CN118010909A

CN118010909A - Fingerprint construction method of traditional Chinese medicine compound containing red paeony root and application thereof

Info

Publication number: CN118010909A
Application number: CN202410262928.0A
Authority: CN
Inventors: 易跃能; 王赞文; 朱亚文; 曾天颖; 付猛
Original assignee: HUNAN YINENG BIOLOGICAL PHARMACEUTICAL CO Ltd
Current assignee: HUNAN YINENG BIOLOGICAL PHARMACEUTICAL CO Ltd
Priority date: 2024-03-07
Filing date: 2024-03-07
Publication date: 2024-05-10

Abstract

The invention discloses a detection method of a traditional Chinese medicine compound containing red paeony root, which comprises the following steps: taking a traditional Chinese medicine compound test sample solution and a reference substance solution for detection, wherein the chromatographic conditions of the detection are as follows: adopting a chromatographic column with octadecyl bonded silica gel as a filler, wherein a mobile phase A is one or more of acetonitrile, methanol and tetrahydrofuran, and a mobile phase B is acid water, alkali water solution and/or buffer saline solution; obtaining component information, or component information and content information of the traditional Chinese medicine compound according to the detection result; wherein the Chinese medicinal composition comprises rehmanniae radix, radix Paeoniae Rubra, radix Sangusorbae, fructus Sophorae, herba Schizonepetae, scutellariae radix, coptidis rhizoma, trichosanthis radix, radix Angelicae sinensis, cimicifugae rhizoma, fructus Aurantii, glycyrrhrizae radix and folium Platycladi, and the reference substance comprises paeoniflorin, naringin and neohesperidin. The invention comprehensively and systematically analyzes the chemical components of the Chinese herbal compound containing the red paeony root, and provides a theoretical basis for the deep study of quality control and pharmacodynamic substance basis.

Description

Fingerprint construction method of traditional Chinese medicine compound containing red paeony root and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a fingerprint construction method of a traditional Chinese medicine compound containing red paeony root and application thereof.

Background

According to the Chinese medicine laws of the people's republic of China, the ancient classical prescription refers to a prescription recorded by ancient Chinese medicine books which are still widely applied, have definite curative effect and obvious characteristics and advantages. The ancient classical prescription has long and rich human histories in China and is applied to the present, and is a summary of clinical practice essence of the traditional Chinese medicine, the ancient classical prescription bears deep accumulation of splendid civilization of the traditional Chinese medicine for thousands of years, the ancient classical prescription is a hammer of the traditional Chinese medicine theory for thousands of years, the ancient clinical experience is a summary of the traditional Chinese medicine great treasury, and the ancient classical prescription is the most essential part of the traditional Chinese medicine great treasury. The traditional Chinese medicine classical prescription is deeply researched and developed, and is a gold key for excavating a traditional Chinese medicine treasury. Particularly in the process of combating novel coronavirus epidemic situation, the traditional Chinese medicine screens out an effective prescription of three medicines on the basis of a classical prescription, for example, the lung-heat clearing and toxin expelling decoction in the three medicines is a classical prescription combination from Zhang Zhongjing Shang Han hybrid disease theory, plays a great role in treating new coronaries and preventing, and highlights the advantages of the traditional Chinese medicine.

The traditional Chinese medicine classical prescription decoction is used as the most common dosage form for clinical medication of traditional Chinese medicine, has the advantages of reasonable prescription, rapid effect, obvious curative effect, easy absorption and the like, and is highly trusted by patients. However, the preparation, carrying, temporary decoction and long-term placement are easy to cause mildew and spoilage, the soup is bitter and has large quantity, the standard is difficult to unify, the clinical curative effect is seriously affected, and the like, so that the preparation cannot meet the living requirements of modern people. In order to maintain the advantages of the decoction and overcome various defects of the decoction, the national drug administration drug review center explicitly indicates that the quality of the traditional Chinese medicine compound preparation managed according to the classical prescription is basically consistent with the quality of a classical prescription reference sample in the guiding principle (trial) of the traditional Chinese medicine compound preparation pharmaceutical research managed according to the classical prescription catalog published by the national drug administration drug review center at the date of 08 and 31 of 2021. The reference sample represents the overall internal mass of the formulation, and is substantially identical to the remaining mass control indicators of the formulation except for the molding process. Therefore, the reference sample is a physical control of the internal quality of the preparation and is a reference object for optimizing the mass production process and formulating the quality standard thereof. The reference sample is a standard of classical formula and even the research and development of all traditional Chinese medicines, and is a reference for ensuring the safety and effectiveness of the medicines. The preparation process route formulation of the classical prescription compound preparation, the optimization of parameters and the quality standard formulation are to take a classical prescription reference sample as a reference. The reference sample is a tie for communicating clinical-enterprise-scientific research, and is a reference for inheriting, applying and developing traditional Chinese medicines.

The fingerprint is based on the knowledge of the overall action of the Chinese medicine substance group, and the spectrum or chromatogram of the chemical components of the Chinese medicine is obtained by means of the spectrum and chromatogram technology, which is a feasible mode for identifying the authenticity of the Chinese medicine, evaluating the quality consistency and the product stability, and has the characteristics of large information quantity, strong characteristics, integrity, ambiguity and the like. The fingerprint spectrum of the traditional Chinese medicine can comprehensively reflect the relative relation of chemical components contained in the medicinal materials, embody the complexity and the relativity of the components of the traditional Chinese medicine, is suitable for the traditional theory of the traditional Chinese medicine, can truly and effectively characterize, comprehensively evaluate and comprehensively control the intrinsic quality of the traditional Chinese medicine, and is particularly suitable for quality control of the traditional Chinese medicine and the traditional Chinese medicine products under the condition that the effective components are not completely clear or are not required to be completely clear. The fingerprint spectrum of the traditional Chinese medicine can be used for examining factors such as the production place, harvesting season, harvesting part, processing, storage time and the like of the traditional Chinese medicine so as to provide the basis for identifying the authenticity and the quality of the raw material medicine before production, and can be used for quality control of the traditional Chinese medicine production process: the change of certain chemical components in the preparation is tracked, and the consistency and stability of the quality between the raw medicinal materials and the finished product and between batches of the finished product are monitored. Compared with the quality analysis method for measuring the content of index components, the fingerprint can reflect the types and the amounts of chemical components of the traditional Chinese medicine more comprehensively, and can realize comprehensive evaluation of the internal quality of the traditional Chinese medicine and effective control of the whole substances of the traditional Chinese medicine under the current situation that the effective components of the traditional Chinese medicine compound preparation are not completely elucidated, thus being one of the effective means for controlling the quality of the traditional Chinese medicine and the preparation thereof at present.

At present, the method for comprehensively analyzing chemical components and researching fingerprints of the Chinese herbal medicine compound containing red paeony root, which is claimed by the invention, is not reported in the literature, in the prior art, only single components in the Chinese herbal medicine compound are subjected to quality analysis, and a comprehensive and systematic quality control method does not reflect the quality conditions of main reference sample components in the Chinese herbal medicine compound and finished products, so that the production process and the product quality of the Chinese herbal medicine compound cannot be effectively controlled, and the clinical curative effect of the Chinese herbal medicine compound cannot be better ensured, so that the key quality of the Chinese herbal medicine compound is required to be controlled by adopting the quality control method of the fingerprints capable of comprehensively controlling the whole quality of the Chinese herbal medicine compound.

Disclosure of Invention

Based on the above, the invention provides a detection method of a traditional Chinese medicine compound containing red paeony root, which comprises the following steps:

detecting the compound sample solution and the reference solution of the traditional Chinese medicine,

The chromatographic conditions for this detection were: adopting a chromatographic column with octadecyl bonded silica gel as a filler, wherein a mobile phase A is one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is acid water, alkali water solution and/or buffer saline solution, the gradient elution process is ：0～6min,0％A;6～15min,0％→5％A;15～20min,5％→10％A;20～31min,10％→12％A;31～39min,12％→14％A;39～42min,14％A;42～52min,14％→16％A;52～62min,16％→17％A;62～64min,17％A;64～70min,17％→21％A;70～80min,21％→30％A;80～85min,30％→45％A;85～88min,45％→0％A;88～100min,0％A;, the flow rate is 0.5-1.5 mL/min, the column temperature is 20-30 ℃, the detection wavelength is 200-300 nm, and the sample injection amount is 1-10 mu l;

obtaining component information, or component information and content information of the traditional Chinese medicine compound according to the detection result;

wherein the Chinese medicinal composition comprises rehmanniae radix, radix Paeoniae Rubra, radix Sangusorbae, fructus Sophorae, herba Schizonepetae, scutellariae radix, coptidis rhizoma, trichosanthis radix, radix Angelicae sinensis, cimicifugae rhizoma, fructus Aurantii, glycyrrhrizae radix and folium Platycladi, and the reference substance comprises paeoniflorin, naringin and neohesperidin.

Further, the information is the content of one or more of the following components in the traditional Chinese medicine compound according to the recorded corresponding peak areas in the chromatogram of the traditional Chinese medicine compound sample solution and the chromatogram of the reference solution and the external standard method: rehmannia root, red paeony root, garden burnet root, pagodatree pod, fineleaf schizonepeta herb, baical skullcap root, golden thread, mongolian snakegourd root, chinese angelica, largetrifoliolious bugbane rhizome, bitter orange, liquoric root and arborvitae twig.

Further, the standard curve of paeoniflorin is y= 6730.5929x-0.6597, and r ² =1.0000.

Further, the standard curve of naringin is y=11572.6279 x+3.1418, and r ² =1.0000.

Further, the standard curve of the neohesperidin is y=11119.1023x+6.6214, and r ² =1.0000.

Further, the preparation method of the compound traditional Chinese medicine sample solution comprises the following steps: weighing a proper amount of Chinese herbal medicine compound, placing into a container with proper volume, adding water for soaking, heating to boil with strong fire, transferring to slow fire, and decocting for a period of time to obtain decoction, and filtering with a filter screen while hot to obtain filtrate; and (3) taking a proper amount of the filtrate, filtering with a microporous filter membrane, and taking a subsequent filtrate to obtain the compound traditional Chinese medicine test sample solution.

