CN113267578B

CN113267578B - Quality control method of peony and licorice decoction

Info

Publication number: CN113267578B
Application number: CN202110535405.5A
Authority: CN
Inventors: 朱广伟; 曲缘章; 李宝国; 马生军; 李刚; 曹丽娟; 康亚娟; 姜恒丽; 王双双; 马向慧
Original assignee: China Medico Corp; Institute of Materia Medica of CAMS
Current assignee: China Medico Corp; Institute of Materia Medica of CAMS
Priority date: 2021-05-17
Filing date: 2021-05-17
Publication date: 2023-02-24
Anticipated expiration: 2041-05-17
Also published as: CN113267578A

Abstract

The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a quality control method of a peony and licorice decoction. The quality control method of the peony and licorice decoction provided by the invention comprises a test solution preparation step, a reference solution preparation step and an ultrahigh liquid chromatography detection step. The quality control method can realize qualitative and quantitative analysis of the index components in the peony licorice decoction, and provides important reference basis for quality evaluation and clinical application of the peony licorice decoction. By optimizing the chromatographic conditions of the ultra-high liquid chromatography detection, the responsiveness of the index component can be improved, the accuracy of the content detection of the index component is further improved, and the attribution and content determination of complex chemical components in the peony and licorice decoction is realized.

Description

Quality control method of peony and licorice decoction

Technical Field

The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a quality control method of a peony and licorice decoction.

Background

In recent years, the demand of traditional Chinese medicines and traditional Chinese medicine preparations is rapidly increased all over the world, and as the essence of the theory of traditional Chinese medicines, the traditional Chinese medicine composition plays a key role in thousands of years of clinical treatment by virtue of the unique compatibility rule and excellent pharmacological curative effect. However, because the traditional Chinese medicine components are complex, the traditional Chinese medicine formula shows special complexity due to the complex effects of antagonism or synergy, dosage difference, four flavors and five flavors, ascending, descending, sinking and floating and the like among different medicines, so that the traditional Chinese medicine lacks an overall quality monitoring technology, and the traditional Chinese medicine formula identification and quality control become important problems influencing the traditional Chinese medicine development.

Peony and licorice Decoction (Shaoyao-Gancao-Decunction, SGD) is originated from Shang Zhong Jing in treatise on typhoid fever and miscellaneous diseases. The prescription is simple and has remarkable curative effect, is popular with doctors all the time, is used up to now, is added or reduced on the basis of the prescription, is used for treating different diseases, and is widely applied to ancient and modern traditional Chinese medicine clinics. Modern pharmacological research and clinical practice also prove that the SGD has obvious effects of spasmolysis, anti-inflammation, analgesia, cough and asthma relief, liver protection, cholagogue, HBV virus resistance, fibrosis resistance, gastric mucosa protection, immunoregulation, antianaphylaxis, constipation relief, achilles tendon tissue protection and the like, and has obvious treatment effects on various spasmodic diseases, painful diseases, inflammatory diseases, bronchial asthma, parkinson disease, constipation and the like. As a first compound selected from 'ancient classic famous prescription catalog', the decoction process and quality standard research of SGD have certain guiding significance for the development of the following classic famous prescriptions.

Chinese patent document CN111624271A discloses a liquid chromatography method, a standard fingerprint and application for detecting corresponding substance of peony-licorice decoction. The method adopts a high performance liquid chromatograph equipped with an ultraviolet detector to carry out detection, and the chromatographic conditions are as follows: mobile phase: acetonitrile (A) -0.05% aqueous phosphoric acid solution (B), gradient elution 0-1min 14-17.5% A,1-5min 17.5-19A, 5-6min 19-20% A,6-7min 20% A,7-8min 20-20.5% A,8-12min 20.5-23% A,12-30min 23-36% A,30-32min 36% A,32-45min 36-43% A; a chromatographic column: venusil XBP C184.6 × 150mm,3 μm; the detection wavelength is 230-240nm; flow rate: 1.0ml/min; the column temperature is 20-30 ℃. The standard fingerprint of the corresponding real object of the peony licorice decoction can be used for quality control of the corresponding real object of the peony licorice decoction.

However, when the liquid chromatography method is used for detecting the sample solution of the peony and licorice decoction, the chromatographic peak heights of the paeoniflorin, liquiritin and glycyrrhizic acid of the sample are all obviously lower than the chromatographic peak heights of the three index components in the reference substance, the responsiveness of the liquid chromatography detection of the index components in the sample is not high, and the accuracy of qualitative or quantitative detection of the index components in the corresponding substance of the peony and licorice decoction is influenced.

Disclosure of Invention

Problems to be solved by the invention

In view of the technical problems in the prior art, for example: the liquid chromatography method for detecting the substance corresponding to the peony and licorice decoction has the problems that the chromatographic peak height of the index components is low, and the accuracy of qualitative or quantitative detection of the index components in the substance corresponding to the peony and licorice decoction is reduced. Therefore, the invention provides a quality control method of the peony and licorice decoction, which can improve the responsiveness of index components by optimizing the chromatographic condition of ultra-high liquid chromatography detection, realize the determination of the attribution and content of chemical components of complex components in the peony and licorice decoction, and improve the accuracy and stability of the quality control of the peony and licorice decoction.

Means for solving the problems

The invention provides a quality control method of a peony and licorice decoction, wherein the method comprises the following steps:

the preparation method of the test solution comprises the following steps: preparing a peony and licorice soup sample solution by taking a peony and licorice soup standard decoction, a peony and licorice soup corresponding substance or a peony and licorice soup granule;

preparing a reference solution: preparing a single-component reference substance to prepare a control solution of the peony and licorice decoction containing the single-component reference substance;

and (3) detecting by using a super high liquid chromatography: detecting the peony and licorice soup sample solution and the reference solution by ultra-high liquid chromatography; wherein, the ultrahigh liquid chromatography detection adopts binary mobile phase to carry out gradient elution, the mobile phase A is 0.1 percent formic acid water solution, and the mobile phase B is acetonitrile.

