter, concentrate, dilute, filter to obtain test solution;S2: preparation of control solution: dissolve target control, filter, and obtain control solution;S3: test solution and control solution are injected into liquid chromatograph respectively, and chromatogram is obtained;The chromatogram is introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system of national pharmacopoeia commission, and the similarity is automatically calculated;The chromatographic conditions are as follows: C18 chromatographic column;Water is used as mobile phase A, and methanol is used as mobile phase B;Flow rate is 0.8-1.2 mL / min;Gradient elution is used, and detection wavelength is 205-280 nm;Injection amount is 5-20 μL;Column temperature is 25-35 DEG C.The fingerprint detection method effectively controls the characteristic components of Artemisia capillaris decoction compound preparation, and the fingerprint has good quality, precision, stability and repeatability.The HPLC detection method established in the patent document when detecting Artemisia capillaris decoction has a detection time of 105 minutes, which is long and low in efficiency;In addition, the positions of peak 1 and peak 2 of the characteristic peaks are close, and there is no experimental data to show whether the separation degree is greater than 1.5, and only the drawing shows that the peak time may be within 5 minutes, which is wrapped in solvent peak.

[0006] In high performance liquid chromatography (HPLC), the reference sample (standard / control) is the core of quantitative analysis, and its quality directly affects the accuracy and reliability of the results. At present, there is no relevant literature reported on the reference sample and quality control method of Yinchen Jia Tang. The preparation of the reference sample of Yinchen Jia Tang and the construction of the characteristic spectrum face the following difficulties:

[0007] (1) Key information investigation: Yinchen Jia Tang belongs to a classic prescription, which is selected from traditional Chinese medicine books, and the compatibility is strict. In order to meet the original intention of Zhang Zhongjing's Shanghan Lun, the key information is investigated by combining ancient and modern literature metrology, history of Yinchen Jia Tang, modern clinical application, etc.

[0008] (2) Complexity of components: The components of traditional Chinese medicine are complex, and the polarity, molecular weight and chemical properties of various compounds are different, which increases the difficulty of separation and detection;

[0009] (3) Identification and attribution of characteristic peaks: In the determination of characteristic spectrum, multiple references are used to identify different characteristic peaks. In addition to evaluating the retention time, relative retention time / relative peak area, etc. of the characteristic peaks, the characteristic peaks identified also need to be confirmed by spectrum. For traditional Chinese medicine compound preparations, the characteristic peaks also need to be attributed according to the chemical components contained in each medicinal material of the prescription and the preparation process. SUMMARY

[0010] The present application relates to a kind of preparation method of Yinchen Jia Tang reference sample and the construction method of high performance liquid characteristic spectrum of Yinchen Jia Tang reference sample.

[0011] The present application provides a kind of preparation method of Yinchen Jia Tang reference sample, it includes the following steps:

[0012] a, weighing each weight ratio of raw material: artemisia capillaris 82.8g, gardenia 12.0g, rhubarb 27.6g;

[0013] b, add water, first cook artemisia capillaris, then add gardenia, rhubarb, decoct, filter, collect the liquid medicine, freeze-drying is obtained by using vacuum freeze-drying machine;

[0014] The freeze-drying program of the vacuum freeze-drying machine is as follows:

[0015] (1) pre-freezing: temperature: -50 DEG C, maintain temperature: 3h;Pressure: normal pressure;

[0016] (2) sublimation drying: temperature: -40 DEG C, maintain temperature: 1h;Pressure: 0.0pa;

[0017] temperature: -30 DEG C, maintain temperature: 1h;Pressure: 0.0pa;

[0018] temperature: -20 DEG C, maintain temperature: 2h;Pressure: 0.0pa;

[0019] Temperature: -10℃, temperature maintenance: 3h; pressure: 0.0pa;

[0020] Temperature: 0℃, temperature maintenance: 3h; pressure: 0.0pa;

[0021] (3) Analysis drying: temperature: 10℃, temperature maintenance: 3h; pressure: 0.0pa;

[0022] Temperature: 20℃, temperature maintenance: 2h; pressure: 0.0pa;

[0023] Temperature: 30℃, temperature maintenance: 2h; pressure: 0.0pa;

[0024] Temperature: 40℃, temperature maintenance: 2h; pressure: 0.0pa.

