A pharmaceutical composition containing "Qingshang Quntong Decoction" and a detection method

Abstract

The present invention discloses a pharmaceutical composition containing "Qingshangjuantong Decoction", and also discloses a quality detection method for Qingshangjuantong Decoction or a pharmaceutical composition containing "Qingshangjuantong Decoction". The present invention discloses a pharmaceutical composition containing "Qingshangjuantong Decoction", which contains stable, comprehensive and uniform medicinal ingredients in "Qingshangjuantong Decoction". The quality detection method of Qingshangjuantong Decoction and its preparation of the present invention has high accuracy, can comprehensively reflect the general appearance of Qingshangjuantong Decoction, and can control the quality of traditional preparations and modern preparations of Qingshangjuantong Decoction as a whole, and has promotion and application value.

Description

Technical Field

[0001] The present invention specifically relates to a pharmaceutical composition containing "Qingshang Quntong Decoction" and a detection method thereof. Background Art

[0002] Qingshang Quntong Decoction comes from Shoushibaoyuan by Gong Yanxian in the Ming Dynasty. The original text states that it is "the main prescription for treating all headaches" and "it is effective for all headaches, regardless of whether they are left or right, partial or straight, new or long-standing." Modern applications include vascular neurosis headache, maxillary sinusitis headache, trigeminal neuralgia, etc.

[0003] The technical difficulty of the research and development of modern preparations of Qingshang Quntong Decoction lies in the equivalence evaluation system of ancient and modern preparations. Since the raw materials, processing of raw materials, extraction technology, and preparation technology of modern preparations of classic prescriptions have an impact on the quality of the final product, and the qualitative identification of index components alone is difficult to reflect its quality, in order to ensure the safety, effectiveness, stability, and controllability of classic prescriptions and their preparations, a more stable and comprehensive full chemical spectrum that reflects its composition is needed as an effective means to control and evaluate the ancient and modern preparations of classic prescriptions. Yan Yuanyuan et al., Study on the Fingerprint of the Classic Prescription Qingshangjuantong Decoction [J], Journal of Nanjing University of Chinese Medicine, 2020.3.36(2), disclosed a fingerprint detection method for the decoction of Qingshangjuantong Decoction. In the fingerprints of different batches of decoction samples obtained by this method, the relative peak areas of the common characteristic peaks vary greatly, making it impossible to accurately control the quality of Qingshangjuantong Decoction and its products. At present, there is no method that can both evaluate the quality control of traditional preparations of Qingshangjuantong Decoction and conduct overall comprehensive quality control of modern preparations of Qingshangjuantong Decoction. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a pharmaceutical composition containing "Qingshangjuantong Decoction", which contains 18 characteristic peaks in its HPLC fingerprint, namely peak 5 liquiritin, peak 7 chuanxiong lactone I, peak 8 isoliquiritin, peak 9 baicalin, peak 13 wogonin, peak 17 baicalin, the peak corresponding to the baicalin reference is S peak, the relative retention time of the remaining characteristic peaks and the S peak is calculated, and the relative retention time should be within ±7% of the specified value, and the specified value is: peak 1 0.098, peak 2 0.196, peak 3 0.249, peak 4 0.358, peak 6 0.499, peak 10 1.194, peak 11 1.270, peak 12 1.325, peak 14 1.551, peak 15 1.597, peak 16 1.636, peak 18 2.088;

[0005] The detection conditions of the HPLC fingerprint are as follows: chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B for gradient elution; the gradient elution procedure is as follows:

[0006]

[0007] Furthermore, the Qingshangjuantong decoction is added with conventional excipients and prepared into a clinically acceptable oral preparation according to conventional processes;

[0008] Furthermore, the oral preparations include but are not limited to decoctions, granules, pills, pellets, mixtures, decoctions, powders or tablets.

[0009] The present invention also provides a method for detecting the quality of Qingshangjuantong Decoction or a pharmaceutical composition containing the Qingshangjuantong Decoction, which adopts HPLC detection and specifically comprises the following steps:

[0010] a. Preparation of reference solution: Take baicalin reference substance, dissolve it in ethanol solution, and use it as reference solution;

[0011] b. Preparation of test solution: Take the sample to be tested, extract it with methanol solution, filter it, evaporate the filtrate to dryness, dissolve the residue in water, then extract it with saturated n-butanol-ethyl acetate mixed solution, evaporate the extract to dryness, dissolve the residue in methanol solution, filter it, and obtain the solution;

[0012] c. Separately aspirate the reference solution and the test solution and inject them into the liquid chromatograph, and detect according to the above chromatographic conditions.

[0013] Furthermore, in step a), the concentration of the reference solution is 0.1-0.3 mg baicalin per 1 ml, preferably 0.2 mg; the concentration of the ethanol solution is 50-80%, v / v; preferably 70%, v / v.