Further, the container is a drug-decocting pot.

Further, the volume of the container was 2L.

Further, the ratio of mass/volume (g/ml) between the compound of the traditional Chinese medicine and the water is 0.05-0.5, for example about 0.11.

Further, the soaking time is 30 to 90 minutes, for example, about 60 minutes.

Further, the voltage of the strong fire is 200 to 300V, for example, about 220V.

Further, the voltage of the slow fire is 150 to 200V, for example, about 175V.

Further, the time of the micro-boiling decoction is 20 to 60 minutes, for example, about 40 minutes.

Further, the ratio of volume/volume (ml/ml) between the water and the decoction is 1-5, for example about 3.

Further, the filter screen is a 120 mesh filter screen.

Further, the preparation method of the reference substance solution comprises the following steps: weighing a proper amount of paeoniflorin, naringin and neohesperidin reference substances, and adding a methanol solution to prepare the reference substance solutions with the paeoniflorin, naringin and neohesperidin concentration of 5-400 mug/ml respectively.

Further, the concentration of paeoniflorin the reference solution is about 60 mug/ml.

Further, the concentration of naringin in the control solution was about 70 μg/ml.

Further, the concentration of neohesperidin in the control solution was about 70. Mu.g/ml.

Further, the flow rate is 0.8 to 1.2ml/min, for example about 1.0ml/min.

Further, the column temperature is 23 to 27 ℃, for example about 25 ℃.

Further, the detection wavelength is 200 to 250nm, for example 230nm.

Further, the amount of the sample is 3 to 7. Mu.l, for example, about 5. Mu.l.

Further, the purity angle of the peak purity of the chromatographic peak corresponding to the control is greater than the purity threshold.

Further, the theoretical plate number of the chromatographic peak corresponding to the reference substance is more than 3000.

Further, the separation degree of the chromatographic peak corresponding to the reference substance is more than 1.5.

Further, the chromatographic column is a HALO AQ-C18 chromatographic column.

Further, the specification of the chromatographic column is: the column length was 150mm, the inner diameter was 4.6mm, and the particle diameter was 2.7. Mu.m.

Further, the mobile phase a is acetonitrile.

Further, the aqueous acid, aqueous base and/or aqueous buffered salt is selected from one or more of weak acids and salts thereof, weak bases and salts thereof of varying concentrations.

Further, the aqueous acid, aqueous base and/or buffered saline solution is selected from the group consisting of formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid at various concentrations.

Further, the aqueous acid solution is 0.08 to 0.12% aqueous acid solution.

Further, the aqueous acid solution is 0.08 to 0.12% of phosphoric acid aqueous solution.

Further, the aqueous acid solution is about 0.1% aqueous phosphoric acid solution.

Further, the buffered saline solution is a phosphate saline solution and/or an acetate saline solution.

Further, the pH of the buffered saline solution is no greater than 7.0.

Further, in the traditional Chinese medicine compound, the mass ratio of rehmannia root, red paeony root, garden burnet root, pagodatree pod, fineleaf schizonepeta herb, baical skullcap root, golden thread, mongolian snakegourd root, chinese angelica, largetrifoliolious bugbane rhizome, bitter orange, liquoric root and arborvitae twig is 7% -19%, 3% -13%, 10% -17%, 3% -8%, 7% -19%, 1% -11%, 1% -4%, 3% -13%, 0.5% -5% and 7% -19%.

Further, in the traditional Chinese medicine compound, the mass ratio of rehmannia root, red paeony root, garden burnet root, pagodatree pod, fineleaf schizonepeta herb, baical skullcap root, golden thread, mongolian snakegourd root, chinese angelica, largetrifoliolious bugbane rhizome, bitter orange, liquoric root and arborvitae twig is about 10.93%, about 5.46%, about 10.93%, about 16.39%, about 5.46%, about 10.93%, about 4.37%, about 8.20%, about 2.74%, about 5.46%, about 2.74% and about 10.93%.

Further, the rehmannia root is rehmannia root.

Further, the radix paeoniae rubra is radix paeoniae rubra decoction pieces.

Further, the garden burnet is garden burnet charcoal.

Further, the sophora fruit is sophora fruit charcoal.

Further, the herba Schizonepetae is charred herba Schizonepetae.

Further, the radix Scutellariae is radix Scutellariae charcoal.

Further, the coptis is stir-fried coptis.

Further, the radix trichosanthis, the Chinese angelica tail and the cimicifugae are prepared into radix trichosanthis decoction pieces, chinese angelica tail decoction pieces and cimicifugae rhizoma decoction pieces.

Further, the fructus Aurantii is bran-parched fructus Aurantii.

Further, the reference solution is a mixed solution of paeoniflorin about 60 mug/ml, naringin about 70 mug/ml and neohesperidin about 70 mug/ml.

According to another aspect of the present invention, there is provided a fingerprint construction method of a Chinese herbal compound including radix Paeoniae Rubra, the construction method comprising the steps of:

Preparation of test solution: weighing a proper amount of Chinese herbal medicine compound, placing into a container with proper volume, adding water for soaking, heating to boil with strong fire, transferring to slow fire, and decocting for a period of time to obtain decoction, and filtering with a filter screen while hot to obtain filtrate; filtering with microporous membrane to obtain filtrate, and collecting the filtrate to obtain the test solution, wherein the Chinese medicinal composition comprises rehmanniae radix, radix Paeoniae Rubra, radix Sangusorbae, fructus Sophorae, herba Schizonepetae, scutellariae radix, coptidis rhizoma, trichosanthis radix, radix Angelicae sinensis, cimicifugae rhizoma, fructus Aurantii, glycyrrhrizae radix and folium Platycladi;

Preparation of a control solution: weighing a proper amount of paeoniflorin, naringin and neohesperidin reference substances, and adding a methanol solution to prepare reference substance solutions with the concentration of paeoniflorin, naringin and neohesperidin of 5-400 mug/ml respectively;

Detecting the results of the sample solution and the reference solution according to the high performance liquid phase to obtain a traditional Chinese medicine compound fingerprint;

The chromatographic conditions of the high performance liquid phase detection are as follows: the chromatographic column with octadecyl bonded silica gel as stuffing is adopted, the mobile phase A is one or several of acetonitrile, methanol and tetrahydrofuran, the mobile phase B is acid water, alkali water solution and/or buffer salt water solution, the gradient elution process is ：0～6min,0％A;6～15min,0％→5％A;15～20min,5％→10％A;20～31min,10％→12％A;31～39min,12％→14％A;39～42min,14％A;42～52min,14％→16％A;52～62min,16％→17％A;62～64min,17％A;64～70min,17％→21％A;70～80min,21％→30％A;80～85min,30％→45％A;85～88min,45％→0％A;88～100min,0％A;, the flow rate is 0.5-1.5 mL/min, the column temperature is 20-30 ℃, the detection wavelength is 200-300 nm, and the sample injection amount is 0.1-10 μl.

Further, the flow rate is 0.8 to 1.2mL/min, for example, about 1.0mL/min.

Further, the column temperature is 23 to 27 ℃, for example about 25 ℃.

Further, the detection wavelength is 250 to 300nm, for example 280nm.

Further, the amount of the sample is 3 to 7. Mu.l, for example, about 5. Mu.l.

Further, when the detection wavelength is 280nm, the fingerprint comprises peaks 1-19, wherein peak 1 belongs to the medicinal flavors of radix paeoniae rubra, sophora fruit and garden burnet; peak 2, peak 4, peak 15, peak 16 are ascribed to coptis medicinal flavor; peak 3 and Peak 6 belong to the medicinal flavor of Sanguisorbae; peak 5 is attributed to the red peony root drug taste; peak 8, peak 10, peak 12, peak 13 are ascribed to the pagodatree pod flavor; peak 7 is ascribed to cimicifuga foetida taste; peak 9 belongs to the flavor of Chinese angelica root; peak 11 and peak 14 are ascribed to bitter orange; peak 17 and peak 18 are ascribed to the scutellaria medicinal taste; peak 19 is attributed to Glycyrrhiza uralensis.

Further, the retention times of peaks 1-19 were 9.7±10％、16.3±10％、19.2±10％、24.1±10％、29.5±10％、31.0±10％、39.5±10％、41.8±10％、43.7±10％、44.8±10％、53.1±10％、55.9±10％、58.9±10％、59.9±10％、67.3±10％、68.3±10％、71.0±10％、83.6±10％ and 85.5.+ -. 10%, respectively.

Further, the peak 1 is a chromatographic peak of gallic acid.

Further, the peak 15 is a chromatographic peak of berberine hydrochloride.

Further, the peak 5 is a chromatographic peak of paeoniflorin.

Further, the peak 12 is a chromatographic peak of sophoricoside.

Further, the peak 7 is a chromatographic peak of isoferulic acid.

Further, the peak 9 is a chromatographic peak of senkyunolide I.

Further, the peak 11 is a chromatographic peak of naringin.

Further, the peak 14 is a chromatographic peak of neohesperidin.

Further, the peak 17 is a chromatographic peak of baicalin.

Further, the peak 18 is a chromatographic peak of wogonin.

Further, the peak 19 is a chromatographic peak of glycyrrhizic acid.

Further, the weight ratio of rehmannia root, red paeony root, garden burnet root, pagodatree pod, fineleaf schizonepeta herb, baical skullcap root, golden thread, mongolian snakegourd root, chinese angelica root, largetrifoliolious bugbane rhizome, bitter orange, liquoric root and arborvitae twig in the traditional Chinese medicine compound is about 10.93%, about 5.46%, about 10.93%, about 16.39%, about 5.46%, about 10.93%, about 4.37%, about 8.20%, about 2.74%, about 5.46%, about 2.74% and about 10.93%, respectively.