[0025] Preferably, the water in step b is 20 times the amount of the water, and the Artemisia capillaris is boiled first to 10 times the amount of water, then the Gardenia and the Rhubarb are added, and boiled to 5 times the amount of water, filtered through a 200 mesh screen, and the medicinal liquid is collected.

[0026] The application also provides the Artemisia capillaris decoction reference sample obtained by the preparation method of the Artemisia capillaris decoction reference sample.

[0027] The application also provides a construction method of the high performance liquid chromatography (HPLC) characteristic map of the Artemisia capillaris decoction reference sample, which comprises the following steps:

[0028] a. Preparation of the test sample solution:

[0029] The Artemisia capillaris decoction reference sample is dissolved by a solvent, extracted, and the test liquid is obtained;

[0030] b. The test liquid is determined by high performance liquid chromatography, and the HPLC characteristic map of the Artemisia capillaris decoction reference sample is obtained; the chromatography conditions of the high performance liquid chromatography are as follows: the chromatography column is a C18 column; the mobile phase A is acetonitrile, and the mobile phase B is 0.1% phosphoric acid, and gradient elution; the gradient elution conditions are as follows:

[0031] 0-6min, the mobile phase A is 2-10%, and the mobile phase B is 98-90%,

[0032] 6-13min, the mobile phase A is 10-15%, and the mobile phase B is 90-85%,

[0033] 13-60min, the mobile phase A is 15-35%, and the mobile phase B is 85-65%.

[0034] The preparation of the test sample solution is as follows: the Artemisia capillaris decoction freeze-dried powder is added into a methanol aqueous solution, ultrasonically treated, cooled to room temperature, filtered, and the continuous filtrate is obtained, and the test sample solution is obtained.

[0035] It also includes the preparation of reference solution: take chlorogenic acid, neochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, gallic acid, catechin and emodin-8-O-β-D-glucoside control, dissolved with methanol, prepared reference solution.

[0036] The chromatographic column is C18 column, the column length is 250mm, the inner diameter is 4.6mm, the silica gel particle size is 5μm; the detector is diode array detector, the detection wavelength is 285nm; the column temperature is 30℃; the flow rate is 1.0mL / min.

[0037] The test product chromatogram contains 14 characteristic peaks, wherein the peak corresponding to the chlorogenic acid reference is the S peak, the relative retention time of each characteristic peak to the S peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value of each peak is: peak 1 0.498, peak 2 0.709, peak 3 0.735, peak 4 0.974, peak 5 is S peak 1.000, peak 6 1.112, peak 7 1.313, peak 8 1.653, peak 9 1.965, peak 10 2.100, peak 11 2.237, peak 12 2.565, peak 13 2.614, peak 14 3.050.

[0038] The peak 1 is gallic acid, the peak 3 is neochlorogenic acid, the peak 4 is catechin, the peak 5 is chlorogenic acid, the peak 10 is isochlorogenic acid B, the peak 11 is isochlorogenic acid A, the peak 12 is isochlorogenic acid C, and the peak 14 is emodin-8-O-β-D-glucoside.

[0039] The application also provides a method for judging the quality of Artemisia capillaris decoction, which comprises the following steps:

[0040] a. Take Artemisia capillaris decoction test product;

[0041] b. The construction method of the high performance liquid chromatography characteristic map of the Artemisia capillaris decoction reference sample detects the test product, and if 14 characteristic peaks are contained at the same time, it is an Artemisia capillaris decoction qualified product.