In some embodiments, the method according to the invention, wherein the ultra high liquid chromatography detection is a UHPLC-DAD detection;

preferably, the chromatographic conditions of the ultra-high liquid chromatography further comprise:

the volume ratio of the mobile phase A to the mobile phase B is (95-5): (5-95);

the flow rate of the binary mobile phase is 0.4 mL-min ^-1 ；

The sample injection amount of the ultra-high liquid chromatography is 5 mu L;

the column temperature of the chromatographic column of the ultra-high liquid chromatography is 30 ℃, and more preferably, the chromatographic column is Thermo Accucore C ₁₈ Column, 150 mm. Times.2.1 mm, 2.6. Mu.m.

In some embodiments, the method according to the present invention, wherein the method further comprises the following step before the test solution preparation step:

identifying the basic source of the medicinal materials: extracting genome DNA of a raw medicinal material of the peony and licorice decoction, carrying out PCR amplification on an ITS2 fragment of the genome DNA, and identifying a medicinal material source according to the ITS2 fragment amplified by the PCR;

preferably, the primer set for PCR amplification comprises:

p3 comprising the nucleotide sequence of the sequence shown as SEQ ID NO.1,

e4, which comprises a nucleotide sequence of the sequence shown as SEQ ID NO. 2;

preferably, the conditions for PCR amplification are: 94 ℃ for 5min,35 cycles (94 ℃ 30s,55 ℃ 30s,72 ℃ 90 s), 72 ℃ for 7min.

In some embodiments, the method according to the present invention, wherein the ultra high liquid chromatography detection step further comprises:

measuring the content of the index components: performing ultra-high liquid chromatography detection on the radix paeoniae alba and licorice root decoction test sample solution and the reference substance solution to obtain respective ultra-high liquid chromatogram, and determining the content of the index component in the radix paeoniae alba and licorice root decoction test sample according to the ultra-high liquid chromatogram; wherein the index components include paeoniflorin, liquiritin and glycyrrhizic acid.

In some embodiments, the method according to the present invention, wherein the method further comprises:

the method comprises the following characteristic map construction steps: performing ultra-high liquid chromatography detection on at least two batches of the peony and licorice soup test sample solution and the reference solution to obtain respective ultra-high liquid chromatogram maps, selecting the chromatogram map of one batch of the peony and licorice soup test sample solution as a reference map, determining a characteristic peak according to the reference map and the chromatogram map of the peony and licorice soup reference solution, determining a common peak of the peony and licorice soup test samples of different batches according to the characteristic peak, and obtaining the characteristic map of the peony and licorice soup;

preferably, the characteristic spectrum of the peony licorice decoction comprises 14 characteristic peaks, more preferably, the liquiritin chromatographic peak in the characteristic spectrum of the peony licorice decoction is an S peak, and the relative retention time of the rest characteristic peaks and the S peak is within +/-5% of a specified value.

In some embodiments, the method according to the present invention, wherein the step of determining the content of the indicator component comprises:

taking radix paeoniae alba decoction pieces and radix glycyrrhizae preparata decoction pieces, determining the content of index components in the radix paeoniae alba decoction pieces and/or the radix glycyrrhizae preparata decoction pieces, and preparing a radix paeoniae alba and radix glycyrrhizae preparata decoction sample solution by using the radix paeoniae alba decoction pieces and the radix glycyrrhizae preparata decoction pieces;

and obtaining the transfer rate of the index component in the peony and licorice decoction sample according to the content of the index component in the peony and licorice decoction pieces and the content of the index component in the peony and licorice decoction sample.

In some embodiments, the method of the present invention, wherein the step of determining the content of the index component in the white peony root decoction pieces comprises:

preparing a white paeony root decoction piece reference substance solution,

preparing a test solution of white paeony root decoction pieces,

carrying out ultra-high liquid chromatography detection on the radix paeoniae alba decoction piece reference solution and the test solution to obtain respective ultra-high liquid chromatogram, and determining the content of paeoniflorin in the radix paeoniae alba decoction pieces according to the ultra-high liquid chromatogram;

and/or the step of measuring the content of the index components in the honey-fried licorice root decoction pieces comprises the following steps:

preparing a honey-fried licorice root decoction piece reference solution,

preparing a test solution of honey-fried licorice root decoction pieces,

and carrying out ultra-high liquid chromatography detection on the honey-fried licorice root decoction piece reference solution and the sample solution to obtain respective ultra-high liquid chromatogram, and determining the content of liquiritin and glycyrrhizic acid in the honey-fried licorice root decoction pieces according to the ultra-high liquid chromatogram.

In some embodiments, the chromatography conditions for performing the hplc detection on the white peony root decoction piece control solution and the test solution are as follows: performing gradient elution by adopting a binary mobile phase, wherein the mobile phase A is 0.1% phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is 43; the chromatographic column is Thermo Accucore C ₁₈ A column, 150mm by 2.1mm,2.6 μm; the sample injection amount is 5 mu L, the column temperature is 30 ℃, and the flow rate is 0.4mL/min; the detection wavelength is 237nm; and/or the presence of a gas in the gas,

the chromatographic conditions for performing the ultra-high liquid chromatography detection on the honey-fried licorice root decoction piece reference solution and the test solution are as follows: performing gradient elution by using a binary mobile phase, wherein the mobile phase A is 0.05% phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is (95-77): (5-23); the chromatographic column is Thermo Accucore C ₁₈ Chromatography column, 150mm × 2.1mm,2.6 μm; the sample injection amount is 3 mu L, the column temperature is 30 ℃, and the flow rate is 0.4mL/min; the detection wavelength was 237nm.