[0014] Furthermore, in step b), the mass volume ratio of the sample to be tested to the methanol solution is 0.05-5 g:25 mL, preferably 0.5-1.5 g:25 mL; the extraction is ultrasonic extraction, with a power of 250 w, a frequency of 40 kHz, and a time of 30 min; the volume of the residue plus water is the same as the volume of the sample to be tested plus the methanol solution.

[0015] Furthermore, the extraction in step b) is performed by adding a water-saturated n-butanol-ethyl acetate mixed solution and shaking it three times, adding an amount equal to the volume of water each time, and combining the upper extracts; the volume ratio of n-butanol-ethyl acetate is 1:2.

[0016] Furthermore, in step b), the volume of the methanol solution added to the residue is 1 / 5 to 1, preferably 4 / 5 of the volume of the methanol solution added to the sample to be tested; the concentration of the methanol solution is 50 to 80%, v / v, preferably 70%, v / v.

[0017] Furthermore, the wavelength of the chromatographic conditions in step c) is 280 nm, and the number of theoretical plates calculated based on baicalin should be no less than 5000.

[0018] Furthermore, the octadecylsilane bonded silica gel chromatographic column in step c) is Welch Ultimate XB C18, 4.6×250 mm, 5 μm, Waters symmetry C18, 4.6×250 mm, 5 μm or Phenomene 4.6×250mm, 5μm.

[0019] The present invention discloses a pharmaceutical composition containing "Qingshangjuantong Decoction", which contains stable, comprehensive and uniform medicinal ingredients in "Qingshangjuantong Decoction". The quality detection method of Qingshangjuantong Decoction and its preparation of the present invention has high accuracy, can comprehensively reflect the general appearance of Qingshangjuantong Decoction, and can control the quality of traditional and modern preparations of Qingshangjuantong Decoction as a whole, and has the value of popularization and application.

[0020] The Qingshang Quntong Decoction described in the present invention is the name of a traditional Chinese medicine prescription, which comes from "Shoushibaoyuan" by Gong Yanxian of the Ming Dynasty. It is composed of one qian (3 grams) of angelica, chuanxiong, angelica dahurica, notopterygium, saposhnikovia, atractylodes, ophiopogon, and duhuo, three fen (0.3 grams) of asarum and licorice, five fen (1.5 grams) of vitex rotundus and chrysanthemum, one qian and five fen (4.5 grams) of scutellaria baicalensis, ginger is used as an introduction, and it is decocted in water for oral administration.

[0021] The Qingshang Quntong Decoction material reference used in the present invention corresponds to the material standard of the Qingshang Quntong Decoction medicinal material prepared based on the Qingshang Quntong Decoction preparation method recorded in the ancient medical book "Shoushi Baoyuan". Except for the molding process, the rest of the preparation method is basically consistent with the record in "Shoushi Baoyuan". The specific preparation method is:

[0022] Weigh the raw materials according to the formula of Qingshang Quntong Decoction: cut the ginger into thick slices (2mm-4mm), crush the other thirteen herbs, pass through a 3mm sieve, put in a casserole, add water and boil twice, the first time add 10 times the amount of water, soak for 30 minutes, boil over high heat, boil over low heat for 30 minutes, filter, and the filtrate is reserved; the second time add 8 times the amount of water, boil over high heat, boil over low heat for 20 minutes, filter, combine with the above filtrate, concentrate under reduced pressure to a weight ratio of the feed amount to the concentrate of about 1g:2g, freeze-dry to obtain a dry paste, package, and obtain the actual object corresponding to the material benchmark of Qingshang Quntong Decoction.

[0023] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Fingerprint search diagram

[0026] Figure 2 Fingerprint peak identification (S1 dry paste powder; S2 baicalin; S3 isoliquiritigenin; S4 liquiritigenin S5 wogonin; S6 wogonin; S7 ligusticum lactone I)

[0027] Figure 3 Fingerprint method determination

[0028] Figure 4 Fingerprint HPLC comparison of blank solvent, reference substance and test substance

[0029] Figure 5 The corresponding physical object (dry paste powder) fingerprint spectrum common pattern diagram (A-common pattern Figure 1 , B-shared mode Figure 2 )

[0030] Figure 6 Comparison fingerprint (18 common peaks: Peak 5: liquiritin, Peak 7: ligusticum lactone I, Peak 8: isoliquiritin, Peak 9 (S): baicalin, Peak 13: wogonin, Peak 17: baicalin) DETAILED DESCRIPTION

[0031] Example 1. Preparation of the substance corresponding to the Qingshang Quntong Decoction