[0042] The application establishes a preparation method of a standard sample of Yinchen Jia Tang, according to the original record, combined with ancient and modern literature metrology to verify the key information, one Han Dynasty Liang is 13.8g of modern, one Shui is 200ml of modern, namely the decoction method of the standard sample of Yinchen Jia Tang is: taking Yinchen 82.8g, Zhizi 12.0g, Dahuang 27.6g, adding 2400ml of water, first decocting Yinchen to 1200ml, then adding Zhizi and Dahuang, decocting to 600ml, filtering, collecting the medicinal liquid, and obtaining the medicinal liquid. The mass ratio of Yinchen, Zhizi and Dahuang is 6.9:1:2.3, and 20 times of water is added for decoction. The taste ratio in the published patent literature is different from the original prescription of Zhang Zhongjing, and aims to prepare a compound preparation, rather than a standard sample of Yinchen Jia Tang.

[0043] At present, there is less research on the characteristic spectrum or fingerprint spectrum of Yinchen Jia Tang, and the application simultaneously adopts a high performance liquid chromatography method to establish a characteristic spectrum detection method of a standard sample of Yinchen Jia Tang. Among the identified components, the organic acid component accounts for 62.5%, and the organic acid is related to the heat-clearing and dampness-drying, and the bile-duct clearing effect of Yinchen, and has obvious antiviral, liver-protecting and bile-duct clearing effects. In addition, the identification of the hydroxyanthraquinone and polyphenol components identifies the key components of Yinchen Jia Tang. In addition, the components of Yinchen Jia Tang are complex, and there are more water-soluble components. In the establishment of the detection method, the initial proportion of the gradient elution water phase is 98%, which can better separate the components with large polarity, and the system has good applicability, and is a more simple and efficient detection method, which has important significance for evaluating the standard sample of Yinchen Jia Tang.

[0044] The beneficial effects of the technical solution are as follows:

[0045] (1) The application first adopts a high performance liquid chromatography method to construct the characteristic spectrum of Yinchen Jia Tang, and the method of the characteristic spectrum can make up the blank of quality control, and comprehensively reflect the quality status of the prescription, so that the quality control is more perfect, and a means is added to the quality standard.

[0046] (2) The method adopts gradient elution by a high performance liquid chromatography method, and has the advantages of short detection time, simple operation, good separation degree, high precision, good repeatability and good stability. Under specific detection conditions, the identification of the characteristic spectrum method of Yinchen Jia Tang can be realized by taking chlorogenic acid as a reference S peak. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 The characteristic spectrum of Yinchen Jia Tang is investigated at different flow rates;

[0048] Figure 2 The characteristic spectrum of Yinchen Jia Tang is investigated at different column temperatures;

[0049] Figure 3Characteristic spectrum of Yinchenhao decoction with different injection amounts;

[0050] Figure 4 Characteristic spectrum of Yinchenhao decoction with different extraction methods;

[0051] Figure 5 Characteristic spectrum of Yinchenhao decoction with different solvents;

[0052] Figure 6 Characteristic spectrum of Yinchenhao decoction with different solvent amounts;

[0053] Figure 7 Characteristic spectrum of Yinchenhao decoction with different extraction times;

[0054] Figure 8 Peak identification chart for detection of Yinchenhao decoction;

[0055] Figure 9 Negative control identification chart for detection of Yinchenhao decoction;

[0056] Figure 10 Characteristic spectrum of Yinchenhao decoction with different instruments;

[0057] Figure 11 Characteristic spectrum of Yinchenhao decoction with different chromatographic columns;

[0058] Figure 12 Characteristic spectrum of Yinchenhao decoction with different chromatographic columns;

[0059] Figure 13 Characteristic spectrum of Yinchenhao decoction with different chromatographic columns; DETAILED DESCRIPTION

[0060] The test instruments and materials involved in the following examples are as follows (% by mass concentration):

[0061] High-performance liquid chromatograph: Agilent 1260 type high-performance liquid chromatograph, Shimadzu LC-20AD type high-performance liquid chromatograph, Waters e2695 type high-performance liquid chromatograph;

[0062] Electronic balance: ME204E / 02, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0063] Ultrapure water machine: Cell-type 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

[0064] Ultrasonic cleaner: KQ5200DB type (600W, 40kHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0065] Chromatographic column: Agilent ZORBAX SB-Aq 250x4.6mm, 5um, Welch AQ-C18 250x4.6mm, 5um, Ecosil 120-5-AQ PLUS 250x4.6mm, 5um;

[0066] Acetonitrile and phosphoric acid were chromatographically pure, water was ultrapure water, and the rest of the reagents were analytically pure.