In some embodiments, the method of the present invention, wherein the step of preparing the peony licorice root decoction test solution comprises:

weighing radix paeoniae alba decoction pieces and radix glycyrrhizae preparata decoction pieces in a mass ratio of 1; precisely adding methanol into the standard decoction of the peony and licorice decoction according to the volume ratio of 1; or,

weighing radix paeoniae alba decoction pieces and radix glycyrrhizae preparata decoction pieces in a mass ratio of 1; precisely absorbing standard decoction of the peony and licorice decoction, and freeze-drying to obtain corresponding substance of the peony and licorice decoction; re-dissolving the corresponding substance in 50% methanol, ultrasonic treating, cooling, diluting to desired volume, centrifuging, collecting supernatant, and filtering to obtain filtrate as the sample solution; or,

precisely adding 50% methanol into the granule, sealing, weighing, ultrasonic treating, cooling, weighing again, adding 50% methanol to make up the weight loss, shaking, and filtering to obtain filtrate as the sample solution;

preferably, the decocting conditions are: heating to slight boiling under the condition of first power, adjusting the power to be second power, and keeping the slight boiling to continue heating for 45-50min; optionally, the first power is 500W, and the second power is 300W;

preferably, the first filtration is filtration with a 150 mesh screen, the second filtration is filtration with a 0.22 μm filter, and the centrifugation is 12000rpm for 5min.

In some embodiments, the method of the present invention, wherein the step of preparing the peony licorice decoction reference solution comprises:

accurately weighing penoniflorin reference substance, adding into solid-liquid ratio (mg: mL) of (1.2-1.4): 1, performing constant volume with methanol to obtain a peony and licorice decoction reference solution containing paeoniflorin;

precisely weighing liquiritin reference substance, adding into a solid-to-liquid ratio (mg: mL) of (1-1.2): 1, performing constant volume with methanol to obtain a peony and liquorice soup reference solution containing liquiritin;

precisely weighing glycyrrhizic acid reference substance, adding into the solution at a solid-to-liquid ratio (mg: mL) of 0.8-1): 1, performing constant volume with methanol to obtain a control solution of the peony and licorice decoction containing glycyrrhizic acid.

ADVANTAGEOUS EFFECTS OF INVENTION

In some embodiments, the quality control method of the peony and licorice decoction provided by the invention can realize qualitative and quantitative analysis of the index components in the peony and licorice decoction, and provide an important reference basis for quality evaluation and clinical application of the peony and licorice decoction. According to the invention, by optimizing the chromatographic conditions of the ultra-high liquid chromatography detection, the responsiveness of the index component can be improved, the peak height of the chromatographic peak is improved, the accuracy of content detection of the index component is further improved, and the attribution and content determination of the chemical components of the complex components in the peony and licorice decoction are realized.

In some embodiments, in the quality control method of the peony and licorice decoction provided by the invention, the UHPLC-DAD is used for performing ultra-high liquid chromatography detection, and the method has the advantages of high stability, good reliability, good reproducibility and the like.

In some embodiments, the quality control method further comprises a step of identifying the source of the drug. The radix paeoniae alba and licorice root soup is traced based on the medicinal material source by PCR amplification, so that the authenticity identification and species distinguishing of the medicinal material are facilitated, the quality control of the radix paeoniae alba and licorice root soup is realized from the medicinal material source, the source consistency of the medicinal material is ensured, and the comprehensiveness and accuracy of the quality control of the radix paeoniae alba and licorice root soup are improved.

In some preferred embodiments, the licorice identified in the step of identifying the medicinal material source is Ural licorice, which has consistency with the medicinal material source of licorice in the ancient prescription.

In some embodiments, the quality control method of the present invention can realize qualitative and quantitative evaluation of complex chemical components in the paeonia lactiflora and glycyrrhiza decoction by constructing the characteristic map of the paeonia lactiflora and glycyrrhiza decoction, and realize overall quality control, can perform method development aiming at the characteristics of each medicine by deeply researching the content detection methods of the index components of paeoniflorin, liquiritin and glycyrrhizic acid, and the like, eliminates the interference of other components, and solves the detection difficulty of each medicine.

In some embodiments, the quality control method provided by the invention can be used for measuring the content of the index component in the radix paeoniae alba decoction pieces and/or the radix glycyrrhizae preparata decoction pieces, and can also be used for measuring the transfer rate of the index component in a sample, so that the quality control and parameter optimization of the preparation process of the radix paeoniae alba and radix glycyrrhizae preparata decoction are realized, and the quality consistency of products prepared in different batches is ensured.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis detection of polybase licorice in the peony licorice decoction amplified by PCR in example 1.

FIG. 2 shows a control characteristic spectrum of standard decoction of Paeoniae radix and Glycyrrhizae radix S01-S15; wherein, peak 3: albiflorin; peak 4: paeoniflorin; peak 6: liquiritin; peak 7: apiosyl liquiritin; peak 9: liquiritigenin; peak 14: glycyrrhizic acid.

FIG. 3 shows a characteristic spectrum of standard decoction of Paeoniae radix and Glycyrrhizae radix S01-S15; wherein, peak 3: albiflorin; peak 4: paeoniflorin; peak 6: liquiritin; peak 7: apiosyl liquiritin; peak 9: liquiritigenin; peak 14: glycyrrhizic acid.

Detailed Description

Various exemplary embodiments, features and aspects of the invention will be described in detail below. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.

All units used in the specification are international standard units unless otherwise stated, and numerical values and numerical ranges appearing in the present invention should be understood to include systematic errors inevitable in industrial production.

In the present specification, the term "may" includes both the case where a certain process is performed and the case where no process is performed.

In the present specification, reference to "some particular/preferred embodiments," "other particular/preferred embodiments," "embodiments," and the like, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

In the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including the end points of numerical values a and B.