[0032] Prescription: 10 parts of wine-washed angelica, 10 parts of Chuanxiong, 10 parts of Angelica dahurica, 3 parts of Asarum, 10 parts of Notopterygium wilfordii, 10 parts of Angelica pubescens, 10 parts of Saposhnikovia divaricata, 5 parts of Chrysanthemum, 5 parts of Vitex rotundifolia, 10 parts of bran-fried Atractylodes lancea, 15 parts of wine-washed Scutellaria baicalensis, 10 parts of Ophiopogon japonicus, 3 parts of Licorice, 5.3 parts of Ginger

[0033] Preparation: For the above fourteen herbs, take 2-4mm slices of ginger, and grind the remaining thirteen herbs, pass through a 3mm sieve, put them in a casserole, add water and boil them twice. The first time, add 10 times the amount of water, soak for 30 minutes, boil over high heat, boil over low heat for 30 minutes, filter, and the filtrate is reserved; the second time, add 8 times the amount of water, boil over high heat, boil over low heat for 20 minutes, filter, combine with the above filtrate, and concentrate under reduced pressure to a weight ratio of the feed amount to the concentrated liquid of about 1g:2g, freeze-dry to obtain a dry paste, and package it to obtain the corresponding physical object.

[0034] Example 2 Detection of the substance corresponding to the Qingshang Quntong Decoction

[0035] 1) Preparation of reference solution:

[0036] Take an appropriate amount of baicalin reference substance, weigh it accurately, and add 70% ethanol to make a solution containing 0.2 mg per 1 ml.

[0037] 2) Preparation of test solution:

[0038] Take 1 g of the substance corresponding to the Qingshang Quntong Decoction prepared in Example 1, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of water to dissolve the residue, shake and extract with a water-saturated n-butanol-ethyl acetate (1:2) mixed solution 3 times, 25 ml each time, combine the upper extracts, evaporate to dryness, add 70% methanol to dissolve the residue, and transfer it to a 20 ml volumetric flask, add 70% methanol to the scale, shake well, filter, and obtain;

[0039] 3) Determination of characteristic spectrum

[0040] Accurately pipette the reference solution and the test solution into the liquid chromatograph to obtain a chromatogram;

[0041] The chromatographic conditions were as follows:

[0042] Column: Phenomene (4.6×250mm, 5μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution as specified in the following table; column temperature is 30°C; flow rate is 1ml per minute; detection wavelength: 280nm, the theoretical plate number calculated based on the baicalin peak should be not less than 5000.

[0043]

[0044] 4) Analyze the characteristic spectrum

[0045] There are 18 characteristic peaks in the characteristic spectrum of the actual object corresponding to the material benchmark of Qingshangjuantong Decoction, peak 5 is liquiritigenin, peak 7 is ligusticum lactone I, peak 8 is isoliquiritigenin, peak 9 is baicalin, peak 13 is wogonin, peak 17 is baicalin, and the peak corresponding to the baicalin reference is the S peak. The relative retention time of the remaining characteristic peaks and the S peak is calculated, and the relative retention time should be within ±7% of the specified value, which is: peak 1 0.098, peak 2 0.196, peak 3 0.249, peak 4 0.358, peak 6 0.499, peak 10 1.194, peak 11 1.270, peak 12 1.325, peak 14 1.551, peak 15 1.597, peak 16 1.636, peak 18 2.088.

[0046] Example 3 Preparation of Qingshang Quntong Decoction Granules

[0047] Prescription: 10 parts of wine-washed angelica, 10 parts of Chuanxiong, 10 parts of Angelica dahurica, 3 parts of Asarum, 10 parts of Notopterygium wilfordii, 10 parts of Angelica pubescens, 10 parts of Saposhnikovia divaricata, 5 parts of Chrysanthemum, 5 parts of Vitex rotundifolia, 10 parts of bran-fried Atractylodes lancea, 15 parts of wine-washed Scutellaria baicalensis, 10 parts of Ophiopogon japonicus, 3 parts of Licorice, 5.3 parts of Ginger

[0048] Preparation: For the above 14 herbs, slice ginger with 2-4 mm diameter. Crush the remaining 13 herbs, pass through a 3 mm sieve, place in a casserole, add water and boil twice. For the first time, add 10 times the amount of water, soak for 30 minutes, boil over high heat, boil over low heat for 30 minutes, filter, and reserve the filtrate. For the second time, add 8 times the amount of water, boil over high heat, boil over low heat for 20 minutes, filter, combine with the above filtrate, concentrate under reduced pressure to a weight ratio of the feed amount to the concentrate of about 1g:2g, freeze-dry, and crush to obtain dry paste powder.

[0049] Take the above dry paste powder, add auxiliary materials to granulate, dry, and sieve to make Qingshang Quntong Decoction granules.

[0050] Example 4 Detection of Qingshang Quntong Decoction Granules

[0051] 1) Preparation of reference solution:

[0052] Take an appropriate amount of baicalin reference substance, weigh it accurately, and add 70% ethanol to make a solution containing 0.2 mg per 1 ml.