[0067] Chlorogenic acid (China Institute for Food and Drug Control, batch number: 110753-202119, content 96.3%);

[0068] Neochlorogenic acid (Chengdu Desit Biotechnology Co., Ltd., batch number: MUST-21030108, content 99.67%);

[0069] Isochlorogenic acid A (Chengdu Desit Biotechnology Co., Ltd., batch number: MUST-18032601, content 98.82%);

[0070] Isochlorogenic acid B (Chengdu Desit Biotechnology Co., Ltd., batch number: DST210823-037, content 98%);

[0071] Isochlorogenic acid C (Chengdu Desit Biotechnology Co., Ltd., batch number: DSTDY003804, content 98%);

[0072] Gallic acid (China Institute for Food and Drug Control, batch number: 110831-202407, content 91.5%);

[0073] Catechin (China Institute for Food and Drug Control, batch number: 110877-202306, content 96.6%);

[0074] Emodin-8-O-β-D-glucoside (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq18082803, content 98%);

[0075] Yinchen (batch number: 010720-2307001);

[0076] Zhizi (batch number: 010533-1901003);

[0077] Dahuang (batch number: 010097-2110001);

[0078] Yinchenhao Decoction (batch number: YCHT-JZYP-01).

[0079] Example 1

[0080] This embodiment relates to Artemisia capillaris soup.

[0081] (1) The preparation of Artemisia capillaris soup freeze-dried powder is as follows: Artemisia capillaris 82.8g, Gardenia 12.0g, and Rhubarb 27.6g are added with 20 times of water, Artemisia capillaris is first boiled to 10 times of water, then Gardenia and Rhubarb are added, and boiling is continued until 5 times of water, 200 mesh screen is used for filtration, medicinal liquid is collected, and vacuum freeze-drying machine is used for freeze-drying to obtain freeze-dried powder.

[0082] The freeze-drying procedure of the vacuum freeze-drying machine is as follows:

[0083] (1) Pre-freezing: temperature: -50℃, temperature maintenance: 3h; pressure: normal pressure;

[0084] (2) Sublimation drying: temperature: -40℃, temperature maintenance: 1h; pressure: 0.0pa;

[0085] Temperature: -30℃, temperature maintenance: 1h; pressure: 0.0pa;

[0086] Temperature: -20℃, temperature maintenance: 2h; pressure: 0.0pa;

[0087] Temperature: -10℃, temperature maintenance: 3h; pressure: 0.0pa;

[0088] Temperature: 0℃, temperature maintenance: 3h; pressure: 0.0pa;

[0089] (3) Desorption drying: temperature: 10℃, temperature maintenance: 3h; pressure: 0.0pa;

[0090] Temperature: 20℃, temperature maintenance: 2h; pressure: 0.0pa;

[0091] Temperature: 30℃, temperature maintenance: 2h; pressure: 0.0pa;

[0092] Temperature: 40℃, temperature maintenance: 2h; pressure: 0.0pa. Freeze-drying technology can better maintain the original form of the material and the nutrients contained therein, and obtain high-quality dried material; the reference sample is the key carrier for bridging traditional Chinese medicine preparations and ancient classical prescriptions, and the selection of the material form will directly affect the key quality attributes of the reference sample and the preparation, and is also the key to “reducing a bowl of soup” in the study of classical prescriptions; therefore, the reference sample is generally prepared by freeze-drying, which maximizes the retention of effective ingredients in classical prescription and yield.

