CN114660218A

CN114660218A - Medicine composition containing 'Qingshanjuantong decoction' and detection method

Info

Publication number: CN114660218A
Application number: CN202011529625.9A
Authority: CN
Inventors: 于娟; 洪绯; 曾金香; 林锦霞; 郑玉清; 吴骅楷; 杨小平
Original assignee: Zhangzhou Pientzehuang Pharmaceutical Co Ltd
Current assignee: Zhangzhou Pientzehuang Pharmaceutical Co Ltd
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2022-06-24

Abstract

The invention discloses a pharmaceutical composition containing 'clear Shandong Juantong decoction' and also discloses a quality detection method of clear Shandong Juan Tong decoction or a pharmaceutical composition containing 'clear Shandong Tong Tang'. The invention relates to a pharmaceutical composition containing 'clear Shangjuantong decoction', which contains stable, comprehensive and uniform active pharmaceutical ingredients in 'clear Shangjuantong decoction'. The quality detection method of the Qingshangtong soup and the preparation thereof has high accuracy, can comprehensively reflect the general view of the Qingshangtong soup, performs quality control on the traditional preparation and the modern preparation of the Qingshangtong soup on the whole, and has popularization and application values.

Description

Medicine composition containing 'clear Shangjuandong decoction' and detection method

Technical Field

The invention relates to a pharmaceutical composition containing 'clear Shangjuan Tang' and a detection method thereof.

Background

The Qingshangtong decoction is derived from Ming & Gong Yangxian (longevity Shi Baoyuan), and the original text records "the main prescription for treating all headaches" and "all headaches are effective for both healthy and new times. The modern application is in angioneurotic headache, maxillary sinusitis headache, trigeminal neuralgia and the like.

The technical difficulty of research and development of modern preparation of Qingshang Juantong decoction is in the system for evaluating the equivalence of the preparation at all times and today. Because the quality of the final product is influenced by a plurality of links of the processing, extraction process and preparation process of the raw materials and the raw materials of the modern preparation of the classical famous prescription, and the quality of the final product is difficult to reflect by the qualitative identification of a simple index component, in order to ensure the safety, effectiveness, stability and controllability of the classical famous prescription and the preparation thereof, a full chemical spectrum which can more stably and comprehensively reflect the composition of the classical famous prescription and the preparation thereof is required to be used as an effective means for controlling and evaluating the ancient and modern preparations of the classical famous prescription. Quality of the face, quality of the girl, etc., classic famous prescription clear Shangtong decoction fingerprint atlas research [ J ], Nanjing university of traditional Chinese medicine, academic newspaper 2020.3.36(2), discloses a fingerprint atlas detection method for clear Shangtong decoction water decoction, and in the fingerprint atlases of different batches of decoction samples obtained by the method, the relative peak areas of all common characteristic peaks are greatly different, so that the quality of clear Shangtong decoction and products thereof cannot be accurately controlled, and no method for performing quality control evaluation on the traditional preparation of clear Shangtong decoction and performing overall and comprehensive quality control on the modern preparation of clear Shangtong decoction is available at present.

Disclosure of Invention

In order to solve the above problems, the present invention provides a pharmaceutical composition containing "Qingshanjuantong decoction", wherein the HPLC fingerprint contains 18 characteristic peaks, namely peak 5 glycyrrhizin, peak 7 senkyunolide I, peak 8 isoliquiritin, peak 9 baicalin, peak 13 wogonoside and peak 17 wogonin, the peak corresponding to a baicalin reference substance is S peak, the relative retention time of the remaining characteristic peaks and the S peak is calculated, the relative retention time is within + -7% of the specified value, and the specified value is: peak 10.098, peak 20.196, peak 30.249, peak 40.358, peak 60.499, peak 101.194, peak 111.270, peak 121.325, peak 141.551, peak 151.597, peak 161.636, peak 182.088;

the detection conditions of the HPLC fingerprint spectrum are as follows: a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: taking acetonitrile as a mobile phase A, and taking a 0.1% phosphoric acid solution as a mobile phase B for gradient elution; the gradient elution procedure was as follows:

furthermore, the Chinese medicinal preparation is prepared by adding conventional auxiliary materials into the Qingshangtong decoction and preparing a clinically acceptable oral preparation according to a conventional process;

further, the oral preparation includes, but is not limited to, decoction, granules, pills, drop pills, mixture, soft extract, powder or tablets.

The invention also provides a quality detection method of the Qingshanjuandong decoction or the pharmaceutical composition containing the Qingshanjuandong decoction, which adopts HPLC detection and specifically comprises the following steps:

a. preparation of reference solutions: dissolving baicalin as reference substance in ethanol solution;

b. preparing a test solution: extracting a sample to be detected with methanol solution, filtering, evaporating the filtrate to dryness, dissolving the residue with water, adding water saturated n-butanol-ethyl acetate mixed solution for extraction, evaporating the extract to dryness, dissolving the residue with methanol solution, and filtering to obtain the final product;

c. and respectively sucking the reference substance solution and the test solution, injecting the reference substance solution and the test solution into a liquid chromatograph, and detecting according to the chromatographic conditions.