Further, the rehmannia root is rehmannia root.

Further, the radix paeoniae rubra is radix paeoniae rubra decoction pieces.

Further, the garden burnet is garden burnet charcoal.

Further, the sophora fruit is sophora fruit charcoal.

Further, the herba Schizonepetae is charred herba Schizonepetae.

Further, the radix Scutellariae is radix Scutellariae charcoal.

Further, the coptis is stir-fried coptis.

Further, the radix trichosanthis, the radix angelicae sinensis and the rhizoma cimicifugae are radix trichosanthis, radix angelicae sinensis and rhizoma cimicifugae decoction pieces.

Further, the fructus Aurantii is bran-parched fructus Aurantii.

Further, the mobile phase a is acetonitrile.

Further, the aqueous acid solution is 0.08 to 0.12% aqueous acid solution.

Further, the aqueous acid solution is 0.08 to 0.12% of phosphoric acid aqueous solution, formic acid aqueous solution or glacial acetic acid aqueous solution.

Further, the pH of the buffered saline solution is no greater than 7.0.

Further, the chromatographic column is a HALO AQ-C18 chromatographic column.

Further, the fingerprint comprises 19 common fingerprint peaks at a detection wavelength of 280nm, wherein 11 # naringin chromatographic peaks are used as reference peaks, the relative retention time of the other 18 common peaks is 0.18+/-10% of No. 1 chromatographic peak, 0.31+/-10% of No. 2 chromatographic peak, 0.36+/-10% of No. 3 chromatographic peak, 0.45+/-10% of No. 4 chromatographic peak, 0.56+/-10% of No. 5 chromatographic peak, 0.59+/-10% of No. 6 chromatographic peak, 0.75+/-10% of No. 7 chromatographic peak, 0.78+/-10% of No. 8 chromatographic peak, 0.83+/-10% of No. 9 chromatographic peak, 0.84+/-10% of No. 10 chromatographic peak, 1.05+/-10% of No. 12 chromatographic peak, 1.11+/-10% of No. 13 chromatographic peak, 1.13+/-10% of No. 14 chromatographic peak, 1.29+/-10% of No. 16 chromatographic peak, 1.30+/-10% of No. 17 chromatographic peak, 1.34+/-10% of No. 18 chromatographic peak, and 1.61+/-10% of No. 19 chromatographic peak.

According to another aspect of the present invention, there is provided a quality control method of a Chinese herbal compound including red peony root, the quality control method comprising the steps of:

(1) The fingerprint construction method establishes a standard fingerprint of a traditional Chinese medicine compound reference sample.

(2) Detecting the solution of the traditional Chinese medicine compound sample according to chromatographic conditions in the fingerprint construction method to obtain a fingerprint of the traditional Chinese medicine compound sample to be detected; and

(3) Comparing the fingerprint of the traditional Chinese medicine compound sample to be detected obtained in the step (2) with the standard fingerprint of the traditional Chinese medicine compound reference sample obtained in the step (1), wherein the traditional Chinese medicine compound reference sample is qualified if meeting the requirements, and is unqualified if not meeting the requirements.

Further, the compliance may include one or more of the following:

(1) The fingerprint of the traditional Chinese medicine compound sample to be detected shows 19 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within +/-10% of the retention time value of the corresponding reference chromatographic peak in the standard fingerprint of the traditional Chinese medicine compound reference sample;

(2) The naringin peak is taken as an S peak, and the relative retention time of each characteristic chromatographic peak and the S peak in the fingerprint of the traditional Chinese medicine compound to be detected sample is within +/-10% of the relative retention time value of each characteristic chromatographic peak of the standard fingerprint of the traditional Chinese medicine compound reference sample; and

(3) According to the similarity evaluation system of the traditional Chinese medicine chromatographic fingerprint, the similarity between the traditional Chinese medicine compound sample fingerprint to be detected and the traditional Chinese medicine compound reference sample standard fingerprint is calculated and is not lower than 0.90.

According to another aspect of the invention, there is provided the use of the above detection method or the above construction method or the above quality control method in quality detection or quality evaluation or quality control of a herbal compound comprising red peony root.

The invention has the beneficial effects that:

In short, the invention provides a fingerprint spectrum measuring and quality control method for a traditional Chinese medicine compound reference sample containing red paeony root, which confirms 19 common characteristic peaks, has simple conditions and short analysis time, solves the problems of difficult separation of fingerprint characteristic peaks and interference of impurity peaks, ensures the chemical composition stability and the use safety of the reference sample, provides an important reference basis for quality control of subsequent preparations, ensures the quality stability of products, ensures the curative effect of the traditional Chinese medicine compound, and ensures that the traditional Chinese medicine compound better serves the life health of human beings.

Specifically, compared with the prior art, the invention has the following beneficial effects:

(1) The fingerprint of the traditional Chinese medicine compound reference sample is established, the defect that the content measurement of a single component is difficult to reflect the whole content is overcome, the intrinsic quality of the traditional Chinese medicine compound reference sample can be controlled integrally and macroscopically, the curative effect of the medicine is ensured, the main means of modern medicine research is utilized, the quality inheritance classical is realized, and the classical name formula is controlled more normally.

(2) The traditional Chinese medicine compound reference sample has complex chemical components, and the difficulty of separating characteristic peaks is high.

(3) In the process of establishing the fingerprint of the traditional Chinese medicine compound reference sample, 19 common characteristic peaks are confirmed, the relative retention time, the relative peak area and the similarity are researched, the chemical composition stability and the use safety of the reference sample are ensured, and important reference basis and quality reference are provided for quality control of subsequent compound preparations.

(4) The fingerprint of each active ingredient in the traditional Chinese medicine compound reference sample is regarded as a whole, the front-back sequence and the correlation of each characteristic peak are focused, the one-sided property of judging the whole quality of the traditional Chinese medicine compound reference sample is avoided by only measuring one and two chemical ingredients, the possibility of artificial treatment for reaching the quality standard is reduced, and a novel method and means are provided for completely and accurately evaluating the quality of the traditional Chinese medicine compound reference sample.

(5) The method has the advantages of good stability, high precision, good repeatability, convenience and easy grasp.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it will be apparent that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings by those skilled in the art without departing from the scope of the claimed invention.

Fig. 1 is a fingerprint of 30 batches of Chinese herbal medicine compound decoction of the application.

Fig. 2 is a fingerprint of a sample solution of a compound decoction of traditional Chinese medicine. Wherein, peak 1: gallic acid, peak 5: paeoniflorin, peak 7: isoferulic acid, peak 9: senkyunolide I, peak 11: naringin (S), peak 12: sophoroside, peak 14: neohesperidin, peak 15: berberine hydrochloride, peak 17: baicalin, peak 18: wogonin, peak 19: glycyrrhizic acid.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains or to which this term applies. Although any methods, conditions, materials, or materials similar or equivalent to those disclosed herein can be used in the practice of the present invention, the preferred methods, conditions, materials, or materials are described herein.

The invention is intended to cover all alternatives, modifications and equivalents, which may be included within the art of the invention as defined by the appended claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The invention is in no way limited to the description of methods and materials.

As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

In the present invention, the term "comprising" is synonymous with "including". The terms "comprising," "including," "having," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

As described in the background art, the comprehensive analysis and fingerprint research of chemical components of the Chinese medicinal composition comprising red paeony root, which are claimed by the invention, have not been reported in the literature. In order to solve the problems, the invention provides a detection method of a traditional Chinese medicine compound containing red paeony root, which comprises the following steps:

In the present invention, when time, flow rate, temperature, wavelength, amount of sample introduced, ratio, voltage, concentration, or other value or parameter is expressed in terms of a range, preferred range, or a range defined by a series of upper preferable values and lower preferable values, this should be understood to specifically disclose all ranges formed by any pair of any upper range limit or preferred value with any lower range limit or preferred value, whether or not the ranges are separately disclosed. For example, when ranges "20-30" are disclosed, the ranges described should be construed to include ranges "20-30", "20-25", "25-30", and the like. When a numerical range is described herein, unless otherwise indicated, the range is intended to include its endpoints and all integers and fractions within the range.

In a preferred embodiment, the information is the content of one or more of the following components in the traditional Chinese medicine compound according to the external standard method according to the recorded corresponding peak areas in the chromatogram of the traditional Chinese medicine compound test solution and the chromatogram of the reference solution: rehmannia root, red paeony root, garden burnet root, pagodatree pod, fineleaf schizonepeta herb, baical skullcap root, golden thread, mongolian snakegourd root, chinese angelica, largetrifoliolious bugbane rhizome, bitter orange, liquoric root and arborvitae twig.

In a preferred embodiment, the standard curve of paeoniflorin is y= 6730.5929x-0.6597, and r ² =1.0000.

In a preferred embodiment, the standard curve for naringin is y=11572.6279 x+3.1418, and r ² =1.0000.

In a preferred embodiment, the standard curve of neohesperidin is y=11119.1023x+6.6214, r ² =1.0000.

In a preferred embodiment, the preparation method of the compound traditional Chinese medicine test solution comprises the following steps: weighing a proper amount of Chinese herbal medicine compound, placing into a container with proper volume, adding water for soaking, heating to boil with strong fire, transferring to slow fire, and decocting for a period of time to obtain decoction, and filtering with a filter screen while hot to obtain filtrate; and (3) taking a proper amount of the filtrate, filtering with a microporous filter membrane, and taking a subsequent filtrate to obtain the compound traditional Chinese medicine test sample solution.

In a preferred embodiment, the container is a drug-decocting pot.

In a preferred embodiment, the volume of the container is 2L.

In a preferred embodiment, the ratio of mass/volume (g/ml) between the herbal compound and the water is 0.05 to 0.5, for example about 0.11.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 0.11" includes ±5% of 0.11, or from 0.1045 to 0.1155.