[0053] 2) Preparation of test solution: Take 5 g of the Qingshang Quntong Decoction Granules prepared in Example 3, accurately weigh, place in a stoppered conical flask, accurately add 25 ml of 70% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of water to dissolve the residue, shake and extract with a water-saturated n-butanol-ethyl acetate (1:2) mixed solution 3 times, 25 ml each time, combine the upper extracts, evaporate to dryness, add 70% methanol to dissolve the residue, and transfer to a 20 ml volumetric flask, add 70% methanol to the scale, shake well, filter, and obtain;

[0054] 3) Determination of characteristic spectrum

[0055] Accurately pipette the reference solution and the test solution into the liquid chromatograph to obtain a chromatogram;

[0056] The chromatographic conditions were as follows:

[0057] Chromatographic column: Waters symmetry C18 (4.6×250mm, 5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table; column temperature 30°C; flow rate 1 ml per minute; detection wavelength: 280nm, the theoretical plate number calculated based on the baicalin peak should not be less than 5000.

[0058]

[0059] 4) Analyze the characteristic spectrum

[0060] There are 18 characteristic peaks in the characteristic spectrum of Qingshangjuantong Decoction decoction, peak 5 is liquiritigenin, peak 7 is ligusticum lactone I, peak 8 is isoliquiritigenin, peak 9 is baicalin, peak 13 is wogonin, peak 17 is baicalin, and the peak corresponding to the baicalin reference is S peak. The relative retention time of the remaining characteristic peaks and the S peak was calculated, and the relative retention time should be within ±7% of the specified value, which is: peak 1 0.098, peak 2 0.196, peak 3 0.249, peak 4 0.358, peak 6 0.499, peak 10 1.194, peak 11 1.270, peak 12 1.325, peak 14 1.551, peak 15 1.597, peak 16 1.636, peak 18 2.088.

[0061] The beneficial effects of the present invention are described below through test examples.

[0062] Test Example 1

[0063] (1) Instruments and reagents

[0064] Liquid chromatograph: Shimadzu LC-20A, Agilent 1260Ⅱ; chromatographic columns: Welch Ultimate XB C18 (4.6×250mm, 5μm); Waters symmetry C18 (4.6×250mm, 5μm); Phenomene (4.6×250mm, 5μm), electronic analytical balance: Changshu Shuangjie Testing Instrument Factory JJ1000 (1 / 100), Sartorius Scientific Instrument (Beijing) Co., Ltd. BSA224S-CW (1 / 10,000), Sartorius Scientific Instrument (Beijing) Co., Ltd. SQP (1 / 10,000), Mettler-Toledo Instrument (Shanghai) Co., Ltd. ME503T (1 / 1,000);

[0065] Methanol was of chromatographic grade and produced by OCEANPAK; acetonitrile was of chromatographic grade and produced by OCEANPAK; phosphoric acid was of chromatographic grade and produced by Shandong Xiya Chemical Co., Ltd.; methanol was of analytical grade and produced by Guangzhou Chemical Reagent Factory; water was ultrapure water, produced by Shanghai Hetai Instrument Co., Ltd.; baicalin reference substance (batch number: 110715-201720, purity: 93.5%), liquiritin reference substance (batch number: 111610-201607, purity: 93.1%), wogonin reference substance (batch number: 112002-201702, purity: 98.5%), and wogonin reference substance (batch number: 11514-201706) were all purchased from China Food and Drug Inspection Institutes. Isoliquiritigenin reference substance (batch number: 17042102, purity: 98%) and ligusticum chuanxiong lactone I reference substance (batch number: 17121105, purity: 98%) were purchased from Chengdu Pufeide Biotechnology Co., Ltd.

[0066] (2) Selection of mobile phase

[0067] Through preliminary experiments, it was found that the mobile phases of acetonitrile-0.1% phosphoric acid and methanol-0.1% phosphoric acid had good separation effects on the components in the actual substance corresponding to the Qingshang Quntong Decoction substance benchmark, so further adjustments were made on this basis.

[0068] ①Acetonitrile-0.1% phosphoric acid system

[0069] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and gradient elution as specified in the following table; the flow rate was 1 ml per minute; the column temperature was 30°C; and the detection wavelength was 280 nm.

[0070]

[0071] Preparation of test solution: Take about 1g of the substance corresponding to the Qingshang Quntong Decoction prepared in Example 1, accurately weigh it, put it in a stoppered conical flask, accurately add 25ml of 70% methanol, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, evaporate it to dryness, add 25ml of water to the residue to dissolve it, shake it with ethyl acetate to extract it 3 times, 25ml each time, combine the ethyl acetate, evaporate it to dryness, add 70% methanol to dissolve the residue, and transfer it to a 20ml volumetric flask, add 70% methanol to the scale, shake it well, filter it, and take the filtrate to obtain it.