Further, the concentration of the reference solution in the step a) is 0.1-0.3 mg of baicalin per 1ml, and preferably 0.2 mg; the concentration of the ethanol solution is 50-80%, and v/v; preferably 70%, v/v.

Further, the mass-to-volume ratio of the sample to be detected in the step b) to the methanol solution is 0.05-5 g:25mL, preferably 0.5-1.5 g:25 mL; the extraction is ultrasonic extraction, the power is 250w, the frequency is 40kHz, and the time is 30 min; the volume of the residue added with water is the same as that of the sample to be detected added with the methanol solution.

Further, in the step b), the extraction is that water-saturated n-butyl alcohol-ethyl acetate mixed solution is added, the mixture is shaken for 3 times, the volume equal to that of water is added for each time, and the upper layer of extraction liquid is combined; the volume ratio of the n-butanol to the ethyl acetate is 1: 2.

Further, the volume of the residue and methanol solution in the step b) is 1/5-1, preferably 4/5 of the sample to be detected and the methanol solution; the concentration of the methanol solution is 50-80%, v/v, preferably 70%, v/v.

Further, the wavelength of the chromatographic condition in the step c) is 280nm, and the number of theoretical plates is not less than 5000 calculated according to baicalin.

Further, the octadecylsilane chemically bonded silica chromatographic column of step C) is Welch Ultimate XB C18, 4.6X 250mm, 5 μm, Waters symmetry C18, 4.6X 250mm, 5 μm or Phenomene

4.6×250mm，5μm。

The invention relates to a pharmaceutical composition containing 'clear Shangjuantong decoction', which contains stable, comprehensive and uniform active pharmaceutical ingredients in 'clear Shangjuantong decoction'. The quality detection method of the Qingshangtong soup and the preparation thereof has high accuracy, can comprehensively reflect the general view of the Qingshangtong soup, performs quality control on the traditional preparation and the modern preparation of the Qingshangtong soup on the whole, and has popularization and application values.

The decoction for clearing away the upper heat and relieving pain is a traditional Chinese medicine prescription name, is named from Ming, Gong Yangxian (longevity's best) and is prepared from Chinese angelica, Szechuan lovage rhizome, dahurian angelica root, incised notopterygium rhizome, divaricate saposhnikovia root, swordlike atractylodes rhizome, dwarf lilyturf tuber and pubescent angelica root, each of which is 3 grams, asarum and liquorice, each of which is three thirds (each of which is 0.3 gram), chastetree fruit and chrysanthemum, each of which is 1.5 grams, each of which is five fifths (4.5 grams), and ginger, which is used as a guide, and is decocted in water for oral administration.

The material standard of the clear Shangjuan pain decoction used in the invention is a standard of medicinal materials of the clear Shangjuan pain decoction prepared according to a preparation method of the clear Shangjuan pain decoction recorded in ancient medical books, shou Shi Bao Yuan, and the preparation methods are basically consistent with the record of the shou Shi Bao Yuan except a molding process. The preparation method comprises the following steps:

weighing the following raw materials according to the formula of clear Shangjutong decoction: slicing rhizoma Zingiberis recens into thick pieces (2 mm-4 mm), pulverizing the rest thirteen materials, sieving with 3mm sieve, placing in casserole, decocting with water twice, adding 10 times of water for the first time, soaking for 30min, boiling with strong fire, decocting with slow fire for 30min, and filtering to obtain filtrate; adding 8 times of water for the second time, boiling with strong fire, decocting with slow fire for 20 minutes, filtering, mixing with the above filtrate, concentrating under reduced pressure until the weight ratio of the fed amount to the concentrated solution is about 1 g: 2g, freeze drying to obtain dry extract, and packaging to obtain the corresponding material of the material standard of the decoction for clearing away the upper part of the body fluid.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a finger print searching chart

FIG. 2 fingerprint spectrum peak identification (S1 dried powder; S2 baicalin; S3 isoliquiritin; S4 liquiritin S5 wogonoside; S6 wogonin; S7 senkyunolide I)

FIG. 3 fingerprint method determination

FIG. 4 shows HPLC contrasts of fingerprint blank solvent, reference substance and sample

FIG. 5 shows a fingerprint spectrum consensus diagram of a corresponding real object (dried extract powder) (A-consensus diagram 1, B-consensus diagram 2)

FIG. 6 shows a comparison fingerprint (18 common peaks, 5 peak: glycyrrhizin, 7 peak: senkyunolide I, 8 peak: isoliquiritin, 9 peak (S): baicalin, 13 peak: wogonoside, 17 peak: wogonin)