[0093] The preparation method of the test solution includes: 0.2g of Artemisia capillaris soup freeze-dried powder is taken, 25mL of 70% methanol is added, ultrasonic treatment (power 600W, frequency 40kHz) is performed, it is cooled to room temperature, 0.45μm filter screen is used for filtration, and the filtrate is taken as the test solution.

[0094] (2) Preparation of the control solution: Take the chlorogenic acid, neochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, gallic acid, catechin and rhein-8-O-β-D-glucoside control samples, dissolve them with methanol to prepare a mixed control solution containing 100 ug per 1 mL of methanol solution.

[0095] (3) The test solution prepared in step (1) and the control solution prepared in step (2) are subjected to high performance liquid chromatography detection respectively to obtain the corresponding characteristic spectrum.

[0096] The high performance liquid chromatography detection conditions meet the following requirements:

[0097] Liquid chromatography column: C18 chromatography column;

[0098] Column temperature: 30℃;

[0099] Flow rate: 1.0 mL / min;

[0100] Detection wavelength: 285 nm;

[0101] Mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid, and gradient elution is performed.

[0102] The gradient elution process is as follows:

[0103] 0-6 min, mobile phase A is 2-10%, mobile phase B is 98-90%,

[0104] 6-13 min, mobile phase A is 10-15%, mobile phase B is 90-85%,

[0105] 13-60 min, mobile phase A is 15-35%, mobile phase B is 85-65%.

[0106] (4) Take the characteristic spectrum of the control solution in step (3) as the reference spectrum, select the common peaks from the characteristic spectrum of the test solution, and construct the characteristic spectrum of Yinchen Jia Tang.

[0107] The constructed characteristic spectrum of Yinchen Jia Tang has 14 characteristic peaks, among which the peak corresponding to the chlorogenic acid reference is S peak. The relative retention time of each characteristic peak and S peak should be within ±10% of the specified value. Peak 10.498, peak 2 0.709, peak 3 0.735, peak 4 0.974, peak 5 1.000, peak 6 1.112, peak 7 1.313, peak 8 1.653, peak 9 1.965, peak 10 2.100, peak 11 2.237, peak 12 2.565, peak 13 2.614, peak 14 3.050.

[0108] Example 2

[0109] System suitability test

[0110] (1) Different flow rate test

[0111] The test sample solution of the Artemisia capillaris decoction described in Example 1 was used as the object, and different flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were compared. It was found that the baseline of the chromatogram was relatively stable when the flow rate was 1.0 ml / min, so the flow rate was 1.0 ml / min, as shown in Figure 1 .

[0112] (2) Different column temperature test

[0113] The test sample solution of the Artemisia capillaris decoction described in Example 1 was used as the object, and different column temperatures of 25℃, 30℃, and 35℃ were compared. It was found that the baseline of the chromatogram was relatively stable when the column temperature was 30℃, so the flow rate was the column temperature of 30℃, as shown in Figure 2 .

[0114] (3) Different injection volume test

[0115] The test sample solution of the Artemisia capillaris decoction described in Example 1 was used as the object, and different injection volumes of 5 μL, 10 μL, and 20 μL were compared. It was found that the baseline of the chromatogram was relatively stable when the injection volume was 10 μL, so the injection volume was 10 μL, as shown in Figure 3 .

[0116] Example 3

[0117] Different extraction methods test

[0118] The test sample solution of the Artemisia capillaris decoction described in Example 1 was used as the object, and different extraction methods of reflux treatment and ultrasonic treatment were compared. It was found that the similarity of the chromatograms of the two extraction methods was high. Since the ultrasonic treatment extraction method was simpler to operate, the extraction method was selected as ultrasonic treatment, as shown in Figure 4 .

[0119] Example 4

[0120] Different extraction solvents test

[0121] The test sample solution of the Artemisia capillaris decoction described in Example 1 was used as the object, and 25 mL of water, 30% methanol, 70% methanol, and methanol were added as different extraction solvents, and ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes. After cooling to room temperature, the filtrate was obtained by filtering with a 0.45 μm filter. It was found that each chromatographic peak was better extracted in 70% methanol, so the extraction solvent was selected as 70% methanol, as shown in Figure 5 .