In a preferred embodiment, the soaking time is 30 to 90 minutes, for example about 60 minutes.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

In a preferred embodiment, the voltage of the strong fire is 200-300V, such as about 220V.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 220" includes ±5% of 220, or from 209 to 231.

In a preferred embodiment, the slow fire has a voltage of 150 to 200V, for example about 175V.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 175" includes + -5% of 175, or from 166.25 to 183.75.

In a preferred embodiment, the time of the micro-boiling decoction is 20 to 60 minutes, for example about 40 minutes.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 40" includes 40.+ -. 5%, or from 38 to 42.

In a preferred embodiment, the ratio of volume/volume (ml/ml) between the water and the decoction is 1-5, for example about 3.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15.

In a preferred embodiment, the screen is a 120 mesh screen.

In a preferred embodiment, the method for preparing the control solution comprises: weighing a proper amount of paeoniflorin, naringin and neohesperidin reference substances, and adding a methanol solution to prepare the reference substance solutions with the paeoniflorin, naringin and neohesperidin concentration of 5-400 mug/ml respectively.

In a preferred embodiment, the concentration of paeoniflorin the control solution is about 60 μg/ml.

In a preferred embodiment, the concentration of naringin in the control solution is about 70 μg/ml.

In a preferred embodiment, the concentration of neohesperidin in the control solution is about 70 μg/ml.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 70" includes ±5% of 70, or from 66.5 to 73.5.

In a preferred embodiment, the flow rate is from 0.8 to 1.2ml/min, for example about 1.0ml/min.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 1.0" includes ±5% of 1.0, or from 0.95 to 1.05.

In a preferred embodiment, the column temperature is from 23 to 27 ℃, such as about 25 ℃.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 25" includes ±5% of 25, or from 23.75 to 26.25.

In a preferred embodiment, the detection wavelength is 200 to 250nm, for example 230nm.

In a preferred embodiment, the sample is introduced in an amount of 3 to 7. Mu.l, for example about 5. Mu.l.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 5" includes + -5% of 5, or from 4.75 to 5.25.

In a preferred embodiment, the purity angle of the peak purity of the chromatographic peak corresponding to the control is greater than the purity threshold.

In a preferred embodiment, the reference corresponds to a chromatographic peak having a theoretical plate number greater than 3000.

In a preferred embodiment, the control corresponds to a chromatographic peak having a resolution of greater than 1.5.

In a preferred embodiment, the chromatography column is a HALO AQ-C18 chromatography column.

In a preferred embodiment, the chromatographic column is of the specification: the column length was 150mm, the inner diameter was 4.6mm, and the particle diameter was 2.7. Mu.m.

In a preferred embodiment, the mobile phase a is acetonitrile.

In a preferred embodiment, the aqueous acid, aqueous base and/or buffered saline solution is selected from one or more of weak acids and salts thereof, weak bases and salts thereof of varying concentrations.

In a preferred embodiment, the aqueous acid, aqueous base and/or buffered saline solution is selected from the group consisting of formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid at various concentrations.

In a preferred embodiment, the aqueous acid is an aqueous acid of 0.08% to 0.12%.

In a preferred embodiment, the aqueous acid solution is an aqueous solution of phosphoric acid at 0.08% to 0.12%.

In a preferred embodiment, the aqueous acid solution is about 0.1% aqueous phosphoric acid.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 0.1%" includes 0.1% of + -5%, or from 0.095% to 0.105%.

In a preferred embodiment, the buffered saline solution is an aqueous phosphate solution and/or an aqueous acetate solution.

In a preferred embodiment, the buffered saline solution has a pH of no greater than 7.0.

In a preferred embodiment, in the traditional Chinese medicine compound, the weight ratio of rehmannia root, red paeony root, garden burnet root, pagodatree pod, fineleaf schizonepeta herb, baical skullcap root, golden thread, mongolian snakegourd root, chinese angelica tail, largetrifoliolious bugbane rhizome, bitter orange, liquoric root and arborvitae twig is 7% -19%, 3% -13%, 10% -17%, 3% -8%, 7% -19%, 1% -11%, 1% -4%, 3% -13%, 0.5% -5% and 7% -19%.

In a preferred embodiment, in the traditional Chinese medicine compound, the weight ratio of rehmannia root, red paeony root, garden burnet root, pagodatree pod, fineleaf schizonepeta herb, baical skullcap root, golden thread, mongolian snakegourd root, chinese angelica tail, largetrifoliolious bugbane rhizome, bitter orange, liquoric root and arborvitae twig is about 10.93%, about 5.46%, about 10.93%, about 16.39%, about 5.46%, about 10.93%, about 4.37%, about 8.20%, about 2.74%, about 5.46%, about 2.74% and about 10.93%.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 10.93%" includes 10.93% plus or minus 5%, or from 10.3835% to 11.4765%; "about 5.46%" includes 5.46% of + -5%, or from 5.187% to 5.733%; "about 16.39%" includes 16.39% of + -5%, or from 15.5705% to 17.2095%; "about 4.37%" includes 4.37% plus or minus 5%, or from 4.1515% to 4.5885%; "about 8.20%" includes 8.20% plus or minus 5%, or from 7.79% to 8.61%; "about 2.74%" includes 2.74% plus or minus 5%, or from 2.603% to 2.877%.

In a preferred embodiment, the rehmannia root is rehmannia root.

In a preferred embodiment, the red peony root is a red peony root decoction piece.

In a preferred embodiment, the garden burnet is burnet char.

In a preferred embodiment, the sophora fruit is sophora fruit charcoal.

In a preferred embodiment, the schizonepeta is schizonepeta charcoal.

In a preferred embodiment, the scutellaria root is scutellaria root charcoal.

In a preferred embodiment, the coptis is a stir-fried coptis.

In a preferred embodiment, the radix Trichosanthis, radix Angelicae sinensis, and rhizoma cimicifugae are radix Trichosanthis decoction pieces, radix Angelicae sinensis decoction pieces, and rhizoma cimicifugae decoction pieces.

In a preferred embodiment, the fructus Aurantii is a bran-fried fructus Aurantii.

In a preferred embodiment, the control solution is a mixed solution of paeoniflorin about 60 μg/ml, naringin about 70 μg/ml, neohesperidin about 70 μg/ml.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63; "about 70" includes + -5% of 70, or from 66.5 to 73.5.

Preparation of test solution: weighing a proper amount of Chinese herbal medicine compound, placing into a container with proper volume, adding water for soaking, heating to boil with strong fire, transferring to slow fire, and decocting for a period of time to obtain decoction, and filtering with a filter screen while hot to obtain filtrate; filtering with microporous membrane to obtain filtrate, and collecting filtrate to obtain the test solution, wherein the Chinese medicinal composition comprises rehmanniae radix, radix Paeoniae Rubra, radix Sangusorbae, fructus Sophorae, herba Schizonepetae, scutellariae radix, coptidis rhizoma, trichosanthis radix, radix Angelicae sinensis, cimicifugae rhizoma, fructus Aurantii, glycyrrhrizae radix and folium Platycladi.

Preparation of a control solution: weighing a proper amount of paeoniflorin, naringin and neohesperidin reference substances, and adding a methanol solution to prepare the reference substance solutions with the paeoniflorin, naringin and neohesperidin concentration of 5-400 mug/ml respectively.

And (3) obtaining the traditional Chinese medicine compound fingerprint according to the results of the high-performance liquid detection of the sample solution and the reference solution.

In a preferred embodiment, the detection wavelength is 250 to 300nm, for example 280nm.

In a preferred embodiment, the fingerprint comprises peaks 1-19 at the detection wavelength of 280nm, wherein peak 1 is assigned to radix Paeoniae Rubra, fructus Sophorae, and radix Sangusorbae; peak 2, peak 4, peak 15, peak 16 are ascribed to coptis medicinal flavor; peak 3 and Peak 6 belong to the medicinal flavor of Sanguisorbae; peak 5 is attributed to the red peony root drug taste; peak 8, peak 10, peak 12, peak 13 are ascribed to the pagodatree pod flavor; peak 7 is ascribed to cimicifuga foetida taste; peak 9 belongs to the flavor of Chinese angelica root; peak 11 and peak 14 are ascribed to bitter orange; peak 17 and peak 18 are ascribed to the scutellaria medicinal taste; peak 19 is attributed to Glycyrrhiza uralensis.

In a preferred embodiment, the retention times for peaks 1-19 are 9.7±10％、16.3±10％、19.2±10％、24.1±10％、29.5±10％、31.0±10％、39.5±10％、41.8±10％、43.7±10％、44.8±10％、53.1±10％、55.9±10％、58.9±10％、59.9±10％、67.3±10％、68.3±10％、71.0±10％、83.6±10％ and 85.5.+ -. 10%, respectively.

In a preferred embodiment, the peak 1 is a chromatographic peak of gallic acid.

In a preferred embodiment, the peak 15 is a chromatographic peak of berberine hydrochloride.

In a preferred embodiment, the peak 5 is a chromatographic peak of paeoniflorin.

In a preferred embodiment, the peak 12 is a chromatographic peak of sophoricoside.

In a preferred embodiment, the peak 7 is a chromatographic peak of isoferulic acid.

In a preferred embodiment, the peak 9 is a chromatographic peak of senkyunolide I.

In a preferred embodiment, the peak 11 is a chromatographic peak of naringin.

In a preferred embodiment, the peak 14 is a chromatographic peak of neohesperidin.

In a preferred embodiment, the peak 17 is a chromatographic peak of baicalin.

In a preferred embodiment, the peak 18 is a chromatographic peak of wogonin.

In a preferred embodiment, the peak 19 is a chromatographic peak of glycyrrhizic acid.

In a preferred embodiment, the weight ratio of rehmannia root, red paeony root, garden burnet root, pagodatree pod, fineleaf schizonepeta herb, baical skullcap root, coptis root, mongolian snakegourd root, chinese angelica, largetrifoliolious bugbane rhizome, bitter orange, liquoric root and arborvitae twig in the traditional Chinese medicine compound is about 10.93%, about 5.46%, about 10.93%, about 16.39%, about 5.46%, about 10.93%, about 4.37%, about 8.20%, about 2.74%, about 5.46%, about 2.74% and about 10.93%, respectively.