Detailed Description

Example 1 preparation of material-based corresponding substance of clear Shangjuan Tang

Prescription: 10 parts of wine-washed angelica, 10 parts of ligusticum wallichii, 10 parts of angelica dahurica, 3 parts of asarum, 10 parts of notopterygium root, 10 parts of radix angelicae pubescentis, 10 parts of divaricate saposhnikovia root, 5 parts of chrysanthemum, 5 parts of fructus viticis, 10 parts of bran-fried rhizoma atractylodis, 15 parts of wine-fried scutellaria baicalensis, 10 parts of radix ophiopogonis, 3 parts of liquorice and 5.3 parts of ginger

Preparation: taking 2-4 mm of ginger, slicing the rest thirteen medicines, crushing, sieving by a 3mm sieve, putting into a casserole, adding water for decocting twice, adding 10 times of water for the first time, soaking for 30 minutes, boiling with strong fire, decocting with slow fire for 30 minutes, and filtering to obtain filtrate for later use; adding 8 times of water for the second time, boiling with strong fire, decocting with slow fire for 20 min, filtering, mixing with the above filtrate, concentrating under reduced pressure until the weight ratio of the feed amount to the concentrate is about 1 g: 2g, lyophilizing to obtain dry extract, and packaging to obtain the corresponding product.

Example 2 detection of substance reference and corresponding object of clear Shangjiang pain soup

1) Preparation of reference solutions:

taking appropriate amount of baicalin as reference, precisely weighing, and adding 70% ethanol to obtain solution containing 0.2mg per 1 ml;

2) preparation of a test solution:

taking 1g of the material standard substance of the Qingshangtong decoction prepared in the example 1, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, filtering, evaporating filtrate to dryness, adding 25ml of water into residue to dissolve, shaking and extracting with a water-saturated n-butyl alcohol-ethyl acetate (1:2) mixed solution for 3 times, 25ml each time, combining upper layer extract liquid, evaporating to dryness, adding 70% methanol into residue to dissolve, transferring to a 20ml measuring flask, adding 70% methanol to scale, shaking uniformly, and filtering to obtain the Qingshangtong decoction;

3) determination of characteristic profiles

Respectively and precisely absorbing the reference substance solution and the test solution, and injecting the reference substance solution and the test solution into a liquid chromatograph to obtain a chromatogram;

the chromatographic conditions were as follows:

a chromatographic column: phenomene

(4.6X 250mm, 5 μm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the column temperature is 30 ℃; the flow rate was 1ml per minute; detection wavelength: 280nm, and the number of theoretical plates is not less than 5000 calculated according to baicalin peak.

4) Analysis of feature maps

The substance standard of the clear upper relieving pain soup corresponds to a physical characteristic map and shows 18 characteristic peaks, wherein the peak 5 is liquiritin, the peak 7 is senkyunolide I, the peak 8 is isoliquiritin, the peak 9 is baicalin, the peak 13 is wogonoside, the peak 17 is wogonin, the peak corresponding to a baicalin reference substance is an S peak, the relative retention time of the rest characteristic peaks and the S peak is calculated, the relative retention time is within +/-7% of a specified value, and the specified value is as follows: peak 10.098, peak 20.196, peak 30.249, peak 40.358, peak 60.499, peak 101.194, peak 111.270, peak 121.325, peak 141.551, peak 151.597, peak 161.636, peak 182.088.

EXAMPLE 3 preparation of clear Shangjuantong decoction granule

Preparation: taking 2-4 mm of ginger, slicing the rest thirteen medicines, crushing, sieving by a 3mm sieve, putting into a casserole, adding water for decocting twice, adding 10 times of water for the first time, soaking for 30 minutes, boiling with strong fire, decocting with slow fire for 30 minutes, and filtering to obtain filtrate for later use; adding 8 times of water for the second time, boiling with strong fire, decocting with slow fire for 20 min, filtering, mixing with the above filtrate, concentrating under reduced pressure until the weight ratio of the feed amount to the concentrate is about 1 g: 2g, lyophilizing, and pulverizing to obtain dry extract powder;

mixing the above dry extract powder with adjuvant, granulating, drying, sieving, and making into QINGSHANGJUANTONG decoction granule.

Example 4 detection of clear Shangjuantong decoction granules

1) Preparation of reference solutions:

2) preparation of a test solution: taking 5g of clear Shangjuandong decoction granules prepared in example 3, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, filtering, evaporating filtrate to dryness, adding 25ml of water into residue to dissolve the residue, shaking and extracting for 3 times by using a water-saturated n-butyl alcohol-ethyl acetate (1:2) mixed solution, 25ml each time, combining upper layer extract solutions, evaporating to dryness, adding 70% methanol into residue to dissolve the residue, transferring to a 20ml measuring flask, adding 70% methanol to a scale, shaking uniformly, and filtering to obtain the medicinal granule;

3) determination of characteristic map

the chromatographic conditions were as follows:

a chromatographic column: waters symmetry C18 (4.6X 250mm, 5 μm); acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the column temperature is 30 ℃; the flow rate was 1ml per minute; detection wavelength: 280nm, and the number of theoretical plates is not less than 5000 calculated according to baicalin peak.