The term "control", as used herein, unless otherwise indicated, refers to a standard substance used in the identification, testing, assay and calibration of assay instrument performance, which is generally approved by the national drug certification authority for approval, and which is not less than the quality standard for the product.

The term "quality control method" as used herein may also be referred to as "quality monitoring method" unless otherwise indicated.

The term "test article" as used herein, unless otherwise specified, refers to an experimental sample used for detection or identification.

As used herein, unless otherwise noted, the term "precisely weigh" means that the weight is weighed to the nearest thousandth of the weight taken, the term "precisely measure" means that the volume is measured to the nearest thousandth of the volume taken, and the term "precisely aspirate" means that the sample is accurately measured by the microsyringe.

The term "secondary filtrate" as used herein, unless otherwise indicated, refers to the filtrate collected continuously after the primary filtrate is discarded during filtration. Compared with the primary filtrate, the secondary filtrate is closer to the real concentration of the sample, because the filtering medium (such as a filter membrane, filter paper and the like) can possibly adsorb the solute, the concentration of the sample in the primary filtrate is lower; in addition, the subsequent filtrate is cleaner, and because the solute adsorbed by the filter medium can form a filter cake, the filter pore size is reduced, and therefore, more tiny particles can be trapped.

As used herein, unless otherwise indicated, the term "binary mobile phase system" refers to a mobile phase consisting of two components, one of which (preferably the former) is generally designated as "mobile phase a" and the other (preferably the latter) is designated as "mobile phase B". For example, in a 0.1% aqueous formic acid-acetonitrile binary mobile phase system, mobile phase a is 0.1% aqueous formic acid and mobile phase B is acetonitrile. However, the above labeling method is not fixed, and acetonitrile is also referred to as mobile phase A, and 0.1% formic acid aqueous solution is referred to as mobile phase B.

The term "ultra performance liquid chromatography" (or "UPLC") as used herein refers to the development of a new technology based on High Performance Liquid Chromatography (HPLC), and has the characteristics of small filler particles, fast detection speed, large analysis flux, high sensitivity, etc., unless otherwise specified. Further, the term "ultra high performance liquid chromatography" as used herein refers to an ultra high performance liquid chromatography-diode array detector detection method (UHPLC-DAD).

The term "retention time" as used herein, unless otherwise indicated, refers to the time that elapses from the start of injection of a separated component until a maximum in the concentration of that component occurs after the column, i.e., the time that elapses from the start of injection until the peak of the chromatographic peak of the separated component occurs; the term "relative retention time" refers to the ratio of the corrected retention time of the separated component to the corrected retention time of the standard; the term "corrected retention time" refers to the retention time of the separated component minus the retention time of air. For example, if the retention time of air is 3s, the retention time of the separated component is 9s, and the retention time of the standard is 15s, then the corrected retention time of the separated component is 6s, the corrected retention time of the standard is 12s, and the relative retention time of the separated component with respect to the standard is 0.5.

The term "transfer rate" as used herein, unless otherwise indicated, refers to the rate at which an indicator ingredient is released from a raw medicinal material into a decoction. The transfer rate in the present invention is calculated by the following method: transfer rate (%) = W (mg)/M (mg) × 100%; wherein: w represents the amount (mg) of index component in the decoction; m represents the amount (mg) of the index component in the decoction pieces.

References herein to concentration percentages of solutions "%" refer to volume percentages, unless otherwise indicated.

Experimental equipment:

1290Infinity II ultra high performance liquid chromatograph, agilent, USA, including automatic sample injection system G7167B, column oven G7166B, diode array detector G7117A (DAD); model BS-210S electronic analytical balance, beijing sidoris balance ltd; model LD510-2 electronic balance, shenyang lungteng electronics ltd; H1650-W model desk-top high speed centrifuge, hunan instruments laboratory Instrument development Co., ltd; LGJ-10E vacuum freeze dryer, tech development ltd of four ring Frey instrument; the HDZ20 type intelligent medicine decocting pot is a delicious family which permanently reaches a basic electrical appliance factory, and has the capacity of 2L, the rated power of 500W and the stable voltage of 220V.

Materials:

reference products of apiosyl liquiritin, liquiritigenin and albiflorin are respectively 18110902, 17091402 and 1701302 in batches, the mass fraction of the reference products is more than or equal to 98 percent, and the reference products are Chengdu pofeld biotechnology limited company; the reference substances, namely paeoniflorin, liquiritin and ammonium glycyrrhizinate, are 17041401, L63P-J7XV and 18012902 respectively, the mass fractions are 98%, 93.1% and 98% in sequence, and the reference substances are purchased from China institute for food and drug assay; the water is the Wahaha purified water, the methanol and the acetonitrile are chromatographically pure, and other reagents are analytically pure. The decoction pieces used by the SGD are provided by Shibataea herbacea GmbH, and the medicinal materials used by the decoction pieces are respectively the dried root of Paeonia lactiflora pall of Paeonia of Ranunculaceae and the dried root and rhizome of Glycyrrhiza uralensis Fisch of Glycyrrhiza of Leguminosae, and the detailed information is shown in Table 1.

TABLE 1

Examples

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: identification of medicinal material primordium

1. The experimental steps are as follows:

wiping the surface of multi-base raw material Glycyrrhrizae radix in the Paeoniae radix Glycyrrhrizae radix decoction with 75% ethanol, scraping off the outer surface, collecting dried rhizome 20-30mg, and grinding with high throughput tissue grinder (Scientz-48, china) for 120s (50 Hz) to obtain powder. When total DNA was extracted using a plant genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., ltd, lot number: DP 305-02), the remaining steps were the same as the kit instructions.