In a preferred embodiment, the rehmannia root is rehmannia root.

In a preferred embodiment, the garden burnet is burnet char.

In a preferred embodiment, the sophora fruit is sophora fruit charcoal.

In a preferred embodiment, the schizonepeta is schizonepeta charcoal.

In a preferred embodiment, the scutellaria root is scutellaria root charcoal.

In a preferred embodiment, the coptis is a stir-fried coptis.

In a preferred embodiment, the radix Trichosanthis, radix Angelicae sinensis, and cimicifugae rhizoma are radix Trichosanthis, radix Angelicae sinensis, and cimicifugae rhizoma decoction pieces.

In a preferred embodiment, the mobile phase a is acetonitrile.

In a preferred embodiment, the aqueous acid solution is an aqueous solution of phosphoric acid, formic acid or glacial acetic acid in an amount of 0.08% to 0.12%.

In a preferred embodiment, the fingerprint comprises 19 common fingerprint peaks at a detection wavelength of 280nm, wherein the 11-peak naringin chromatographic peak is taken as a reference peak, and the relative retention time of the other 18 common peaks is sequentially 0.18+/-10% of the 1 st chromatographic peak, 0.31+/-10% of the 2 nd chromatographic peak, 0.36+/-10% of the 3 rd chromatographic peak, 0.45+/-10% of the 4 th chromatographic peak, 0.56+/-10% of the 5 th chromatographic peak, 0.59+/-10% of the 6 th chromatographic peak, 0.75+/-10% of the 7 th chromatographic peak, 0.78+/-10% of the 8 th chromatographic peak, 0.83+/-10% of the 9 th chromatographic peak, 0.84+/-10% of the 10 th chromatographic peak, 1.05+/-10% of the 12 th chromatographic peak, 1.11+/-10% of the 13 th chromatographic peak, 1.13+/-10% of the 15 th chromatographic peak, 1.29+/-10% of the 16 th chromatographic peak, 1.30+/-10% of the 17 th chromatographic peak, 1.34+/-10% of the 18 th chromatographic peak, 1.57+/-10% of the 19 th chromatographic peak.

In a preferred embodiment, the compliance includes one or more of the following:

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are generally according to conventional conditions or conditions suggested by manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

The above-mentioned features of the invention, or of the embodiments, may be combined in any desired manner. All of the features disclosed in this patent specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

Examples

1. Instrument, reagent and reference substance

The apparatus and manufacturer used in the present application are shown below.

Model of ultra-high performance liquid chromatograph: waters UPLC Waters technologies limited; model of ultra-high performance liquid chromatograph: agilent 1290_dad Agilent technologies limited; chromatographic column model: HALO AQ-C18.6X105 mm 2.7 μm; sequence number: USAQT001502; chromatographic column model: HALO AQ-C18.6X105 mm 2.7 μm; sequence number: USAQT001519; chromatographic column model: HALO AQ-C18.6X105 mm 2.7 μm; sequence number: USAQT001446.

The reference substances used in the present application are shown in Table 1.

TABLE 1 control used in the present application

Methanol, acetonitrile, phosphoric acid, formic acid, glacial acetic acid are from national pharmaceutical group chemical reagent limited.

2. Preparation method of traditional Chinese medicine compound decoction

7.46G of rehmannia root, 3.73g of red peony root, 7.46g of garden burnet root, 11.19g of sophora fruit, 3.73g of schizonepeta herb, 3.73g of baikal skullcap root, 7.46g of fried coptis root, 2.98g of radix trichosanthis, 5.60g of Chinese angelica tail, 1.87g of cimicifuga foetida, 3.73g of bitter orange fried with bran, 1.87g of liquorice, 7.46g of biota oriental arborvitae, placing the above thirteen medicinal herbs in a 2L multifunctional decocting pot, adding 600ml of water, covering, soaking for 60 minutes, placing a temperature-regulating heating plate, heating with strong fire (220V) until boiling, transferring to soft fire (175V), keeping micro-boiling, decocting for about 40 to 200ml, and filtering with a 120-mesh filter screen while the materials are hot.

3. Investigation of elution conditions

3.1 Elution condition 1

Chromatographic conditions and system suitability test: octadecyl bonded silica gel as filler (column: HALOAQ-C18.6X105 mm 2.7 μm); acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, and gradient elution was performed as specified in table 2; the flow rate is 1ml per minute; the column temperature is 35 ℃; scanning the whole wavelength and detecting the whole wavelength; the characteristic detection wavelength is 280nm, and the content detection wavelength is 230nm.

Table 2 Chinese herbal medicine Compound decoction characteristic spectrum chromatographic condition-gradient 1

Reference solution preparation: taking a proper amount of paeoniflorin reference substance, naringin reference substance and neohesperidin reference substance, precisely weighing, and respectively adding methanol to prepare mixed solutions containing 60 mug of paeoniflorin, 70 mug of naringin and 70 mug of neohesperidin per 1ml of mixed solution serving as reference substance solutions of the reference substances.

Sample solution preparation: taking about 5g of the Chinese herbal medicine compound decoction, precisely weighing, placing into a 25ml measuring flask, adding a proper amount of 50% methanol, performing ultrasonic treatment (power 500W, frequency 40 kHz) for 10 minutes, cooling, adding 50% methanol to dilute to scale, shaking uniformly, filtering, and taking subsequent filtrate.

Assay: precisely sucking 5 μl of reference solution and sample solution, and injecting into liquid chromatograph for measurement.

The result shows that under the condition of gradient 1, the chromatogram of the traditional Chinese medicine compound decoction sample has relatively poor separation of each peak, more mixed peaks and overlong analysis time, more peak information within 50-60 min of peak-out time, and is optimized subsequently, and gradient 2 is designed.

3.2 Elution condition 2

Chromatographic conditions and system suitability test: octadecyl bonded silica gel as filler (column: HALOAQ-C18.6X105 mm 2.7 μm); acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, and gradient elution was performed as specified in table 3; the flow rate is 1ml per minute; the column temperature is 35 ℃; scanning the whole wavelength and detecting the whole wavelength; the characteristic detection wavelength is 280nm, and the content detection wavelength is 230nm.

Table 3 Chinese herbal Compound decoction characteristic spectrum chromatographic condition-gradient 2

The result shows that under the condition of gradient 2, the chromatogram of the traditional Chinese medicine compound decoction sample has relatively poor separation of each peak, more mixed peaks and longer time, and is optimized subsequently, so that gradient 3 is designed.

3.3 Elution conditions 3 (Table 4 shows)

Table 4 Chinese herbal medicine Compound decoction characteristic spectrum chromatographic condition-gradient 3

Time (min)	Acetonitrile (%)	0.1% Phosphoric acid (%)
			0～6	0	100
6～15	0→5	100→95
			15～20	5→14	95→86
20～42	14	86
			42～52	14→16	86→84
52～62	16→17	84→83
			62～64	17	83
64～70	17→21	83→79
			70～80	21→30	79→70
80～85	30→45	70→55
			85～88	45→0	55→100
88～100	0	100

The result shows that under the condition of gradient 3, the chromatogram of the test sample of the Chinese herbal compound decoction has relatively poor separation of each peak within 15-20 min of the peak-exiting time, and gradient 4 is designed.

3.4 Elution conditions 4

Chromatographic conditions and System applicability test octadecyl bonded silica gel was used as filler (column: HALO AQ-C18.6X250 mm 2.7 μm); gradient elution was performed as specified in table 5 with acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; the flow rate is 1ml per minute; the column temperature is 25 ℃; full wavelength detection; the characteristic detection wavelength is 280nm, and the content detection wavelength is 230nm.

Table 5 Chinese herbal medicine Compound decoction characteristic spectrum chromatographic condition-gradient 4

Time (min)	Acetonitrile (%)	0.1% Phosphoric acid (%)
			0～6	0	100
6～15	0→5	100→95
			15～20	5→10	95→90
20～31	10→12	90→88
			31～39	12→14	88→86
39～42	14	86
			42～52	14→16	86→84
52～62	16→17	84→83
			62～64	17	83
64～70	17→21	83→79
			70～80	21→30	79→70
80～85	30→45	70→55
			85～88	45→0	55→100
88～100	0	100

The results show that under gradient 4 conditions, there are more chromatographic peaks, more complex components, and better resolution than gradient 1, gradient 2, and gradient 3.

4. Column temperature investigation

Taking 1 part of sample solution of the traditional Chinese medicine compound decoction to be tested in 3.2, filtering the sample solution by a microporous filter membrane, and examining the influence of the column temperature of 23 ℃, 25 ℃ and 28 ℃ on the characteristic map of the gradient 4.

The results show that the liquid chromatogram of the sample solution of the traditional Chinese medicine compound decoction is generally similar under different column temperatures (23 ℃,25 ℃ and 28 ℃) with gradient 4; the peak time of each characteristic peak is slightly advanced with the rise of the column temperature, wherein the color spectrum is more and the separation degree is better when the column temperature is 25 ℃ compared with other column temperatures, and the peak type of each color spectrum is more excellent.

5. Flow rate investigation

Taking 1 part of sample solution of the traditional Chinese medicine compound decoction related in 3.2, filtering the sample solution by a microporous filter membrane, and examining the influence of the flow rates of 0.8ml/min, 1.0ml/min and 1.2ml/min on the gradient 4 characteristic spectrum.

The result shows that under the condition of gradient 4 of different flow rates (0.8 ml/min, 1.0ml/min and 1.2 ml/min), the peak-out time of each characteristic peak is slightly advanced along with the rise of the flow rate, wherein when 1.2ml/min, the liquid chromatogram of the traditional Chinese medicine compound decoction sample solution has poor separation degree, and the liquid chromatograms of 0.8ml/min and 1.0ml/min are generally similar, and the separation distance of each characteristic peak is increased; the flow rate was chosen to be 1.0ml/min considering the durability of the chromatographic column.