4) Analysis of feature maps

The clear Shangjuan decoction feature chromatogram shows 18 feature peaks, wherein the peak 5 is liquiritin, the peak 7 is senkyunolide I, the peak 8 is isoliquiritin, the peak 9 is baicalin, the peak 13 is wogonoside, the peak 17 is wogonin, the peak corresponding to a baicalin reference substance is an S peak, the relative retention time of the rest feature peaks and the S peak is calculated, the relative retention time is within +/-7% of a specified value, and the specified value is as follows: peak 10.098, peak 20.196, peak 30.249, peak 40.358, peak 60.499, peak 101.194, peak 111.270, peak 121.325, peak 141.551, peak 151.597, peak 161.636, peak 182.088.

The advantageous effects of the present invention are described below by way of test examples.

Test example 1

(1) Instrument and reagent

Liquid chromatograph: shimadzu LC-20A, Agilent 1260 II; a chromatographic column: welch Ultimate XB C18 (4.6X 250mm, 5 μm); waters symmetry C18 (4.6X 250mm, 5 μm); phenomene

(4.6X 250mm, 5 μm), electronic analytical balance: double Jie test instrumentation works in Heiguan JJ1000 (hundredth), Saydorshi scientific instruments (Beijing) Inc. BSA224S-CW (one in ten thousand), Saydorshi scientific instruments (Beijing) Inc. SQP (one in ten thousand), Metler-Torilat instruments (Shanghai) Inc. ME503T (one in thousand);

the methanol is chromatographically pure, and is produced by OCEANPAK; acetonitrile is chromatographically pure, and is produced by OCEANPAK; phosphoric acid is chromatographically pure, and is produced by Shandong Xiya chemical Co., Ltd; the methanol is analytically pure and is produced by Guangzhou chemical reagent factories; water is ultrapure water, Shanghai and Tai instruments Inc.; baicalin reference substances (batch No. 110715-201720, purity: 93.5%), liquiritin reference substances (batch No. 111610-201607, purity: 93.1%), wogonin reference substances (batch No. 112002-201702, purity: 98.5%) and wogonin reference substances (batch No. 11514-201706) were purchased from China institute for testing and determining food and drug. Isoliquiritin reference substance (lot number: 17042102, purity: 98%), and senkyunolide I reference substance (lot number: 17121105, purity: 98%), were all purchased from Dupont Biotechnology Co., Ltd.

(2) Selection of mobile phase

Earlier experiments show that mobile phase acetonitrile-0.1% phosphoric acid and methanol-0.1% phosphoric acid have good separation effect on components in corresponding real objects of the material standard of the clear Shangjiang pain soup, so that the method is further adjusted on the basis of the mobile phase acetonitrile-0.1% phosphoric acid and the methanol-0.1% phosphoric acid.

Acetonitrile-0.1% phosphoric acid system

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 280 nm.

Preparing a test solution: taking about 1g of the substance standard substance of the Qingshangtong decoction prepared in example 1, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, filtering, evaporating to dryness, adding 25ml of water into residues to dissolve, shaking up and extracting for 3 times with ethyl acetate, 25ml each time, combining ethyl acetate solutions, evaporating to dryness, adding 70% methanol into residues to dissolve, transferring to a 20ml measuring flask, adding 70% methanol to the scale, shaking up, filtering, and taking the subsequent filtrate to obtain the Qingshangtong decoction.

The determination method comprises precisely sucking 5 μ l of sample solution, injecting into liquid chromatograph, determining, and recording chromatogram. The results are shown in FIG. 1.

② methanol-0.1 percent phosphoric acid system

Taking the test solution under the item 'i', changing the mobile phase into methanol-0.1% phosphoric acid, and carrying out the determination result under the condition that other detection conditions are not changed: the methanol-0.1 percent phosphoric acid mobile phase system has less peak information, so the acetonitrile-0.1 percent phosphoric acid solution system is selected for testing.