[0072] Determination method: Accurately pipette 5μl of the test solution, inject it into the liquid chromatograph, measure, and record the chromatogram. Figure 1 .

[0073] ②Methanol-0.1% phosphoric acid system

[0074] Take the test solution under item "①", change the mobile phase to methanol-0.1% phosphoric acid, and keep other detection conditions unchanged, and perform the measurement results: methanol-0.1% phosphoric acid mobile phase system has less peak information, so the acetonitrile-0.1% phosphoric acid solution system is selected for the test.

[0075] (3) Selection of test sample preparation method

[0076] ① Investigation of extraction solvent

[0077] a 70% methanol solution ultrasonic

[0078] Take 1 g of the substance corresponding to the Qingshang Quntong Decoction prepared in Example 1, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh it, ultrasonically treat it (250W, 40KHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0079] b Extraction by shaking water-saturated n-butanol solution

[0080] Take 1 g of the substance corresponding to the Qingshang Quntong Decoction prepared in Example 1, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh it, ultrasonically treat it (250 W, 40 kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, evaporate the filtrate to dryness, add 25 ml of water to dissolve the residue, shake and extract it 3 times with a saturated n-butanol solution with water, 25 ml each time, combine the n-butanol solutions, evaporate it to dryness, add 70% methanol to dissolve the residue, and transfer it to a 20 ml volumetric flask, add 70% methanol to the scale, shake it well, filter it, and obtain it.

[0081] c Ethyl acetate solution shaking extraction

[0082] Take 1 g of the substance corresponding to the material standard of the Qingshang Quntong Decoction prepared in Example 1, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh it, ultrasonically treat it (250 W, 40 KHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, evaporate the filtrate to dryness, add 25 ml of water to dissolve the residue, shake it with ethyl acetate for 3 times, 25 ml each time, combine the ethyl acetate, evaporate it to dryness, add 70% methanol to dissolve the residue, and transfer it to a 20 ml volumetric flask, add 70% methanol to the scale, shake it well, filter it, and obtain it.

[0083] According to the chromatographic conditions determined above, the chromatograms of the test solution extracted with different solvents were recorded. The results showed that the test solution treated with 70% methanol ultrasonically had more impurities and poor peak separation; the number of chromatographic peaks extracted by shaking with water-saturated n-butanol was less than that by ultrasonic treatment with 70% methanol, and the peak area of ​​baicalin chromatographic peak accounted for more than 50%; the chromatographic peaks extracted by shaking with ethyl acetate were less than that by shaking with water-saturated n-butanol, but the baseline was more stable. Comprehensive analysis, taking into account the baseline and chromatographic peak information, requires the investigation of different proportions of ethyl acetate and n-butanol.

[0084] ②Inspection of solvent ratio

[0085] Take 1 g of the substance corresponding to the Qingshang Quntong Decoction prepared in Example 1, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, take it out, cool it, weigh it, make up the lost weight with 70% methanol, shake it well, filter it, evaporate the filtrate to dryness, add 25 ml of water to dissolve the residue, shake and extract it with a mixed solution of saturated n-butanol and ethyl acetate (1:1, 1:2, 2:1) respectively, combine the extracts, evaporate it to dryness, add 70% methanol to dissolve the residue, and transfer it to a 20 ml volumetric flask, add 70% methanol to the scale, shake it well, filter it, and obtain it. According to the chromatographic conditions determined above, the chromatograms of the test sample solutions extracted with different proportions of solvents were recorded. The results showed that the number of chromatographic peaks in the test sample solutions with the three solvent proportions was basically the same, taking into account the baseline and the proportion of the baicalin chromatographic peak area. The proportion of baicalin peak in the test sample solution extracted by shaking with water-saturated n-butanol-ethyl acetate (1:2) was more moderate, and the baseline was relatively stable. Therefore, n-butanol-ethyl acetate (1:2) was selected as the solvent for preparing the test sample.

[0086] (4) Selection of detection wavelength

[0087] Using a diode array detector, the test solution was scanned at a wavelength of 190-400nm. At a wavelength of 280nm, the information content of each chromatographic peak was large, the response value was moderate, the separation was good, and the baseline was relatively stable. Therefore, 280nm was selected as the detection wavelength.

[0088] (5) Peak attribution and reference selection

[0089] ① Attribution of drug flavor peaks

[0090] According to the determined preparation method, the single-flavor drug test solution was prepared, and the determination was carried out according to the determined chromatographic conditions. At the same time, the attribution of each peak in the material reference fingerprint was determined by mass spectrometry (Table 1). Among them, peaks 9, 11, and 12 are the exclusive characteristic peaks of Huangqin wine, and the other peaks are all common chromatographic peaks of multiple drugs.