6. Chromatographic column inspection

Taking 1 part of sample solution of the traditional Chinese medicine compound decoction to be tested in 3.2, filtering the sample solution by a microporous filter membrane, and examining the influence of different numbered chromatographic columns HALO AQ-C18 on the characteristic spectrum of the gradient 4.

The results show that the liquid chromatogram of the sample solution of the traditional Chinese medicine compound decoction under the condition of different chromatographic column numbers (HALO AQ-C18) with gradient 4 is generally similar, which indicates that the chromatographic conditions are stable, and the selected chromatographic column is HALO AQ-C18.

7. Investigation of acid species

Taking 1 part of sample solution of the traditional Chinese medicine compound decoction related in 3.2, filtering the sample solution by a microporous filter membrane, and examining the influence of 0.1% phosphoric acid, 0.1% formic acid and 0.1% glacial acetic acid on the characteristic spectrum of the gradient 4.

The result shows that under the gradient 4, the liquid chromatogram baseline of the traditional Chinese medicine compound decoction sample solution is unstable under the condition of 0.1 percent formic acid and 0.1 percent glacial acetic acid; the overall spectrum was better at 0.1% phosphoric acid, so 0.1% phosphoric acid was determined as chromatographic condition flowability B.

8. Investigation of the extraction solvent

Taking 10 parts of traditional Chinese medicine compound decoction, and respectively examining the influence of pure water, 10% methanol, 25% methanol, 50% methanol and 75% methanol serving as extraction solvents on the content measurement of a sample solution of a test sample of the traditional Chinese medicine compound decoction.

Taking 6 parts of Chinese medicinal compound decoction, respectively weighing about 5g, placing into 25ml measuring flask, respectively adding 25% methanol, 50% methanol and 75% methanol, processing with ultrasound (500W, 40 kHz) for 10 min, cooling, fixing volume, shaking, filtering, collecting filtrate, and sampling. The results are shown in Table 6.

TABLE 6 summary of solution content detection information of test sample solutions of compound Chinese medicinal decoction processed by different extraction solvents

Extraction solvent	Paeoniflorin (mg/g)	Naringin (mg/g)	New hesperidin (mg/g)
				25% Methanol	0.28	0.35	0.35
50% Methanol	0.29	0.36	0.37
				75% Methanol	0.29	0.37	0.37

The result shows that the relative average deviation of paeoniflorin, naringin and neohesperidin content is less than or equal to 3.0%, which indicates that different extraction solvents have no influence on paeoniflorin, naringin and neohesperidin content, and the conditions of each peak of the characteristic spectrum are synthesized, so 50% methanol is selected as the extraction solvent.

9. Sample size investigation

Taking 6 parts of the traditional Chinese medicine compound decoction, and respectively examining the influence of the sampling amount of 2.5g, 5.0g and 10.0g on the content measurement of the sample solution of the traditional Chinese medicine compound decoction.

About 2.5g, 5.0g and 10.0g of the traditional Chinese medicine compound decoction are taken, 2 parts of each decoction are precisely weighed, placed in a 25ml measuring flask, added with a proper amount of 50% methanol, treated for 10 minutes by ultrasound (power 500W and frequency 40 kHz), cooled, diluted to a scale by adding 50% methanol, shaken uniformly, filtered, and the subsequent filtrate is taken and sampled, and the result is shown in a table 7.

Table 7 summary of detection information of different sampling amounts and contents of Chinese herbal medicine compound decoction

Sampling amount	Paeoniflorin (mg/g)	Naringin (mg/g)	New hesperidin (mg/g)
				2.5g	0.28	0.35	0.37
5.0g	0.28	0.38	0.37
				10.0g	0.28	0.38	0.37

The result shows that the contents of paeoniflorin and neohesperidin tend to be stable, the naringin content slightly increases along with the increase of the sampling amount, the sampling amount is 5.0g consistent with the naringin content of 10.0g, the peak conditions of the characteristic spectrum are synthesized, and the sampling amount is 5.0g as the sampling amount of a sample to be tested.

10. Detection method determination

Octadecyl bonded silica gel as filler (column: HALOAQ-C18.6X105 mm2.7 μm); gradient elution was performed as specified in table 5 with acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; the flow rate is 1ml per minute; the column temperature is 25 ℃; full wavelength detection; the characteristic detection wavelength is 280nm, and the content detection wavelength is 230nm.

Taking 30 batches of Chinese herbal compound decoction. The preparation method of the traditional Chinese medicine compound decoction comprises the following steps: 7.46g of rehmannia root, 3.73g of red peony root, 7.46g of garden burnet root, 11.19g of sophora fruit, 3.73g of schizonepeta herb, 3.73g of baikal skullcap root, 7.46g of fried coptis root, 2.98g of radix trichosanthis, 5.60g of Chinese angelica tail, 1.87g of cimicifuga foetida, 3.73g of bitter orange fried with bran, 1.87g of liquorice, 7.46g of biota oriental arborvitae, placing the above thirteen medicinal herbs in a 2L multifunctional decocting pot, adding 600ml of water, covering, soaking for 60 minutes, placing a temperature-regulating heating plate, heating with strong fire (220V) until boiling, transferring to soft fire (175V), keeping micro-boiling, decocting for about 40 to 200ml, and filtering with a 120-mesh filter screen while the materials are hot.

The Chinese herbal medicine compound decoction is prepared into a sample solution and subjected to fingerprint detection, and the results are shown in fig. 1 and 2.

Feature map detection-detection wavelength 280nm.

Content measurement and detection-detection wavelength 230nm (paeoniflorin, naringin, neohesperidin).

And precisely sucking 5 μl of the sample solution by the measuring method, injecting into a liquid chromatograph, and measuring to obtain the final product.

11. Characteristic spectrum peak assignment

As shown in chromatograms of fig. 1 and 2, the characteristic chromatogram peaks of the traditional Chinese medicine compound decoction disclosed by the invention mainly show 19 chromatographic peaks, and peak 1 is classified as red paeony root, sophora fruit and garden burnet, and is gallic acid; peak 2, peak 4, peak 15, peak 16 are ascribed to coptis medicinal flavor, wherein peak 15 is berberine hydrochloride; peak 3 and Peak 6 belong to radix Sanguisorbae flavor, peak 5 belongs to radix Paeoniae Rubra flavor, and is paeoniflorin; peak 8, peak 10, peak 12, peak 13 are ascribed to the sophorae medicinal taste, wherein peak 12 is sophoricoside; peak 7 belongs to cimicifugae rhizoma flavor, and is isoferulic acid; peak 9 belongs to the flavor of Chinese angelica and is senkyunolide I; peak 11 is naringin, peak 14 is neohesperidin, all of which are attributed to fructus Aurantii; peak 17 is baicalin, peak 18 is wogonin, and is ascribed to the medicinal flavor of radix Scutellariae; peak 19 belongs to Glycyrrhrizae radix, and is glycyrrhizic acid.

And (3) carrying out sample injection analysis on each medicine negative and single control solution of the blank solvent, the control solution and the traditional Chinese medicine compound reference sample solution, wherein the blank solvent and the negative control solution have no interference at the peak positions of paeoniflorin, naringin and neohesperidin and the peak positions of the set characteristic peaks. Specific chromatographic peak assignment medicinal flavors in the chromatograms are shown in tables 8 and 9.

Specific chromatographic peak belonging to medicinal taste-1 in Table 8 chromatogram

Peak number/medicinal taste

Radix Angelicae sinensis

Radix paeoniae rubra

Cimicifugae rhizoma

Sophora japonica fruit

Rehmannia root

Radix Sanguisorbae

Coptis chinensis Franch

Peak 1

-

+

-

+

-

+

-

Peak 2

-

+

Peak 3

-

+

-

Peak 4

-

+

Peak 5

-

+

-

Peak 6

-

+

-

Peak 7

-

+

-

Peak 8

-

+

-

Peak 9

+

-

Peak 10

-

+

-

Peak 11

-

Peak 12

-

+

-

Peak 13

-

+

-

Peak 14

-

Peak 15

-

+

Peak 16

-

+

Peak 17

-

Peak 18

-

Peak 19

-

The symbols: + represent the characteristic peak, -represent the absence of the characteristic peak.

Table 9 specific chromatographic peak belonging to medicinal taste-2 in the chromatogram

Note that: + is indicated as having this characteristic peak, -is indicated as not having this characteristic peak.

At the detection wavelength of 230nm, the content measurement peak paeoniflorin, naringin and neohesperidin in the traditional Chinese medicine compound decoction is not interfered by the comparison of the measurement spectrum of the single-medicine negative decoction, and the specificity is high, so that the paeoniflorin, naringin and neohesperidin in the traditional Chinese medicine compound decoction are determined as the content measurement index.

12. Methodological verification of content determination methods

1) Precision (content detection repeatability, intermediate precision, instrument precision)

The content RSD of paeoniflorin, neohesperidin and naringin is less than or equal to 3 percent.

2) Accuracy of content detection

The recovery rate RSD of paeoniflorin, naringin and neohesperidin is less than or equal to 3%, and the recovery rates are all within the range of 90% -108%; according to the analysis method verification guiding principle of the Chinese pharmacopoeia 2020 edition 9101, the method is proved to have better accuracy.

3) Sensitivity of content detection method

Detection limit refers to the minimum amount (concentration) in a sample that can be qualitatively detected, but does not require accurate quantification. In chromatographic analysis, the detection limit is generally defined by using a signal-to-noise ratio (S/N), which is a ratio of a response value of a target substance in a sample to a baseline noise, and it is generally considered that a signal-to-noise ratio of 3 or more is useful for qualitative purposes and a signal-to-noise ratio of 10 or more is useful for quantitative purposes. The quantitative limit of paeoniflorin is 0.2997 mug/ml, the quantitative limit of naringin is 0.3219 mug/ml, the quantitative limit of neohesperidin is 0.2856 mug/ml, and the RSD of the three is less than or equal to 10%; the detection limit of paeoniflorin is 0.09373 mug/ml, the detection limit of naringin is 0.05101 mug/ml, and the detection limit of neohesperidin is 0.1055 mug/ml.