(3) Selection of test article preparation method

First, solvent extraction and investigation

a 70% methanol solution sonication

Taking 1g of the material standard substance of the clear Shangjiang pain relieving soup prepared in example 1, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, ultrasonically treating (250W, 40KHz) for 30 minutes, taking out, cooling, weighing again, complementing the lost weight with 70% methanol, shaking uniformly, filtering, and taking out the subsequent filtrate.

b extracting with water saturated n-butanol solution by shaking

Taking 1g of the Qingshangtong decoction prepared in example 1 according to the quality standard, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, carrying out ultrasonic treatment (250W and 40kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the weight loss by 70% methanol, shaking up, filtering, evaporating to dryness, adding 25ml of water into residues for dissolving, shaking up and extracting for 3 times by using a water saturated n-butyl alcohol solution, 25ml each time, combining the n-butyl alcohol solutions, evaporating to dryness, adding 70% methanol into residues for dissolving, transferring to a 20ml measuring flask, adding 70% methanol to the scale, shaking up and filtering to obtain the Qingshangtong decoction.

c extracting with ethyl acetate solution by shaking

Taking 1g of the clear Shangjiang pain-relieving soup prepared in example 1 according to the quality standard, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, carrying out ultrasonic treatment (250W, 40KHz) for 30 minutes, taking out, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, filtering, evaporating to dryness, adding 25ml of water into residues for dissolving, shaking up and extracting for 3 times with ethyl acetate, each time 25ml, combining ethyl acetate solutions, evaporating to dryness, adding 70% methanol into residues for dissolving, transferring to a 20ml measuring flask, adding 70% methanol to the scale, shaking up and filtering to obtain the clear Shangjiang pain-relieving soup.

Measuring according to the determined chromatographic conditions, recording the chromatogram of the test solution extracted by different solvents, and obtaining the result: the sample solution subjected to the ultrasonic treatment by 70% methanol has more peaks and poorer peak separation degree; the number of chromatographic peaks of a sample solution extracted by shaking water-saturated n-butanol is less than that of the chromatographic peaks of the 70% methanol ultrasonic treatment, and the ratio of the peak area of the baicalin chromatographic peak is more than 50%; the chromatographic peak of the ethyl acetate shaking extraction is less than that of the water saturated n-butyl alcohol shaking extraction, but the baseline is more stable, the comprehensive analysis is carried out, the information of the baseline and the chromatographic peak is considered, and the investigation needs to be carried out on different proportions of the ethyl acetate and the n-butyl alcohol in mixing.

② investigation of solvent ratio

Taking 1g of the clear Shangjuan decoction prepared in example 1, accurately weighing the decoction, placing the decoction in a conical flask with a plug, accurately adding 25ml of 70% methanol, weighing the weight, carrying out ultrasonic treatment (the power is 250W and the frequency is 40KHz) for 30 minutes, taking out, cooling, weighing the weight, complementing the lost weight with 70% methanol, shaking up, filtering, evaporating to dryness the filtrate, adding 25ml of water to the residue to dissolve, shaking up and extracting the residue with a mixed solution (1:1, 1:2 and 2:1) of water saturated n-butyl alcohol and ethyl acetate respectively, combining the extracting solutions, evaporating to dryness, adding 70% methanol to the residue to dissolve, transferring the residue to a 20ml measuring flask, adding 70% methanol to scale, shaking up and filtering to obtain the clear Shangjuan analgesic decoction. Measuring according to the determined chromatographic conditions, recording the chromatogram of the test solution extracted by the menstruum with different proportions, and obtaining the result that: the chromatographic peak numbers in the test solution with three solvent ratios are basically consistent, the ratio of the peak area of the baseline to the peak area of the baicalin chromatographic peak is considered, the baicalin peak ratio in the test solution extracted by shaking water saturated n-butyl alcohol-ethyl acetate (1:2) is moderate, the baseline is relatively stable, so the n-butyl alcohol-ethyl acetate (1:2) is selected as the solvent for preparing the test.

(4) Selection of detection wavelength

By adopting a diode array detector, a sample solution is scanned at the wavelength of 190-400 nm, and under the wavelength of 280nm, the information content of each chromatographic peak is large, the response value is moderate, the separation degree is good, and the base line is stable. Therefore, 280nm is selected as the detection wavelength.

(5) Peak assignment and selection of reference

Attribution of medicinal flavor peaks

According to a determined preparation method, a single medicine test solution is prepared, the determination is carried out according to determined chromatographic conditions, and simultaneously the attribution of each peak in a substance reference fingerprint is determined through mass spectrum detection (Table 1). Wherein peaks 9, 11 and 12 are special characteristic peaks of Scutellariae radix processed with wine, and other peaks are common chromatographic peaks of multiple medicines.

TABLE 1 attribution table of herbs and flavors

② chromatographic peak identification and reference selection

Through literature research and comparison of 6 known components (wogonin, senkyunolide I, baicalin, liquiritin, wogonoside and isoliquiritin) with reference substances, it is determined that the 6 components can be identified in common peaks. The retention time of the baicalin chromatographic peak in the chromatogram is moderate, the response value is high, and baseline separation is achieved, so that baicalin is selected as a reference peak of a reference substance, the baicalin chromatographic peak is marked as a peak S, and meanwhile, the baicalin reference substance is used as a reference substance. The results are shown in FIG. 2.