[0091] Table 1: Table of medicinal taste

[0092]

[0093]

[0094] ② Chromatographic peak identification and reference material selection

[0095] Through literature research and comparison with reference substances, six known components (wogonin, chuanxiong lactone I, baicalin, liquiritin, wogonin, and isoliquiritin) were determined to be able to be identified in the common peak. The retention time of the baicalin chromatographic peak in the chromatogram is moderate, the response value is high, and the baseline separation is achieved. Therefore, baicalin was selected as the reference peak of the reference substance, and the baicalin chromatographic peak was marked as peak S. At the same time, the baicalin reference substance was used as a reference substance. The results are shown in Figure 2 .

[0096] (6) Preliminary determination of the method

[0097] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2015 edition, Part IV, General Rules 0512).

[0098] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and gradient elution as specified in the following table; the detection wavelength was 280 nm, the flow rate was 1 ml per minute; the column temperature was 30°C; the theoretical plate number calculated based on the baicalin peak should be no less than 5000.

[0099]

[0100] Preparation of reference solution: Take an appropriate amount of baicalin reference substance, weigh it accurately, and add 70% ethanol to make a solution containing 0.2 mg per 1 ml.

[0101] Preparation of test solution: Take 1g of the actual dry powder of the substance standard of Qingshang Quntong Decoction, weigh it accurately, put it in a stoppered conical flask, add 25ml of 70% methanol, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, filter it, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, shake and extract it 3 times with a mixed solution of saturated n-butanol-ethyl acetate (1:2) with water, 25ml each time, combine the extracts, evaporate to dryness, add 70% methanol to dissolve the residue, and transfer it to a 20ml volumetric flask, add 70% methanol to the scale, shake well, filter it, and measure it. Accurately aspirate 5μl of the reference solution and the test solution respectively, inject it into the liquid chromatograph, measure it, and record the chromatogram to obtain the result. See the results. Figure 3 .

[0102] (7) Methodological review

[0103] ① Specificity test and overall investigation

[0104] Through the specificity and integrity test, the blank solvent has no interference and the specificity is good; there is no chromatographic peak within the extended 30 minutes, and the integrity is good. Figure 4 .

[0105] ②Instrument precision test

[0106] Take about 1 g of the actual substance corresponding to the Qingshang Quntong Decoction material standard prepared in Example 1, accurately weigh it, and operate according to the preparation and determination method of the test solution under the "Preliminary Determination of the Method", inject it continuously for 6 times, calculate the relative retention time of each characteristic peak and the S peak, compare it with the control fingerprint spectrum generated by repeatability, calculate the similarity, and the measurement results are shown in Table 2.

[0107] Table 2 Relative retention time results of fingerprint precision test (n=6)

[0108]

[0109]

[0110] The results showed that the fingerprint of the test sample showed the same chromatographic peak retention time as the reference chromatographic peak, and showed 18 main chromatographic peaks. The relative retention time RSD was less than 3.0%, and the similarity was 1.000 when compared with the control fingerprint generated by repeatability, indicating that the instrument had good precision.

[0111] ③Stability test

[0112] Take 1 g of the actual object corresponding to the material standard of Qingshang Quntong Decoction prepared in Example 1, accurately weigh it, and operate according to the preparation and determination method of the test solution under the item "Preliminary Determination of the Method". After preparation, place it for 0, 3, 6, 9, 12, 24, and 36 hours, respectively, and measure it. Calculate the relative retention time of each characteristic peak and the S peak, compare it with the control fingerprint spectrum generated by repeatability, and evaluate the similarity. The results are shown in Table 3.

[0113] Table 3 Relative retention time results of stability test (n=6)

[0114]

[0115] The results showed that the fingerprint of the test sample showed chromatographic peaks with the same retention time as the reference chromatographic peaks, and 18 main chromatographic peaks. The relative retention time RSD was less than 5.0%, and the similarity was greater than 0.99 when compared with the control fingerprint generated by repeatability, indicating that the test solution had good stability within 36 hours.

[0116] ④Repeatability test

[0117] Take 1 g (6 portions in total) of the actual substance corresponding to the Qingshang Quntong Decoction material benchmark prepared in Example 1, accurately weigh it, and operate according to the preparation and determination method of the test solution under the item "Preliminary Determination of the Method", calculate the relative retention time of each characteristic peak and the S peak, and compare it with the control fingerprint spectrum generated by repeatability to evaluate the similarity. The results are shown in Table 4.

[0118] Table 4 Relative retention time results of repeatability test (n=6)

[0119]

[0120] The results showed that the fingerprint of the test sample showed the same chromatographic peak retention time as the reference chromatographic peak, and showed 18 main chromatographic peaks. The relative retention time RSD was less than 3.0%, and the similarity was 1.000 when compared with the self-generated control fingerprint, indicating that the method had good repeatability.