[0122] Example 5

[0123] Investigation of different solvent dosages

[0124] Using the Artemisia capillaris decoction sample solution described in Example 1 as the test sample, different solvent volumes of 10 mL, 25 mL, and 50 mL of 70% methanol were added, respectively. The mixture was ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, cooled to room temperature, and filtered through a 0.45 μm filter. The filtrate was collected. The results showed that the separation of each chromatographic peak was better when 25 mL of 70% methanol was added for extraction. Therefore, 25 mL of solvent was selected. Figure 6 As shown.

[0125] Example 6

[0126] Examination of different extraction times

[0127] Using the Artemisia capillaris decoction sample solution described in Example 1 as the test sample, 25 mL of 70% methanol was added, and the mixture was ultrasonically treated (600 W power, 40 kHz frequency) for 20 minutes, 30 minutes, and 40 minutes respectively. After cooling to room temperature, the solution was filtered through a 0.45 μm filter, and the filtrate was collected. The results showed that the extraction time had little effect on the chromatographic peaks; therefore, an extraction time of 30 minutes was selected. Figure 7 As shown.

[0128] Example 7

[0129] Identification of chromatographic peaks

[0130] Using the test solution of Artemisia capillaris decoction described in Example 1 as the object, different reference standards (chlorogenic acid reference solution, neochlorogenic acid reference solution, isochlorogenic acid A reference solution, isochlorogenic acid B reference solution, isochlorogenic acid C reference solution, gallic acid reference solution, catechin reference solution, and emodin-8-O-β-D-glucoside reference solution) were compared. Peak 1 was identified as gallic acid, peak 3 as neochlorogenic acid, peak 4 as catechin, peak 5 as chlorogenic acid, peak 10 as isochlorogenic acid B, peak 11 as isochlorogenic acid A, peak 12 as isochlorogenic acid C, and peak 14 as emodin-8-O-β-D-glucoside. Figure 8 As shown.

[0131] Example 8

[0132] Identification of negative controls

[0133] Using the test solution of Yin Chen Hao Tang described in Example 1 as the object, and employing the same preparation method, negative control sample solutions (Yin Chen negative control, Gardenia negative control, and Rhubarb negative control) lacking one of the herbs were prepared for comparison. It was found that the chromatographic peaks exhibited by this method mainly originated from the principal herb, Yin Chen, further demonstrating the significance of this method for the quality evaluation of Yin Chen Hao Tang. Figure 9 As shown.

[0134] Example 9

[0135] Investigation of precision

[0136] 0.2 g of Yinchengao Decoction lyophilized powder was precisely weighed, and the test sample solution was prepared according to the method described in Example 1, and injected into the liquid chromatograph. The relative retention time RSD% of each characteristic peak was calculated for 6 consecutive injections, which was less than 2%, indicating that the instrument precision was good.

[0137] Table 1 Investigation of precision ~ relative retention time

[0138]

[0139]

[0140] Example 10

[0141] Investigation of repeatability

[0142] 0.2 g of Yinchengao Decoction lyophilized powder was precisely weighed, and the test sample solution was prepared according to the method described in Example 1, and injected into the liquid chromatograph. The relative retention time RSD% of each characteristic peak was calculated for 6 consecutive injections, which was less than 2%, indicating that the instrument precision was good.

[0143] Table 2 Investigation of repeatability ~ relative retention time

[0144]

[0145] Example 11

[0146] Investigation of stability

[0147] 0.2 g of Yinchengao Decoction lyophilized powder was precisely weighed, and the test sample solution was prepared according to the method described in Example 1. The relative retention time RSD% of each characteristic peak was calculated at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, which was less than 5%, indicating that the test sample solution was stable within 24 hours.