The sample DNA is used as a template, the ITS2 sequence is subjected to PCR amplification by selecting a universal primer, the PCR reaction conditions and the universal primer are shown in the table, the PCR reaction system is mix 12.5 mu l, the forward and reverse primers are respectively 1 mu l, the double distilled water is 6.5 mu l, the DNA is 4 mu l, and the total volume is 25 mu l. The PCR product was sent to Biotechnology Co., ltd, beijing Optimus Alco, for bidirectional sequencing.

TABLE 2 primers and reaction conditions

2. The experimental results are as follows:

the result of agarose gel electrophoresis detection of the PCR amplification product is shown in figure 1, the sequencing result of the sequence shown in SEQ ID NO.3-SEQ ID NO.17 is obtained after sequencing the PCR amplification fragment, and all the medicinal materials of the liquorice are Ural liquorice after comparing the sequences shown in SEQ ID NO.3-SEQ ID NO. 17.

According to the research and development requirements of the classical name, for one medicinal material from different sources, the medicinal material needs to be determined to have a uniform medicinal material primitive, and cannot be mixed with different primitives. The liquorice adopted in the embodiment is identified by the medicinal material primitive and is unified into the glycyrrhiza uralensis. The Wularg licorice is a common herb material source in the ancient compound of the peony licorice decoction, and the peony licorice decoction prepared by using the Wularg licorice can realize the aim of consistent key quality attributes in the ancient compound and fully exert the medicinal effect. Meanwhile, the glycyrrhiza uralensis is used as a raw material, so that the resource is rich, and the requirement of mass preparation is met.

Example 2: content determination of index components in white peony root decoction pieces and honey-fried licorice root decoction pieces

1. The experimental steps are as follows:

1.1 preparing reference solution of radix Paeoniae alba decoction pieces and radix Glycyrrhizae Preparata decoction pieces

1) Paeoniflorin: taking a paeoniflorin reference substance, precisely weighing 9.30mg in a 10mL volumetric flask, and fixing the volume with methanol to obtain the paeoniflorin.

2) Liquiritin: taking a liquiritin reference substance, precisely weighing 10.3mg in a 10mL volumetric flask, and carrying out constant volume on methanol (adding DMSO0.5mL and then carrying out constant volume on methanol) to obtain the liquiritin.

3) Glycyrrhizic acid: precisely weighing glycyrrhizic acid reference substance 9.114mg in a 10mL volumetric flask, and diluting with methanol to constant volume to obtain the glycyrrhizic acid reference substance.

1.2 preparing test solution for radix Paeoniae alba decoction pieces and radix Glycyrrhizae Preparata decoction pieces

1) White peony root decoction piece test solution: taking about 0.1g of the powder in the product, precisely weighing, placing in a 50ml volumetric flask, precisely adding 35ml of 50% ethanol, sealing, performing ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooling, fixing the volume with 50% ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the product.

2) Honey-fried licorice root decoction piece test solution: weighing about 0.1g of the powder (sieved by a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% ethanol, sealing the plug, weighing, ultrasonically treating (with the power of 250W and the frequency of 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product.

1.3 chromatographic conditions

1) White peony root

A chromatographic column: thermo Accucore C ₁₈ Chromatography column (2.1 mm. Times.150mm, 2.6. Mu.m); mobile phase: water of phosphate a (0.1%) -acetonitrile B;

TABLE 3 gradient elution conditions

Time (min)	A％	B％
			0	86.00	14.00
15.00	86.00	14.00

Column temperature: 30 ℃; flow rate: 0.4mL/min; detection wavelength: 237nm.

The sample injection volume of the test solution is 3 mu L.

2) Prepared licorice root

And (3) chromatographic column: thermo Accucore C ₁₈ Chromatography column (2.1 mm. Times.150mm, 2.6 μm);

mobile phase: water of phosphate a (0.05%) -acetonitrile B;

TABLE 4 gradient elution conditions

Time (min)	A％	B％
			6.00	95.00	5.00
10.00	85.00	15.00
			23.50	79.00	21.00
26.50	78.00	22.00
			30.50	77.00	23.00

Column temperature: 30 ℃; flow rate: 0.4mL/min; detection wavelength: 237nm. The sample injection volume of the test solution is 3 mu L.

1.4 Standard Curve

1) White peony root decoction pieces

Paeoniflorin standard curve: precisely sucking 0.1mL, 0.2mL, 0.3mL, 0.4mL and 0.5mL of the reference substance solution, respectively adding into a 5mL volumetric flask, and fixing the volume to the scale. Sampling 3 mu L of each concentration, taking the sampling concentration (mu g/mg) of the paeoniflorin reference substance solution as an abscissa (X), taking the peak area under the wavelength of 237nm as an ordinate (Y), and calculating a regression equation, wherein Y =7759.9x +2.7061; r2=0.9978.

2) Prepared licorice root decoction pieces

Liquiritin standard curve: precisely sucking the reference substance solution 0.1mL, 0.2mL, 0.3mL, 0.4mL and 0.5mL, respectively, adding into a 5mL volumetric flask, and diluting to constant volume. Injecting 2 mu L of each concentration, taking the injection concentration (mu g/mg) of the liquiritin reference substance solution as an abscissa (X) and the peak area under the wavelength of 237nm as an ordinate (Y), and calculating a regression equation, wherein Y =9945.4x +13.658; r is ² ＝0.9994；

Ammonium glycyrrhizinate standard curve: precisely sucking the reference substance solution 0.3mL, 0.5mL, 0.8mL, 1mL and 2mL respectively, adding into a 5mL volumetric flask, and fixing the volume to the scale. Sampling 2 μ L of each concentration, calculating a regression equation by taking the sampling concentration (μ g/mg) of the ammonium glycyrrhizinate reference solution as an abscissa (X) and the peak area under the wavelength of 237nm as an ordinate (Y), wherein Y =2417.2X-42.478; r is ² ＝0.997。

2. The experimental results are as follows:

TABLE 5 determination of paeoniflorin in white peony root decoction pieces

TABLE 6 measurement results of liquiritin in licorice decoction pieces

TABLE 7 measurement results of glycyrrhizic acid in licorice decoction pieces

Example 3: content and transfer rate determination of index components in peony and licorice decoction sample

1. The experimental steps are as follows:

1.1 peony and licorice decoction control solution preparation

1) Paeoniflorin: taking a paeoniflorin reference substance, precisely weighing 13.8mg in a 10mL volumetric flask, and fixing the volume with methanol to obtain the paeoniflorin.