[0121] ⑤Durability test

[0122] The durability of the chromatographic conditions under different flow rates, column temperatures, acid concentrations, chromatographic columns and instruments was investigated, and the fingerprints were compared with those generated by repeatability to calculate the similarity. The results showed that the results were basically consistent under different flow rates, column temperatures, chromatographic columns and instrument conditions. There were 18 main chromatographic peaks in the fingerprints of the test samples, which corresponded to the characteristic peaks in the chromatographic peaks of the reference sample, and the similarities were not less than 0.90. The peak corresponding to the baicalin reference was the S peak. The relative retention time of the unidentified characteristic peaks and the S peak was calculated, and the relative retention time should be within ±7% of the specified value, which is: peak 1 0.098, peak 2 0.196, peak 3 0.249, peak 4 0.358, peak 6 0.499, peak 10 1.194, peak 11 1.270, peak 12 1.325, peak 14 1.551, peak 15 1.597, peak 16 1.636, peak 18 2.088. When different acidities were investigated, 0.05% phosphoric acid had poor separation, while 0.1% phosphoric acid and 0.2% phosphoric acid had good separation effects, and the fixed acid ratio was 0.1% phosphoric acid. This shows that the method has good durability for different flow rates, different column temperatures, different chromatographic columns, and different instruments.

[0123] (8) Determination of fingerprint spectrum determination method

[0124] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the detection wavelength was 280nm, the flow rate was 1ml per minute, and the column temperature was 30℃. The theoretical plate number calculated based on the baicalin peak should be no less than 5000.

[0125]

[0126] Preparation of reference solution: Take an appropriate amount of baicalin reference substance, weigh it accurately, and add 70% ethanol to make a solution containing 0.2 mg per 1 ml.

[0127] Preparation of test solution: Take about 1g of the substance corresponding to the standard of the clear Quntong Decoction, accurately weigh it, put it in a stoppered conical flask, accurately add 25ml of 70% methanol, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, shake and extract 3 times with a mixed solution of water-saturated n-butanol-ethyl acetate (1:2), 25ml each time, combine the extracts, evaporate to dryness, add 70% methanol to dissolve the residue, and transfer it to a 20ml volumetric flask, add 70% methanol to the scale, shake well, filter, and obtain.

[0128] Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and record the chromatogram.

[0129] (9) Establishment of fingerprint

[0130] ① Common peak calibration

[0131] According to the determined method, 15 batches (30 portions) of the Qingshang Quntong Decoction prepared in Example 1 were measured, and the obtained fingerprints were analyzed ( Figure 5 ),from Figure 5 It can be seen that the material benchmark of Qingshang Quntong Decoction prepared by the present invention has good homogeneity and stable quality. The chromatographic peaks with good stability and appropriate response values ​​in the fingerprints of 15 batches (30 copies) of samples are selected as common peaks, and a total of 18 common peaks are calibrated.

[0132] ② Establishment of reference map

[0133] The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System 2012 Edition issued by the Chinese Pharmacopoeia Committee was used to automatically match the chromatographic peaks of 15 batches (30 copies) of Qingshang Quntong Decoction substance benchmarks to the actual substance (dry paste powder) to form a common pattern map and establish a reference map. The results are shown in Figure 6 .

[0134] ③ Calculation of similarity

[0135] The similarity evaluation software system of Chinese medicine chromatographic fingerprint was used to calculate the similarity between the corresponding physical objects (dry paste powder) of 15 batches (30 copies) of material benchmarks and the reference fingerprints based on the common peaks. According to the similarity calculated by the fingerprints, the similarity between the 15 batches (30 copies) of material benchmarks and the reference fingerprints of Qingshang Quntong Decoction was compared, and the measured range was 0.951-1.000, all greater than 0.90, as shown in Table 5.

[0136] Table 5 Similarity between the fingerprints of 15 batches (30 copies) of the material benchmark (dry paste powder) and the reference

[0137] Dry paste powder batch number Similarity with the control map Dry paste powder batch number Similarity with the 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

[0138] (Note: Every two adjacent batches of dry paste powder are from the same batch)

[0139] The fingerprint of the test sample should show a chromatographic peak with the same retention time as the chromatographic peak of the reference. The chromatogram of the test sample should be basically consistent with the reference fingerprint, with 18 corresponding common peaks. According to the similarity evaluation system of Chinese medicine chromatographic fingerprints, the similarity is calculated based on the common peaks. The similarity between the fingerprint of the test sample and the reference fingerprint shall not be less than 0.90.