[0148] Table 3 Investigation of stability ~ relative retention time

[0149]

[0150] Example 12

[0151] Investigation of delay

[0152] The test sample solution of Yinchengao Decoction described in Example 1 was used as the object, and on the basis of the original elution gradient, the isocratic elution of mobile phase A 35% and mobile phase B 65% was added from 60 min to 120 min for delay investigation. The results showed that no chromatographic peak was eluted after 60 minutes of delay, indicating that the original gradient had good elution effect on Yinchengao Decoction.Figure 10 as shown.

[0153] Example 13

[0154] Investigation of durability

[0155] (1) Investigation of different personnel and time

[0156] 0.2 g of the freeze-dried powder of Yinchengao Decoction was precisely weighed by different personnel (A, B) at different times (T1, T2), and each two portions were prepared to prepare the test sample for determination. The relative retention time RSD% of each characteristic peak was calculated to be <5%, indicating that the method had good stability.

[0157] Table 4 Investigation of personnel and time ~ relative retention time

[0158]

[0159] (2) Investigation of different instruments

[0160] The test sample solution of Yinchengao Decoction described in Example 1 was taken as the object, and was compared in Agilent 1260 type high performance liquid chromatograph, Shimadzu LC-20AD type high performance liquid chromatograph, and Waters e2695 type high performance liquid chromatograph, respectively, as shown in Table 2. Figure 11

[0161] (3) Investigation of different chromatographic columns

[0162] The test sample solution of Yinchengao Decoction described in Example 1 was taken as the object, and was compared in different chromatographic columns Agilent ZORBAX SB-Aq, Welch AQ-C18, and Ecosil 120-5-AQ PLUS with column length of 250 mm, inner diameter of 4.6 mm, and silica particle size of 5 μm, respectively, as shown in Table 3. Figure 12

[0163] Example 14

[0164] Investigation of Yinchengao Decoction

[0165] The characteristic chromatogram method was investigated for Yinchengao Decoction and mixed control solution, and the corresponding characteristic chromatogram was obtained, as shown in Table 4. Figure 13

[0166] From the above Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 and Figure 9 ​​​It can be known that the application establishes a method of characteristic spectrum of Yinchen Jia Tang by analyzing HPLC spectrum, 14 characteristic peaks are detected in the chromatogram of Yinchen Jia Tang, the method is used for evaluating Yinchen Jia Tang, the detection of 14 characteristic peaks is within 60 minutes, and the methodological results are good, which reflects the blank of the prescription in the quality evaluation, and provides another means for the quality control.

[0167] The above specific embodiments further specifically describe the purposes, technical solutions and beneficial effects of the application, and it should be understood that the above description is only for specific embodiments of the application and is not used to limit the application, and any modification, equivalent replacement, improvement, etc. within the spirit and principle of the application should be included in the protection scope of the application.

Claims

1. A method for preparing a reference sample of Artemisia capillaris tea, characterized by: It comprises the following steps: a. Weigh each weight ratio of raw materials: 82.8g of Artemisia capillaris, 12.0g of Gardenia, 27.6g of Rhubarb; b. Add water, first cook Artemisia capillaris, then add Gardenia and Rhubarb, decoct, filter, collect the medicinal liquid, and freeze dry with a vacuum freeze dryer to obtain freeze-dried powder; The freeze-drying program of the vacuum freeze dryer is as follows: (1) Pre-freezing: temperature: -50℃, temperature maintenance: 3h; pressure: normal pressure; (2) Sublimation drying: temperature: -40℃, temperature maintenance: 1h; Pressure: 0.0pa; Temperature: -30℃, temperature maintenance: 1h; Pressure: 0.0pa; Temperature: -20℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: -10℃, temperature maintenance: 3h; Pressure: 0.0pa; Temperature: 0℃, temperature maintenance: 3h; Pressure: 0.0pa; (3) Desorption drying: temperature: 10℃, temperature maintenance: 3h; Pressure: 0.0pa; Temperature: 20℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: 30℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: 40℃, temperature maintenance: 2h; Pressure: 0.0pa.