4) Content detection method linearity

7 Control solutions of different concentration levels were prepared for the assay. The measured response signal is plotted as a function of the concentration of the test object, and linear regression is performed by the least square method. The correlation coefficient R ² is not less than 0.9990, which indicates that the linearity of the method is good.

Linear regression of paeoniflorin: y= 6730.5929x-0.6597, r ² =1.0000;

Linear regression of naringin: y=11572.6279x+3.1418, r ² =1.0000;

Linear regression of neohesperidin: y=11119.1023x+6.6214, r ² =1.0000.

5) Durability of

Flow rate, column temperature, different acid concentrations, different chromatographic columns, stability (72 h)

The durability of the flow rate (0.9 ml/min, 1.0ml/min, 1.1 ml/min) was examined, and the fixed flow rate was 1.0ml/min;

The durability of the column temperature (23 ℃,25 ℃ and 28 ℃) is examined, and the column temperature is 25 ℃ to 28 ℃ with certain durability;

The content RSD of paeoniflorin, naringin and neohesperidin with different acid concentrations, different chromatographic columns and stability is less than or equal to 4%, and the durability is good.

13. Methodological verification of feature maps

1) Precision (feature map detection repeatability, intermediate precision, instrument precision)

The repeatability of the characteristic spectrum, the intermediate precision and the similarity of the spectrum of the instrument precision are all more than 0.90.

2) Durability of

① Flow rate durability

The result shows that when the flow rate is 0.9ml/min and 1.0ml/min, the relative retention time RSD of the characteristic peaks of the characteristic patterns is less than or equal to 3.0 percent; the similarity among the patterns is larger than 0.90, and when the flow rate is 1.1ml/min, the separation degree of paeoniflorin chromatographic peak 5 (t _R = 29.646) is poor, which indicates that the method has certain durability under the flow rate of 0.9-1.0 ml/min, so that experiments under the flow rate of 0.9-1.0 ml/min are recommended.

② Column temperature durability

The result shows that the relative retention time RSD of characteristic peaks of the characteristic patterns is less than or equal to 3.0 percent when the column temperature is between 23 and 28 ℃; the similarity among the patterns is larger than 0.90, which indicates that the method has certain durability at the column temperature of 23-28 ℃.

③ Durability at different acid concentrations

The result shows that when the concentration of different acids is 0.08 to 0.12 percent, the relative retention time RSD of characteristic peaks of the characteristic spectrum is less than or equal to 3.0 percent; the similarity among the patterns is larger than 0.90, which indicates that the method has certain durability under the condition of different acid concentrations of 0.08-0.12%.

④ Different column durability

The result shows that when the chromatographic columns of different batches are replaced, the relative retention time RSD of characteristic peaks of the characteristic patterns is less than or equal to 3.0 percent; the similarity among all the patterns is larger than 0.90, which indicates that the chromatographic columns of different batches of the method have certain durability.

⑤ Stability of test solution

The result shows that the RSD of the relative retention time of each characteristic peak in the stability experiment is less than or equal to 3 percent; the relative peak area has an RSD range of 1.5% -11.6%; the peak area percentage of the peak 5 and the peak 18 is less than 10 percent, so that the method can be properly widened; RSD of the relative peak areas of other chromatographic peaks is less than or equal to 5%; and the similarity of each characteristic peak is more than 0.90. Therefore, the method is shown to have better stability within 0-72 hours.

The foregoing has outlined rather broadly the more detailed description of embodiments of the invention in order that the detailed description of the principles and embodiments of the invention may be implemented in conjunction with the detailed description of embodiments of the invention that follows. Meanwhile, based on the idea of the present invention, those skilled in the art can make changes or modifications on the specific embodiments and application scope of the present invention, which belong to the protection scope of the present invention. In view of the foregoing, this description should not be construed as limiting the invention.

Claims

1. The detection method of the Chinese herbal compound containing red paeony root is characterized by comprising the following steps of:

The chromatographic conditions of the detection are: adopting a chromatographic column with octadecyl bonded silica gel as a filler, wherein a mobile phase A is one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is acid water, alkali water solution and/or buffer saline solution, the gradient elution process is ：0～6min,0％A;6～15min,0％→5％A;15～20min,5％→10％A;20～31min,10％→12％A;31～39min,12％→14％A;39～42min,14％A;42～52min,14％→16％A;52～62min,16％→17％A;62～64min,17％A;64～70min,17％→21％A;70～80min,21％→30％A;80～85min,30％→45％A;85～88min,45％→0％A;88～100min,0％A;, the flow rate is 0.5-1.5 mL/min, the column temperature is 20-30 ℃, the detection wavelength is 200-300 nm, and the sample injection amount is 1-10 mu l;

wherein the Chinese medicinal compound comprises rehmanniae radix, radix Paeoniae Rubra, radix Sangusorbae, fructus Sophorae, herba Schizonepetae, scutellariae radix, coptidis rhizoma, trichosanthis radix, radix Angelicae sinensis, cimicifugae rhizoma, fructus Aurantii, glycyrrhrizae radix and folium Platycladi, and the reference substance comprises paeoniflorin, naringin and neohesperidin.

2. The detection method according to claim 1, wherein the information is the content of one or more of the following components in the traditional Chinese medicine compound according to the external standard method according to the recorded corresponding peak areas in the chromatogram of the traditional Chinese medicine compound sample solution and the chromatogram of the reference solution: rehmannia root, red paeony root, garden burnet root, pagodatree pod, fineleaf schizonepeta herb, baical skullcap root, golden thread, mongolian snakegourd root, chinese angelica, largetrifoliolious bugbane rhizome, bitter orange, liquoric root and arborvitae twig;

more preferably, the standard curve of paeoniflorin is y= 6730.5929x-0.6597, and r ² =1.0000;

More preferably, the standard curve of naringin is y=11572.6279 x+3.1418, and r ² =1.0000;

More preferably, the standard curve of the neohesperidin is y=11119.1023x+6.6214, and r ² =1.0000;

still preferably, the preparation method of the compound traditional Chinese medicine sample solution comprises the following steps: weighing a proper amount of Chinese herbal medicine compound, placing into a container with proper volume, adding water for soaking, heating to boil with strong fire, transferring to slow fire, and decocting for a period of time to obtain decoction, and filtering with a filter screen while hot to obtain filtrate; taking a proper amount of the filtrate, filtering with a microporous filter membrane, and taking a subsequent filtrate to obtain the compound traditional Chinese medicine sample solution;

still preferably, the container is a drug-decocting pot;

Still preferably, the volume of the container is 2L;

Still preferably, the ratio of mass/volume (g/ml) between the herbal compound and the water is 0.05-0.5, for example about 0.11;

Still preferably, the soaking time is 30 to 90 minutes, for example about 60 minutes;

still preferably, the voltage of the strong fire is 200-300V, such as about 220V;

still preferably, the voltage of the slow fire is 150-200V, for example about 175V;

Still preferably, the time of the micro-boiling decoction is 20 to 60 minutes, for example, about 40 minutes;

still preferably, the ratio of volume/volume (ml/ml) between the water and the decoction is between 1 and 5, for example about 3;

Still preferably, the filter screen is a 120 mesh filter screen;

Particularly preferably, the preparation method of the reference substance solution comprises the following steps: weighing a proper amount of paeoniflorin, naringin and neohesperidin reference substances, and adding a methanol solution to prepare reference substance solutions with the concentration of paeoniflorin, naringin and neohesperidin of 5-400 mug/ml respectively;

particularly preferably, the concentration of paeoniflorin the control solution is about 60 μg/ml;

Particularly preferably, the concentration of naringin in the control solution is about 70 μg/ml;

particularly preferably, the concentration of neohesperidin in the control solution is about 70 μg/ml;

Particularly preferably, the flow rate is from 0.8 to 1.2ml/min, for example about 1.0ml/min;

Particularly preferably, the column temperature is from 23 to 27 ℃, for example about 25 ℃;

particularly preferably, the detection wavelength is 200 to 250nm, for example 230nm;

particularly preferably, the sample loading is 3 to 7. Mu.l, for example about 5. Mu.l;

Still more preferably, the purity angle of the peak purity of the chromatographic peak corresponding to the control is greater than a purity threshold;

still more preferably, the theoretical plate number of the chromatographic peak corresponding to the reference substance is greater than 3000;

Still more preferably, the separation degree of the chromatographic peak corresponding to the reference substance is more than 1.5;

still more particularly preferably, the chromatography column is a HALO AQ-C18 chromatography column;

still more preferably, the specification of the chromatographic column is: the column length was 150mm, the inner diameter was 4.6mm, and the particle diameter was 2.7. Mu.m.