[0140] In summary, the pharmaceutical composition containing "Qing Shang Juan Tong Decoction" of the present invention comprises stable medicinal ingredients in "Qing Shang Juan Tong Decoction", and the confirmed ingredients include liquiritigenin, ligusticum chuanxiong lactone I, isoliquiritigenin, baicalin, wogonin and baicalein, and the relative retention times of the characteristic peaks of the other 12 ingredients in the chromatogram and the chromatographic peak of baicalin are also confirmed, which are 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, respectively. The pharmaceutical composition containing "Qingshang Quntong Decoction" of the present invention has good uniformity of effective ingredients and high stability; the quality detection method of the present invention has high accuracy, can comprehensively reflect the general appearance of Qingshang Quntong Decoction, and conduct quality control of traditional preparations and modern preparations of Qingshang Quntong Decoction as a whole, and has promotion and application value.

Claims

1. A quality detection method for Qingshangjuantong Decoction, characterized in that: It is detected by HPLC, which includes the following steps: a. Preparation of reference solution: Take baicalin reference substance, dissolve it in ethanol solution, and use it as reference solution; b. Preparation of test solution: Take the sample to be tested, extract it with methanol solution, filter it, evaporate the filtrate to dryness, dissolve the residue in water, then extract it with saturated n-butanol-ethyl acetate mixed solution, evaporate the extract to dryness, dissolve the residue in methanol solution, filter it, and obtain the solution; c. Pipette the reference solution and the test solution into the liquid chromatograph respectively. The chromatographic conditions are as follows: Chromatographic column: octadecylsilane bonded silica gel as filler, specification 4.6×250mm, 5μm; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B for gradient elution; wavelength 280nm; gradient elution program is as follows: The fingerprint spectrum detected by HPLC contains 18 characteristic peaks, namely peak 5 liquiritin, peak 7 chuanxiong lactone I, peak 8 isoliquiritin, peak 9 baicalin, peak 13 wogonin, peak 17 baicalin, and the peak corresponding to the baicalin reference is the S peak. The relative retention time of the remaining characteristic peaks and the S peak is calculated, and the relative retention time should be within ±7% of the specified value. The specified value is: peak 1 0.098, peak 2 0.196, peak 3 0.249, peak 4 0.358, peak 6 0.499, peak 10 1.194, peak 11 1.270, peak 121.325, peak 14 1.551, peak 15 1.597, peak 16 1.636, peak 18 2.

088.

2. The detection method according to claim 1, characterized in that: The preparation method of the Qingshangjuantong Decoction is: Weigh the raw materials according to the formula of Qingshang Quntong Decoction; take ginger, slice it into 2-4mm slices, crush the other raw materials through a 3mm sieve, mix them, boil and filter, concentrate the filtrate under reduced pressure to a weight ratio of the feed amount to the concentrated liquid of 1g:1.5-2.5g, freeze-dry and obtain the product; The formula of the Qingshang Quntong Decoction is as follows by weight: 10 parts of angelica washed with wine, 10 parts of chuanxiong, 10 parts of angelica dahurica, 3 parts of asarum, 10 parts of notopterygium root, 10 parts of duhuo, 10 parts of siler, 5 parts of chrysanthemum, 5 parts of vitex rotundus, 10 parts of atractylodes stir-fried with bran, 15 parts of scutellaria baicalensis washed with wine, 10 parts of ophiopogon japonicus, 3 parts of licorice, and 5.3 parts of ginger.

3. The detection method according to claim 1, characterized in that: Step a) The concentration of the reference solution is 0.1-0.3 mg baicalin per 1 ml; the concentration of the ethanol solution is 50-80%, v / v.

4. The detection method according to claim 1, characterized in that: Step b) the mass volume ratio of the sample to be tested to the methanol solution is 0.5-5 g:25 mL; the extraction is ultrasonic extraction, with a power of 250 W, a frequency of 40 kHz, and a time of 30 min; the volume of the residue plus water is the same as the volume of the sample to be tested plus the methanol solution.

5. The detection method according to claim 1, characterized in that: The extraction in step b) is performed by adding a water-saturated n-butanol-ethyl acetate mixed solution and shaking it 1 to 3 times, adding an amount equal to the volume of water each time, and combining the upper extracts; the volume ratio of n-butanol to ethyl acetate is 1:

2.

6. The detection method according to claim 3, characterized in that: In step b), the volume of the methanol solution added to the residue is 1 / 5 to 1 of the volume of the methanol solution added to the sample to be tested; the concentration of the methanol solution is 50 to 80%, v / v.

7. The detection method according to claim 1, characterized in that: The theoretical plate number of the chromatographic conditions in step c) should be no less than 5000 calculated based on baicalin.

8. The detection method according to claim 1, characterized in that: Step c) The octadecylsilane bonded silica gel chromatographic column is Welch Ultimate XB C18, 4.6×250mm, 5μm, Waters symmetry C18, 4.6×250mm, 5μm or Phenomene NX-C18, 4.6×250mm, 5μm.

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