(6) Preliminary determination of the method

The measurement is carried out by high performance liquid chromatography (0512 in the four-department general regulation of the 2015 edition in China pharmacopoeia).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 280nm, and the flow rate is 1ml per minute; the column temperature is 30 ℃; the number of theoretical plates is not less than 5000 calculated according to baicalin peak.

Preparation of reference solutions: taking appropriate amount of baicalin reference substance, precisely weighing, and adding 70% ethanol to obtain solution containing 0.2mg per 1 ml.

Preparation of a test solution: taking 1g of dry extract powder of the matter standard of clear supernatant pain relieving soup corresponding to the real object, precisely weighing, placing in a conical flask with a plug, adding 25ml of 70% methanol, weighing, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, filtering, evaporating filtrate, dissolving residue in 25ml of water, shaking and extracting for 3 times with 25ml of water saturated n-butyl alcohol-ethyl acetate (1:2) mixed solution, combining extracting solutions, evaporating to dryness, dissolving residue in 70% methanol, transferring to a 20ml measuring flask, adding 70% methanol to scale, shaking uniformly, filtering, precisely absorbing 5 mu l of reference solution and sample solution respectively by measuring method, injecting into a liquid chromatograph, measuring, recording chromatogram to obtain the product. The results are shown in FIG. 3.

(7) Methodology investigation

First, a special test and an integrity survey

Through the investigation of specificity and integrity tests, the blank solvent has no interference and good specificity; no chromatographic peak was present for the extended 30 minutes, and the integrity was good. The results are shown in FIG. 4.

Instrument precision test

About 1g of the material standard of the Qingshangtong decoction prepared in example 1 is taken, precisely weighed, the preparation and the measurement operation of the sample solution under the item of 'preliminary determination of the method' are carried out for 6 times of continuous sample introduction, the relative retention time of each characteristic peak and an S peak is calculated, the comparison with a reference fingerprint spectrum generated repeatedly is carried out, the similarity is calculated, and the measurement result is shown in table 2.

Table 2 fingerprint precision test relative retention time results (n ═ 6)

The result shows that the fingerprint of the test sample presents chromatographic peaks with the same retention time as the chromatographic peaks of the reference substance and presents 18 main chromatographic peaks. The relative retention time RSD is less than 3.0%, the similarity is 1.000 when compared with the reference fingerprint spectrum which is repeatedly generated, and the precision of the instrument is good.

Stability test

1g of the material standard corresponding to 1g of the Qingshangtong decoction prepared in example 1 is precisely weighed, the preparation and the measurement operation of the test solution under the item of 'preliminary determination of the method' are carried out, the test solution is respectively placed for 0, 3, 6, 9, 12, 24 and 36 hours after the preparation for sample injection measurement, the relative retention time of each characteristic peak and an S peak is calculated, the S peak is compared with a reference fingerprint spectrum generated repeatedly, the similarity is evaluated, and the result is shown in Table 3.

Table 3 stability test relative retention time results (n ═ 6)

The result shows that the fingerprint of the test sample presents chromatographic peaks with the same retention time as the chromatographic peaks of the reference substance and presents 18 main chromatographic peaks. The relative retention time RSD is less than 5.0%, and the similarity is more than 0.99 by comparison of the reference fingerprints generated repeatedly, which shows that the stability of the test solution is good within 36 hours.

Repeat test

1g (total 6 parts) of the substance standard corresponding to the Qingshangtong decoction prepared in example 1 is precisely weighed, the preparation and measurement operation of the test solution under the item of 'preliminary determination of the method' is carried out, the relative retention time of each characteristic peak and the S peak is calculated, the retention time is compared with a reference fingerprint spectrum generated repeatedly, and the similarity is evaluated, and the result is shown in Table 4.

Table 4 repeatability test relative retention time results (n ═ 6)

The result shows that the fingerprint of the test sample presents chromatographic peaks with the same retention time as the chromatographic peaks of the reference substance and presents 18 main chromatographic peaks. The relative retention time RSD is less than 3.0%, and compared with the control fingerprint spectrum generated by the self, the similarity is 1.000, which shows that the method has good repeatability.

Durability test

The durability of different flow rates, different column temperatures, different concentrations of acid, different chromatographic columns and different instruments on the chromatographic conditions is considered, the durability is compared with a reference fingerprint generated repeatedly, the similarity is calculated, the result shows that the measurement results under the conditions of different flow rates, different column temperatures, different chromatographic columns and different instruments are basically consistent, 18 main chromatographic peaks are presented in the fingerprint of the sample to be tested, the main chromatographic peaks correspond to characteristic peaks in the chromatographic peaks of a reference substance of the sample, the sample to calculate the sample of the reference substance of different flow rate of different flow rates of the reference substance of different flow rates of the reference substance of: peak 10.098, peak 20.196, peak 30.249, peak 40.358, peak 60.499, peak 101.194, peak 111.270, peak 121.325, peak 141.551, peak 151.597, peak 161.636, peak 182.088. When different acidity is examined, the separation is poor in the 0.05% phosphoric acid proportion, the separation effect is good in the 0.1% phosphoric acid and 0.2% phosphoric acid proportion, and the fixed acid proportion is 0.1% phosphoric acid. The method is proved to have good durability to different flow rates, different column temperatures, different chromatographic columns and different instruments.