2) Liquiritin: taking a liquiritin reference substance, precisely weighing 10.3mg in a 10mL volumetric flask, and fixing the volume with methanol to obtain the liquiritin reference substance.

3) Glycyrrhizic acid: precisely weighing glycyrrhizic acid reference substance 9.00mg in a 10mL volumetric flask, and diluting with methanol to constant volume to obtain the glycyrrhizic acid reference substance.

1.2 preparation of peony and licorice root decoction sample solution

1) Test solution of standard decoction of peony and licorice decoction

Placing 12g of white peony root and 12g of honey-fried licorice root in a decoction pot, adding 600mL of water, placing the decoction pot on a heating plate, regulating the power to 500W for decocting till slight boiling (about 15 min), regulating the power to 300W, keeping the slight boiling for continuously decocting for 48min, and filtering by using a 150-mesh screen to obtain about 300mL of liquid medicine, namely the standard decoction of the peony and licorice root decoction, which is numbered from S01 to S15.

Precisely sucking 0.8ml of standard decoction of Paeoniae radix and Glycyrrhrizae radix decoction, precisely adding 0.8ml of methanol, vortex, mixing, centrifuging at 12000rpm for 5min, collecting supernatant, filtering with 0.22 μm filter membrane, and collecting filtrate to obtain test solution of Paeoniae radix and Glycyrrhrizae radix decoction.

2) Test solution of peony and licorice soup corresponding substance

Precisely sucking 5mL of standard decoction of the peony and licorice decoction, placing the standard decoction into a 10mL screw bottle, placing the screw bottle into a freeze dryer, after complete freeze drying, redissolving the standard decoction by using 50% methanol, transferring the redissolved decoction into a 10mL volumetric flask, carrying out ultrasonic treatment, cooling, carrying out constant volume treatment, centrifuging the solution at 12000rpm for 5min, taking supernatant, filtering the supernatant by using a 0.22 mu m filter membrane, and taking subsequent filtrate as a test solution of a corresponding substance of the peony and licorice decoction.

3) Test solution of peony and licorice decoction particles

Taking the peony and liquorice soup granules, placing the peony and liquorice soup granules in a conical flask with a plug, precisely adding 50% methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 20min, cooling, weighing again, complementing the lost weight with 50% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the sample solution of the peony and liquorice soup granules.

1.3 chromatographic conditions

The chromatographic column is Thermo Accucore C ₁₈ Chromatography column (2.1 mm. Times.150mm, 2.6. Mu.m);

mobile phase: 0.1% aqueous formic acid A-acetonitrile B;

TABLE 8 gradient elution conditions

2. Results of the experiment

2.1 precisely absorbing the test sample solution, injecting sample according to chromatographic conditions, measuring peak area, and calculating the contents of paeoniflorin, liquiritin and glycyrrhizic acid in the test sample according to an external standard method.

TABLE 9 ingredient contents of radix Paeoniae and radix Glycyrrhizae decoction

Numbering	Paeoniflorin mg/ml	Liquiritin mg/ml	Glycyrrhizic acid mg/ml
				SGT1	0.5938	0.2168	0.4914
SGT2	0.7226	0.2471	0.4349
				SGT3	0.6980	0.1889	0.4479
SGT4	0.6244	0.2236	0.4243
				SGT5	0.6736	0.2336	0.4668
SGT6	0.5626	0.1255	0.3241
				SGT7	0.6022	0.2333	0.3051
SGT8	0.5442	0.2536	0.4668
				SGT9	0.7984	0.1542	0.5935
SGT10	0.6455	0.3104	0.4711
				SGT11	0.7215	0.3013	0.5747
SGT12	0.6891	0.2528	0.4397
				SGT13	0.8286	0.2514	0.4701
SGT14	0.6069	0.3173	0.5993
				SGT15	0.6426	0.2863	0.5769

2.2 index component content of peony and licorice decoction corresponding to the real object

Taking the sample solution of the corresponding substance of the peony and licorice decoction, measuring the peak area under the same chromatographic condition as the standard decoction content, and calculating the content of the index component.

TABLE 10 peony-licorice root decoction corresponding to the contents of the index components

Numbering	Paeoniflorin mg/ml	Liquiritin mg/ml	Glycyrrhizic acid mg/ml
				SGT1	0.5822	0.2104	0.4768
SGT2	0.7418	0.2473	0.4266
				SGT3	0.6571	0.1828	0.4242
SGT4	0.6036	0.2125	0.4113
				SGT5	0.6818	0.2328	0.4599
SGT6	0.5801	0.1265	0.3298
				SGT7	0.6105	0.2391	0.3119
SGT8	0.5357	0.2417	0.5501
				SGT9	0.7655	0.1494	0.5383
SGT10	0.6436	0.3033	0.4634
				SGT11	0.7356	0.3057	0.5810
SGT12	0.6985	0.2559	0.4410
				SGT13	0.8473	0.2565	0.4781
SGT14	0.6458	0.3400	0.6424
				SGT15	0.6492	0.2844	0.5666

Table 9 and Table 10 show the contents of the index components in the peony licorice root decoction and the peony licorice root decoction