2. The method of preparing the reference sample of Artemisia capillaris tea according to claim 1, characterized by: b. The water added in step b is 20 times the amount, first cook Artemisia capillaris until the water is 10 times the amount, then add Gardenia and Rhubarb, cook until the water is 5 times the amount, filter through a 200-mesh screen, and collect the medicinal liquid.

3. The Artemisia capillaris decoction reference sample prepared by the preparation method of the Artemisia capillaris decoction reference sample of claim 1 or 2.

4. The method for constructing the high performance liquid chromatography (HPLC) fingerprint of the reference sample of the Artemisia capillaris tea according to claim 3, characterized in that: It comprises the following steps: a. Preparation of test solution: Take the Artemisia capillaris decoction reference sample, dissolve it with a solvent, extract it, and obtain the test liquid; b. Determine the HPLC characteristic spectrum of the Artemisia capillaris decoction reference sample by high-performance liquid chromatography; the chromatographic conditions of the high-performance liquid chromatography are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, and the mobile phase B is 0.1% phosphoric acid, gradient elution; the gradient elution conditions are as follows: 0-6min, mobile phase A is 2-10%, mobile phase B is 98-90%, 6-13min, mobile phase A is 10-15%, mobile phase B is 90-85%, 13-60min, mobile phase A is 15-35%, mobile phase B is 85-65%.

5. The method for constructing the high performance liquid chromatography fingerprint of the Artemisia capillaris tea reference sample according to claim 4, characterized in that: The preparation of the test solution is as follows: take the freeze-dried powder of Artemisia capillaris decoction, add methanol solution, ultrasonic treatment, cool to room temperature, filter, and take the filtrate to obtain the test solution.

6. The method for constructing the high performance liquid chromatography fingerprint of the Artemisia capillaris tea reference sample according to claim 4, characterized in that: It also includes the preparation of a reference solution: take chlorogenic acid, neochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, gallic acid, catechin, and emodin-8-O-β-D-glucoside reference substances, dissolve them with methanol to prepare the reference solution.

7. The method for constructing the high performance liquid chromatography fingerprint of the Artemisia capillaris tea reference sample according to claim 4, characterized in that: The chromatographic column is a C18 column with a length of 250mm, an inner diameter of 4.6mm, and a silica gel particle size of 5μm; the detector is a diode array detector with a detection wavelength of 285nm; the column temperature is 30℃; and the flow rate is 1.0mL / min.

8. The method for constructing the high performance liquid chromatography fingerprint of the Artemisia capillaris tea reference sample according to claim 4 or 5, characterized in that: The test product chromatograph contains 14 characteristic peaks, wherein the peak corresponding to the chlorogenic acid reference is the S peak, the relative retention time of each characteristic peak to the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified value of each peak is: peak 1 0.498, peak 2 0.709, peak 3 0.735, peak 4 0.974, peak 5 is the S peak 1.000, peak 6 1.112, peak 7 1.313, peak 8 1.653, peak 9 1.965, peak 10 2.100, peak 11 2.237, peak 12 2.565, peak 13 2.614, peak 14 3.

050.

9. The method for constructing the high performance liquid chromatography fingerprint of the Artemisia capillaris tea reference sample according to claim 8, characterized in that: The peak 1 is gallic acid, the peak 3 is neochlorogenic acid, the peak 4 is catechin, the peak 5 is chlorogenic acid, the peak 10 is isochlorogenic acid B, the peak 11 is isochlorogenic acid A, the peak 12 is isochlorogenic acid C, and the peak 14 is emodin-8-O-β-D-glucoside.

10. A method for judging the quality of a yinchenhao decoction, characterized by: It comprises the following steps: a. Take the test product of Artemisia capillaris soup; b. Detect the test product according to the construction method of the high performance liquid chromatography characteristic spectrum of the Artemisia capillaris soup reference sample according to any one of claims 4-9, if it contains 14 characteristic peaks at the same time, it is a qualified product of Artemisia capillaris soup.

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