(8) Determination of fingerprint determination method

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 280nm, and the flow rate is 1ml per minute; the column temperature is 30 ℃; . The number of theoretical plates is not less than 5000 calculated according to baicalin peak.

Preparation of a test solution: taking about 1g of clear supernatant pain relieving soup material standard corresponding to real object, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, performing ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, filtering, evaporating filtrate to dryness, dissolving residue with 25ml of water, shaking and extracting with water saturated n-butyl alcohol-ethyl acetate (1:2) mixed solution for 3 times, 25ml each time, combining extract solutions, evaporating to dryness, dissolving residue with 70% methanol, transferring to a 20ml measuring flask, adding 70% methanol to scale, shaking uniformly, and filtering to obtain the final product.

The determination method comprises the following steps: precisely absorbing 5 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, measuring, and recording chromatogram.

(9) Establishment of fingerprint

Calibration of common peaks

According to the determined method, 15 batches (30 parts) of the substance reference corresponding object of the clear Shangjutong soup prepared in example 1 are measured, the obtained fingerprint spectrum is analyzed (figure 5), as can be seen from figure 5, the substance reference corresponding object of the clear Shangjutong soup prepared by the invention has good uniformity and stable quality, chromatographic peaks with good stability and appropriate response value in 15 batches (30 parts) of sample fingerprint spectra are selected as common peaks, and 18 common peaks are calibrated in total.

Establishment of contrast atlas

The method comprises the steps of automatically matching 15 batches (30 parts) of Chinese medicinal chromatographic fingerprint similarity evaluation software system 2012 edition issued by the State pharmacopoeia Committee with HPLC (high Performance liquid chromatography) chromatographic peak of corresponding real object (dry extract powder) chromatographic spectrum of the supernatant pain decoction to form a common pattern diagram, and establishing a comparison diagram, wherein the result is shown in figure 6.

Calculating similarity

A traditional Chinese medicine chromatogram fingerprint similarity evaluation software system is adopted to calculate the similarity of 15 batches (30 parts) of substance reference corresponding real objects (dry paste powder) and a reference fingerprint by using a common peak. The similarity calculated according to the fingerprint spectrum is compared with the similarity of the fingerprint spectrum of comparison of 15 batches (30 parts) of material and Qingshanjuantong decoction, the actual measurement range is 0.951-1.000, and the actual measurement ranges are all more than 0.90, which is shown in Table 5.

Table 515 shows the similarity between the standard substance (30 parts) and the fingerprint of the reference substance

Dried paste powder batch number	Similarity to control map	Dried paste powder batch number	Similarity to control map
				D-181127-01	1.000	D-181127-02	0.999
D-181127-03	0.999	D-181127-04	0.999
				D-181128-05	0.999	D-181128-06	0.999
D-181128-07	0.999	D-181128-08	0.999
				D-181130-09	0.998	D-181130-10	0.998
D-181202-11	0.951	D-181202-12	0.957
				D-181204-13	0.999	D-181204-14	0.999
D-181204-15	0.999	D-181204-16	0.999
				D-181205-17	0.999	D-181205-18	0.999
D-181205-19	0.999	D-181205-20	0.999
				D-181205-21	0.999	D-181205-22	0.999
D-181206-23	0.998	D-181206-24	0.998
				D-181206-25	0.997	D-181206-26	0.996
D-181207-27	0.999	D-181207-28	0.998
				D-181207-29	0.999	D-181207-30	0.999

(note: each two adjacent dry paste powder batches are the same batch)

The sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 18 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90.

In summary, the pharmaceutical composition of the present invention comprises stable effective components in the "clear juantong decoction", wherein the identified components comprise liquiritin, senkyunolide I, isoliquiritin, baicalin, wogonoside and wogonin, and the retention time of characteristic peaks and baicalin chromatographic peaks in chromatograms of the other 12 components are respectively 0.098, 0.196, 0.249, 0.358, 0.499, 1.194, 1.270, 1.325, 1.551, 1.597, 1.636 and 2.088. The medicine composition containing the 'clear Shangjuantong decoction' has good uniformity of effective components and high stability; the quality detection method has high accuracy, can comprehensively reflect the general view of clear ShangShangtong decoction, performs quality control on the traditional preparation and the modern preparation of the clear ShangTong decoction on the whole, and has popularization and application values.