3. The detection method according to claim 1 or 2, wherein the mobile phase a is acetonitrile;

Preferably, the aqueous acid, aqueous base and/or aqueous buffered salt is selected from one or more of weak acids and salts thereof, weak bases and salts thereof of varying concentrations;

preferably, the aqueous acid, aqueous base and/or buffered saline solution is selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid;

More preferably, the aqueous acid solution is 0.08% -0.12% aqueous acid solution;

more preferably, the aqueous acid solution is 0.08% -0.12% aqueous phosphoric acid solution;

more preferably, the aqueous acid solution is about 0.1% aqueous phosphoric acid;

more preferably, the buffered saline solution is a phosphate saline solution and/or an acetate saline solution;

more preferably, the PH of the buffered saline solution is no greater than 7.0;

Still preferably, in the traditional Chinese medicine compound, the weight ratio of rehmannia root, red paeony root, garden burnet root, pagodatree pod, fineleaf schizonepeta herb, baical skullcap root, golden thread, mongolian snakegourd root, chinese angelica, largetrifoliolious bugbane rhizome, bitter orange, liquoric root and arborvitae twig is 7% -19%, 3% -13%, 10% -17%, 3% -8%, 7% -19%, 1% -11%, 1% -4%, 3% -13%, 0.5% -5% and 7% -19%;

Still preferably, in the traditional Chinese medicine compound, the weight ratio of rehmannia root, red paeony root, garden burnet root, pagodatree pod, fineleaf schizonepeta herb, baical skullcap root, golden thread, mongolian snakegourd root, chinese angelica, largetrifoliolious bugbane rhizome, bitter orange, liquoric root and arborvitae twig is about 10.93%, about 5.46%, about 10.93%, about 16.39%, about 5.46%, about 10.93%, about 4.37%, about 8.20%, about 2.74%, about 5.46%, about 2.74% and about 10.93%;

Particularly preferably, the rehmannia root is rehmannia root;

particularly preferably, the radix paeoniae rubra is radix paeoniae rubra decoction pieces;

particularly preferably, the garden burnet is carbonized garden burnet;

particularly preferably, the sophora fruit is sophora fruit charcoal;

Particularly preferably, the schizonepeta is schizonepeta charcoal;

particularly preferably, the baikal skullcap root is baikal skullcap root charcoal;

Particularly preferably, the coptis is fried coptis;

particularly preferably, the radix trichosanthis, the Chinese angelica tail and the cimicifugae are prepared from radix trichosanthis decoction pieces, chinese angelica tail decoction pieces and cimicifugae rhizoma decoction pieces;

Particularly preferably, the fructus Aurantii is bran-fried fructus Aurantii;

particularly preferably, the reference solution is a mixed solution of paeoniflorin about 60 mug/ml, naringin about 70 mug/ml, neohesperidin about 70 mug/ml.

4. The fingerprint spectrum construction method of the Chinese herbal medicine compound containing red paeony root is characterized by comprising the following steps of:

preparation of test solution: weighing a proper amount of Chinese herbal medicine compound, placing into a container with proper volume, adding water for soaking, heating to boil with strong fire, transferring to slow fire, and decocting for a period of time to obtain decoction, and filtering with a filter screen while hot to obtain filtrate; filtering the filtrate with microporous membrane, and collecting filtrate to obtain the sample solution, wherein the Chinese medicinal composition comprises rehmanniae radix, radix Paeoniae Rubra, radix Sangusorbae, fructus Sophorae, herba Schizonepetae, scutellariae radix, coptidis rhizoma, trichosanthis radix, radix Angelicae sinensis, cimicifugae rhizoma, fructus Aurantii, glycyrrhrizae radix and folium Platycladi;

obtaining a traditional Chinese medicine compound fingerprint according to the results of high-performance liquid phase detection of the sample solution and the reference solution;

5. The method of claim 4, wherein the flow rate is 0.8-1.2 mL/min, such as about 1.0mL/min;

preferably, the column temperature is from 23 to 27 ℃, such as about 25 ℃;

Preferably, the detection wavelength is 250 to 300nm, for example 280nm;

Preferably, the sample loading is 3 to 7. Mu.l, for example about 5. Mu.l;

Still preferably, the preparation method of the reference substance solution comprises the following steps: weighing a proper amount of paeoniflorin, naringin and neohesperidin reference substances, and adding a methanol solution to prepare reference substance solutions with the concentration of paeoniflorin, naringin and neohesperidin of 5-400 mug/ml respectively;

still preferably, the concentration of paeoniflorin the control solution is about 60 μg/ml;

Still preferably, the concentration of naringin in the control solution is about 70 μg/ml;

still preferably, the concentration of neohesperidin in the control solution is about 70 μg/ml;

Particularly preferably, the purity angle of the peak purity of the chromatographic peak corresponding to the control is greater than a purity threshold;

Particularly preferably, the theoretical plate number of the chromatographic peak corresponding to the reference substance is more than 3000;

Particularly preferably, the separation degree of the chromatographic peak corresponding to the reference substance is more than 1.5.

6. The construction method according to claim 4, wherein the fingerprint comprises peaks 1-19 at the detection wavelength of 280nm, wherein peak 1 is attributed to red peony root, pagodatree pod and garden burnet root; peak 2, peak 4, peak 15, peak 16 are ascribed to coptis medicinal flavor; peak 3 and Peak 6 belong to the medicinal flavor of Sanguisorbae; peak 5 is attributed to the red peony root drug taste; peak 8, peak 10, peak 12, peak 13 are ascribed to the pagodatree pod flavor; peak 7 is ascribed to cimicifuga foetida taste; peak 9 belongs to the flavor of Chinese angelica root; peak 11 and peak 14 are ascribed to bitter orange; peak 17 and peak 18 are ascribed to the scutellaria medicinal taste; peak 19 is ascribed to licorice;

preferably, the retention times of peaks 1-19 are 9.7±10％、16.3±10％、19.2±10％、24.1±10％、29.5±10％、31.0±10％、39.5±10％、41.8±10％、43.7±10％、44.8±10％、53.1±10％、55.9±10％、58.9±10％、59.9±10％、67.3±10％、68.3±10％、71.0±10％、83.6±10％ and 85.5.+ -. 10%, respectively;

preferably, the peak 1 is a chromatographic peak of gallic acid;

preferably, the peak 15 is a chromatographic peak of berberine hydrochloride;

Preferably, the peak 5 is a chromatographic peak of paeoniflorin;

preferably, the peak 12 is a chromatographic peak of sophoricoside;

preferably, the peak 7 is a chromatographic peak of isoferulic acid;

preferably, the peak 9 is a chromatographic peak of senkyunolide I;

Preferably, the peak 11 is a chromatographic peak of naringin;

preferably, the peak 14 is a chromatographic peak of neohesperidin;

Preferably, the peak 17 is a chromatographic peak of baicalin;

Preferably, the peak 18 is a chromatographic peak of wogonin;

Preferably, the peak 19 is a chromatographic peak of glycyrrhizic acid;

Preferably, the weight ratio of rehmannia root, red paeony root, garden burnet root, pagodatree pod, fineleaf schizonepeta herb, baical skullcap root, golden thread, mongolian snakegourd root, chinese angelica, largetrifoliolious bugbane rhizome, bitter orange, liquoric root and arborvitae twig in the traditional Chinese medicine compound is about 10.93%, about 5.46%, about 10.93%, about 16.39%, about 5.46%, about 10.93%, about 4.37%, about 8.20%, about 2.74%, about 5.46%, about 2.74% and about 10.93%, respectively;

More preferably, the rehmannia root is rehmannia root;

More preferably, the red peony root is red peony root decoction pieces;

more preferably, the garden burnet is garden burnet char;

more preferably, the sophora fruit is sophora fruit charcoal;

More preferably, the schizonepeta is schizonepeta charcoal;

more preferably, the baikal skullcap root is baikal skullcap root charcoal;

More preferably, the coptis chinensis is fried coptis chinensis;

more preferably, the radix trichosanthis, the radix angelicae sinensis and the rhizoma cimicifugae are radix trichosanthis, radix angelicae sinensis and rhizoma cimicifugae decoction pieces;

More preferably, the fructus Aurantii is bran-parched fructus Aurantii.

7. The method of claim 4, wherein mobile phase a is acetonitrile;

More preferably, the aqueous acid solution is 0.08-0.12% phosphoric acid aqueous solution, formic acid aqueous solution or glacial acetic acid aqueous solution;

more preferably, the PH of the buffered saline solution is no greater than 7.0;

still preferably, the chromatography column is a HALO AQ-C18 chromatography column;

still preferably, the chromatographic column has the following specifications: the column length is 150mm, the inner diameter is 4.6mm, and the grain diameter is 2.7 mu m;

Particularly preferably, the fingerprint comprises 19 common fingerprint peaks, 11-peak naringin chromatographic peaks are taken as reference peaks, and the relative retention time of the other 18 common peaks is sequentially 0.18+/-10% of No. 1 chromatographic peak, 0.31+/-10% of No. 2 chromatographic peak, 0.36+/-10% of No. 3 chromatographic peak, 0.45+/-10% of No. 4 chromatographic peak, 0.56+/-10% of No.5 chromatographic peak, 0.59+/-10% of No.6 chromatographic peak, 0.75+/-10% of No.7 chromatographic peak, 0.78+/-10% of No.8 chromatographic peak, 0.83+/-10% of No.9 chromatographic peak, 0.84+/-10% of No. 10 chromatographic peak, 1.05+/-10% of No. 12 chromatographic peak, 1.11+/-10% of No. 13 chromatographic peak, 1.13+/-10% of No. 14 chromatographic peak, 1.29+/-10% of No. 15 chromatographic peak, 1.30+/-10% of No. 16 chromatographic peak, 1.34+/-10% of No. 18 chromatographic peak, 1.57+/-10% of No. 19 chromatographic peak.

8. The quality control method of the Chinese herbal compound containing red paeony root is characterized by comprising the following steps of:

(1) Establishing a standard fingerprint of a traditional Chinese medicine compound reference sample according to the fingerprint establishing method of any one of claims 4 to 7;

(2) Taking a traditional Chinese medicine compound sample solution, and detecting according to chromatographic conditions in the fingerprint construction method of any one of claims 4 to 7 to obtain a traditional Chinese medicine compound sample fingerprint to be detected; and

9. The quality control method of claim 8, wherein the compliance includes one or more of:

(3) And according to a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, calculating the similarity between the traditional Chinese medicine compound sample fingerprint to be detected and the traditional Chinese medicine compound reference sample standard fingerprint, wherein the similarity is not lower than 0.90.

10. Use of the detection method according to any one of claims 1 to 3 or the construction method according to any one of claims 4 to 7 or the quality control method according to claim 8 or 9 in quality detection or quality evaluation or quality control of a chinese herbal compound comprising red peony root.