Table 9 shows the content of index components in the peony licorice decoction reference, and the content of three index components, i.e., paeoniflorin, liquiritin, and glycyrrhizic acid in 15 batches of peony licorice decoction references including SGT1 to SGT15, can be used to evaluate the quality stability of raw material herbs and the stability of the decoction process. If the content of the index components in individual batches is larger or smaller, the quality stability of the raw material medicinal materials is poor, and the raw material medicinal materials are not suitable for being used as the raw materials for preparing the peony and licorice decoction; or the decocting process needs further optimization. As can be seen from the data in table 9, the fluctuation of the content of the index component in the substance standard of the paeonia lactiflora and glycyrrhiza uralensis decoction prepared in the invention between different batches is small, which indicates that the stability and the process stability of the medicinal material are good, the content intervals of paeoniflorin, liquiritin and glycyrrhizic acid can be respectively determined according to the index component contents of SGT1 to SGT15, and the quality of the paeonia lactiflora and glycyrrhiza uralensis decoction is evaluated by judging whether the content of the index component in the paeonia lactiflora and glycyrrhiza uralensis decoction falls within the interval in the subsequent quality evaluation.

Table 10 shows the content of the index component in the peony-licorice decoction corresponding to the real object, which reflects the condition of the index component after the conversion from the decoction to the lyophilized powder. In addition, the influence of the freeze-drying equipment and equipment parameters on the index components can be reflected, and the method is used for evaluating the stability of each factor in the freeze-drying process. By comparing the content of index components in the corresponding substance of the peony and licorice decoction with the content of index components in the standard decoction, the stability of the index components under the conditions of low temperature, vacuum and the like can be evaluated. If a certain index component in a certain batch is unstable under the conditions of low temperature, vacuum and the like, the content of the component in the obtained freeze-dried powder is greatly changed compared with that of decoction.

2.3 index component transfer Rate measurement

According to the content measurement results of the index components in the decoction pieces and the standard decoction, the transfer rate of the index components is calculated according to a transfer rate calculation formula, wherein the transfer rate calculation formula is as follows: transfer rate (%) = W (mg)/M (mg) × 100% (W represents the content of index component in standard decoction, and M represents the content of index component in decoction pieces).

TABLE 11 measurement of transfer rate of index component

Table 11 shows the measurement results of the transfer rate of the index component, which can be used to evaluate the solubility of the index component and the stability of the decoction process. For example, the index components have better solubility, and the amount of the index components dissolved in the decoction is large, but the index components can be decomposed into other components after the decoction is carried out for too long time, and the technological parameters for preparing the peony and licorice decoction can be effectively controlled and the technological process can be optimized by measuring the transfer rate of the index components.

Example 4: construction of characteristic spectrum of peony and licorice decoction

1. The experimental steps are as follows:

the sample solutions of numbers S01 to S15 in example 3 and the reference solution were precisely aspirated, the sample solution of number 01 was used as a reference solution, detection was performed under the chromatographic conditions in example 3, and a UHPLC-DAD chromatogram was recorded.

Inputting the chromatographic information of S01-S15 into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) for analysis, setting the chromatogram of S01 as a reference chromatogram (figure 2), and matching full spectrum peaks, as shown in figure 3. The results showed a total of 21 peaks, 6 assigned. Similarity between the 15 batches of peony and licorice decoction standard decoction samples and the reference characteristic spectrum is more than 0.9, and the similarity requirement of the fingerprint spectrum is met.

2. The experimental results are as follows:

FIG. 3 shows that there should be 14 characteristic peaks in the characteristic spectrum of Paeoniae radix and Glycyrrhizae radix decoction, wherein 6 peaks should be respectively the same as the retention time of the corresponding control peak, and the No. 6 peak corresponding to the glycyrrhizin control is S peak, and the relative retention time of each characteristic peak and S peak is calculated and is within + -5% of the specified value. The specified values are: 0.29 (peak 1), 0.54 (peak 2), 0.62 (peak 3), 0.71 (peak 4), 0.92 (peak 5), 1.00 (peak 6), 1.07 (peak 7), 1.78 (peak 8), 1.81 (peak 9), 2.66 (peak 10), 2.88 (peak 11), 2.93 (peak 12), 3.11 (peak 13), 3.24 (peak 14).

The characteristic map is constructed to provide reference basis for the quality evaluation of the peony and licorice decoction, realize qualitative and quantitative analysis of complex components in the peony and licorice decoction, and realize effective control of the drug effect of the peony and licorice decoction.

The above examples are intended only to illustrate several embodiments of the invention, which are described in greater detail and detail, but are not to be construed as imposing any limitation on the scope thereof. It should be clear that a person skilled in the art can make several variations and modifications without departing from the inventive concept, which fall within the scope of protection of the present invention.

Sequence listing

<110> institute of traditional Chinese medicine of Chinese academy of traditional Chinese medicine

Shinshengbai herbal medicine Co Ltd

<120> quality control method of peony and licorice decoction

<130> 6700-2133102I

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 1

gactctcggc aacggata 18

<210> 2

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 2

gtttctttcc tccgctta 18

<210> 3

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 3

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 4

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 4

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 5

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 5

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 6

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 6

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 7

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 7

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 8

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 8

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 9

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 9

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 10

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 10

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 11

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 11

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 12

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 12

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 13

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 13

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 14

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 14

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 15

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 15

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 16

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 16

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

<210> 17

<211> 223

<212> DNA

<213> Glycyrrhiza uralensis

<400> 17

cagaccgttg cccgatgcca attgcctcgc gataggtact ttggttgtgc agggtgaatg 60

ttggcttccc gtgagcattg cggcctcacg gttggctcaa aactgagtcc atggtagggt 120

ttggcatgat cgatggtggt tgagtgacgc tcgagaccaa tcatgtgtga ctccactgag 180

tttgggctct gtaaccaata ggcgtctttg aacgctcgtg atg 223

Claims

1. A quality control method of a peony and licorice decoction is characterized by comprising the following steps:

preparing a reference solution: preparing a single-component reference substance, and preparing a peony and licorice decoction reference substance solution containing the single-component reference substance;

and (3) detecting by using a super high liquid chromatography: detecting the peony and licorice soup sample solution and the reference solution by ultra-high liquid chromatography; wherein the ultrahigh liquid phase chromatography detection adopts binary mobile phase to carry out gradient elution, the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is acetonitrile;

the method further comprises the following steps prior to the test solution preparation step:

identifying the basic source of the medicinal materials: extracting genome DNA of raw medicinal materials of the peony and licorice decoction, carrying out PCR amplification on ITS2 fragments of the genome DNA, and identifying medicinal material primordium according to the ITS2 fragments amplified by the PCR.