Claims

1. A medicine composition containing 'clear Shangjuantong decoction' is characterized in that: its HPLC fingerprint contains 18 characteristic peaks, which are respectively peak 5 glycyrrhizin, peak 7 senkyunolide I, peak 8 isoliquiritin, peak 9 baicalin, peak 13 wogonoside, peak 17 wogonin, the peak corresponding to baicalin reference substance is S peak, the relative retention time of the other characteristic peaks and S peak is calculated, the relative retention time should be within + -7% of the specified value, the specified value is: peak 10.098, peak 20.196, peak 30.249, peak 40.358, peak 60.499, peak 101.194, peak 111.270, peak 121.325, peak 141.551, peak 151.597, peak 161.636, peak 182.088;

the detection conditions of the HPLC fingerprint spectrum are as follows: and (3) chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: taking acetonitrile as a mobile phase A, and taking a 0.1% phosphoric acid solution as a mobile phase B for gradient elution; the gradient elution procedure was as follows:

2. the pharmaceutical composition of claim 1, wherein: the Chinese medicinal preparation is prepared by adding conventional auxiliary materials into clear ShangShangtong decoction and preparing a clinically acceptable oral preparation according to a conventional process; the oral preparation includes, but is not limited to, decoction, granules, pills, dripping pills, mixture, soft extract, powder or tablets.

3. The pharmaceutical composition of claim 2, wherein: the preparation method of the decoction for clearing away clear heat and relieving pain comprises the following steps:

weighing raw material medicines according to a formula of clear Shangjutong decoction; slicing ginger into slices of 2-4 mm, crushing the rest raw material medicines, sieving the crushed raw material medicines with a 3mm sieve, mixing, decocting, filtering, concentrating the filtrate under reduced pressure until the weight ratio of the fed material to the concentrated solution is 1 g: 1.5-2.5 g, and freeze-drying to obtain the ginger tea;

the formula of the clear Shangjuantong decoction comprises the following components in parts by weight: 10 parts of wine-washed angelica, 10 parts of ligusticum wallichii, 10 parts of radix angelicae, 3 parts of asarum, 10 parts of notopterygium root, 10 parts of radix angelicae pubescentis, 10 parts of radix sileris, 5 parts of chrysanthemum, 5 parts of fructus viticis, 10 parts of bran-fried rhizoma atractylodis, 15 parts of wine-fried scutellaria baicalensis, 10 parts of radix ophiopogonis, 3 parts of liquorice and 5.3 parts of ginger.

4. A quality detection method for Qingshanjuandong decoction or a pharmaceutical composition containing Qingshanjuandong decoction is characterized in that: it adopts HPLC detection, and concretely comprises the following steps:

b. preparation of a test solution: extracting a sample to be detected with methanol solution, filtering, evaporating the filtrate, dissolving the residue with water, extracting with water saturated mixed solution of n-butanol and ethyl acetate, evaporating the extractive solution, dissolving the residue with methanol solution, and filtering;

c. sucking reference solution and test solution separately, injecting into liquid chromatograph, and detecting according to the chromatographic condition of claim 1.

5. The detection method according to claim 4, characterized in that: the concentration of the reference substance solution in the step a) is 0.1-0.3 mg of baicalin per 1ml, and preferably 0.2 mg; the concentration of the ethanol solution is 50-80%, and v/v; preferably 70%, v/v.

6. The detection method according to claim 4, characterized in that: the mass-to-volume ratio of the sample to be detected in the step b) to the methanol solution is 0.5-5 g:25mL, preferably 0.5-1.5 g:25 mL; the extraction is ultrasonic extraction, the power is 250w, the frequency is 40kHz, and the time is 30 min; the volume of the residue added with water is the same as that of the sample to be detected added with the methanol solution.

7. The detection method according to claim 4, characterized in that: adding water-saturated n-butanol-ethyl acetate mixed solution into the extraction in the step b), shaking for 1-3 times, preferably 3 times, adding an equal volume of water for each time, and combining the upper-layer extraction liquid; the volume ratio of the n-butanol to the ethyl acetate is 1: 2.

8. The detection method according to claim 4, characterized in that: the volume of the residue methanol solution in the step b) is 1/5-1, preferably 4/5, of the sample to be detected methanol solution; the concentration of the methanol solution is 50-80%, v/v, preferably 70%, v/v.

9. The detection method according to claim 4, characterized in that: the wavelength of the chromatographic condition in the step c) is 280nm, and the number of theoretical plates is not less than 5000 calculated according to baicalin.

10. The detection method according to claim 4, characterized in that: the chromatographic column of octadecylsilane chemically bonded silica in the step C) is Welch Ultimate XB C18, 4.6X 250mm, 5 μm, Waters symmetry C18, 4.6X 250mm, 5 μm or Phenomene

NX-C18，4.6×250mm，5μm。