2. The method of claim 1, wherein the ultra high liquid chromatography detection is a UHPLC-DAD detection.

3. The method of claim 2, wherein the chromatographic conditions of the ultra-high liquid chromatography further comprise:

the volume ratio of the mobile phase A to the mobile phase B is (95-5): (5-95);

the flow rate of the binary mobile phase is 0.4 mL-min ^-1 ；

The sample injection amount of the ultra-high liquid chromatography is 5 mu L;

the column temperature of the chromatographic column of the ultra-high liquid chromatography is 30 ℃.

4. The method of claim 3, wherein the chromatography column is Thermo Accucore C ₁₈ Column, 150 mm. Times.2.1 mm, 2.6. Mu.m.

5. The method of any one of claims 1 to 4, wherein the primer set for PCR amplification comprises:

p3 comprising the nucleotide sequence of the sequence shown as SEQ ID NO.1,

e4 comprising the nucleotide sequence of the sequence shown as SEQ ID No. 2.

6. The method of any one of claims 1 to 4, wherein the PCR amplification conditions are: 94 ℃ for 5min,35 cycles, 72 ℃ for 7min; wherein the cycle is: 30s at 94 ℃, 30s at 55 ℃ and 90s at 72 ℃.

7. The method of any one of claims 1-4, wherein the ultra-high liquid chromatography detection step further comprises:

8. The method according to any one of claims 1-4, further comprising:

the method comprises the following characteristic map construction steps: performing ultra-high liquid chromatography detection on at least two batches of the peony and licorice root decoction test sample solution and the reference solution to obtain respective ultra-high liquid chromatogram, selecting the chromatogram of one batch of the peony and licorice root decoction test sample solution as a reference chromatogram, determining a characteristic peak according to the reference chromatogram and the chromatogram of the peony and licorice root decoction reference solution, and determining a common peak of different batches of the peony and licorice root decoction test samples according to the characteristic peak to obtain the characteristic chromatogram of the peony and licorice root decoction.

9. The method of claim 8, wherein the feature map of the peony licorice decoction comprises 14 feature peaks.

10. The method of claim 9, wherein the characteristic spectrum of the peony licorice decoction comprises an S peak, and the retention time of the remaining characteristic peaks relative to the S peak is within ± 5% of the predetermined value.

11. The method of claim 7, wherein the step of determining the content of the target component comprises:

12. The method of claim 11, wherein the step of determining the content of the index constituents in the white peony root slices comprises:

preparing a white paeony root decoction piece reference substance solution,

preparing a white paeony root decoction piece test solution,

and/or the step of measuring the content of the index component in the honey-fried licorice root decoction pieces comprises the following steps:

preparing a honey-fried licorice root decoction piece reference solution,

preparing a test solution of honey-fried licorice root decoction pieces,

13. The method of claim 12, wherein the chromatography conditions for performing the hplc detection on the radix paeoniae alba decoction piece control solution and the test solution are as follows: performing gradient elution by adopting a binary mobile phase, wherein the mobile phase A is 0.1% phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is 43; the chromatographic column is Thermo Accucore C ₁₈ Chromatography column, 150mm × 2.1mm,2.6 μm; the sample injection amount is 5 mu L, the column temperature is 30 ℃, and the flow rate is 0.4mL/min; the detection wavelength is 237nm; and/or the presence of a gas in the atmosphere,

the chromatographic conditions for performing the ultra-high liquid chromatography detection on the honey-fried licorice root decoction piece reference solution and the test solution are as follows: performing gradient elution by using binary mobile phases, wherein the mobile phase A is 0.05% phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is (95-77): (5-23); the chromatographic column is Thermo Accucore C ₁₈ Chromatography column, 150mm × 2.1mm,2.6 μm; the sample injection amount is 3 mu L, the column temperature is 30 ℃, and the flow rate is 0.4mL/min; the detection wavelength was 237nm.

14. The method as claimed in any one of claims 1 to 4, wherein the step of preparing the peony licorice root decoction test solution comprises:

weighing radix paeoniae alba decoction pieces and radix glycyrrhizae preparata decoction pieces in a mass ratio of 1; precisely sucking standard decoction of the peony and licorice decoction, and freeze-drying to obtain corresponding substance of the peony and licorice decoction; re-dissolving the corresponding substance in 50% methanol, ultrasonic treating, cooling, diluting to desired volume, centrifuging, collecting supernatant, and filtering to obtain filtrate as the sample solution; or,

precisely adding 50% methanol into the peony and licorice decoction particles, sealing, weighing, ultrasonically treating, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, and filtering to obtain filtrate, namely the peony and licorice decoction test solution.

15. The method of claim 14, wherein the conditions of said decocting are: heating to slight boiling under the condition of first power, adjusting the power to second power, and keeping the slight boiling to continue heating for 45-50min.

16. The method of claim 15, wherein the first power is 500W and the second power is 300W.

17. The method as claimed in claim 14, wherein the first filtration is filtration with a 150 mesh screen, the second filtration is filtration with a 0.22 μm filter, and the centrifugation is 12000rpm for 5min.

18. The method of any one of claims 1 to 4, wherein the step of preparing the peony licorice decoction control solution comprises: