A medicinal composition containing "Shentong Zhuyu Decoction" and a fingerprint detection method thereof

Abstract

The present invention discloses a pharmaceutical composition containing "Shen Tong Zhu Yu Tang", and also discloses a fingerprint spectrum detection method of Shen Tong Zhu Yu Tang or a pharmaceutical composition containing "Shen Tong Zhu Yu Tang". The present invention discloses a pharmaceutical composition containing "Shen Tong Zhu Yu Tang", which contains stable, comprehensive and uniform medicinal ingredients in "Shen Tong Zhu Yu Tang". The fingerprint spectrum detection method of Shen Tong Zhu Yu Tang and its preparation of the present invention has high spectral separation and strong characteristic, and can accurately, comprehensively and stably reflect the chemical quality profile of the classic prescription Shen Tong Zhu Yu Tang, realize the quality control of the traditional preparation and modern preparation of Shen Tong Zhu Yu Tang, and has the value of promotion and application.

Description

Technical Field

[0001] The present invention specifically relates to a pharmaceutical composition containing "Shen Tong Zhu Yu Tang" and a fingerprint spectrum detection method thereof. Background Art

[0002] Shentong Zhuyu Decoction is the name of a Chinese medicine prescription, which is composed of 12 traditional Chinese medicines such as Qinjiao, Chuanxiong, and peach kernel. It comes from "Yi Lin Gaicuo" by Wang Qingren in the Qing Dynasty. This prescription has the effects of promoting blood circulation and qi, dispelling wind and dampness, relieving numbness and relieving pain. It is often used to treat symptoms of qi and blood obstruction in the meridians, such as shoulder pain, arm pain, low back pain, leg pain, and body pain.

[0003] In recent years, Shentong Zhuyu Decoction has been widely used clinically for arthritis, lumbar spinal stenosis, lumbar muscle strain, radiculopathy of cervical spondylosis, and lumbar disc herniation, with remarkable results. At the same time, taking Shentong Zhuyu Decoction as the basic prescription, adding or subtracting medicinal ingredients according to clinical symptoms is also used in the treatment of some difficult and complicated diseases, such as arteriosclerosis occlusive disease, rheumatism and blood stasis, and it also plays a good role. The clinical application scope of Shentong Zhuyu Decoction is constantly expanding, but the research on its quality control method is relatively lagging behind. Zhu Zhifei et al., optimized the UPLC fingerprint of the classic prescription Shentong Zhuyu Decoction based on the principle of information entropy maximization [J]. Chinese Herbal Medicine, 2020.6.51(12), disclosed a fingerprint detection method for Shentong Zhuyu Decoction decoction. This method optimizes the fingerprint conditions of Shentong Zhuyu Decoction by using the principle of information entropy maximization. The total number of chromatographic peaks obtained is large and the amount of information is large. However, only 8 chromatographic peaks with good separation can be calibrated from the information obtained. The separation effect of the remaining multiple components is not ideal, making it difficult to achieve the purpose of comprehensive quality control evaluation. Therefore, the chromatographic conditions optimized by the principle of information entropy maximization have certain limitations. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a pharmaceutical composition containing "Shentong Zhuyu Decoction", characterized in that: its HPLC fingerprint contains 12 characteristic peaks, namely peak 1 protocatechuic acid, peak 4 caffeic acid, peak 5 gentiopicroside, peak 7 p-coumaric acid, peak 8 ferulic acid, peak 9 neoliquiritin, peak 10 liquiritin, peak 11 kaempferol-3-O-rutinoside, peak 12 ligusticolide I, peak 13 ligusticolide H, peak 15 isoliquiritigenin, peak 16 neoliquiritigenin;

[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 12 characteristic peaks are calculated with the ferulic acid chromatographic peak as the reference peak, and the relative retention time of each characteristic peak is calculated, and the relative retention time should be within ±7% of the specified value, and the specified value is: peak 1 protocatechuic acid 0.349, peak 4 caffeic acid 0.637, peak 5 gentiopicroside 0.675, peak 7 p-coumaric acid 0.880, peak 8 ferulic acid 1.000, peak 9 neoliquiritigenin 1.028, peak 10 liquiritigenin 1.063, peak 11 kaempferol-3-O-rutinoside 1.278, peak 12 ligusticum lactone I 1.440, peak 13 ligusticum lactone H 1.570, peak 15 isoliquiritigenin 1.727, peak 16 neoisoliquiritigenin 1.820.

[0008] Furthermore, the HPLC fingerprint also contains 5 characteristic peaks. Taking the ferulic acid chromatographic peak as the reference peak, the relative retention time of each characteristic peak and the reference peak is calculated, and the relative retention time should be within ±7% of the specified value. The specified value is: peak 2 0.509, peak 3 0.607, peak 6 0.83, peak 14 1.667, peak 17 2.066.

[0009] Furthermore, conventional auxiliary materials are added to the Shentong Zhuyu Decoction and prepared into a clinically acceptable oral preparation according to conventional processes; the oral preparation includes but is not limited to decoctions, granules, pills, pellets, mixtures, decoctions, powders or tablets.

[0010] Furthermore, the preparation method of the Shentong Zhuyu Decoction is:

[0011] Weigh the raw materials according to the formula of Shentong Zhuyu Decoction; add water, cover and boil twice, filter while hot, combine the filtrate, concentrate under reduced pressure, freeze-dry and grind to obtain the material standard of Shentong Zhuyu Decoction; the weight parts of the Shentong Zhuyu Decoction formula are: 2-4 parts of Gentiana macrophylla, 6-8 parts of Chuanxiong, 10-12 parts of peach kernel, 10-12 parts of safflower, 6-8 parts of liquorice, 2-4 parts of Notopterygium wilfordii, 6-8 parts of vinegar myrrh, 10-12 parts of angelica, 6-8 parts of vinegar aconiti lobata, 2-4 parts of Cyperus rotundus, 10-12 parts of Achyranthes bidentata and 6-8 parts of earthworm;

[0012] Preferably, 3.73 parts of Gentiana macrophylla, 7.46 parts of Chuanxiong, 11.19 parts of peach kernel, 11.19 parts of safflower, 7.46 parts of liquorice, 3.73 parts of Notopterygium wilfordii, 7.46 parts of vinegar myrrh, 11.19 parts of angelica sinensis, 7.46 parts of vinegar aconiti lobata, 3.73 parts of Cyperus rotundus, 11.19 parts of Achyranthes bidentata, and 7.46 parts of earthworm.

[0013] Furthermore, the concentration of the reference solution in step a) is 0.1-0.3 mg of ferulic acid per 1 ml, preferably 0.16 mg of ferulic acid.

[0014] Furthermore, in step b), the mass volume ratio of the sample to be tested to water is 0.5-5 g:25 mL, preferably 1 g:25 mL.

[0015] Furthermore, the extraction in step b) is performed by adding ethyl acetate and shaking 1 to 3 times, preferably 3 times, each time adding 1 to 3 times the volume of water, preferably 2 times, and combining the ethyl acetate solutions.

[0016] Furthermore, in step b), the amount of methanol added to the residue is 1 / 6 to 1 / 2 of the amount of water added to the sample to be tested, v / v, ml / ml; preferably 1 / 5, v / v, ml / ml.

[0017] Furthermore, the wavelength of the chromatographic conditions in step c) is 254 nm, and the number of theoretical plates calculated based on ferulic acid should be no less than 3000.

[0018] Furthermore, the octadecylsilane bonded silica gel chromatographic column in step c) is Ultimate XB-C18, 4.6×250 mm, 5 μm; Agilent Zorbax SB-C18, 4.6×250 mm, 5 μm; Waters symmetry-C18, 4.6×250 mm, 5 μm.

[0019] The present invention discloses a pharmaceutical composition containing "Shentong Zhuyu Decoction", which contains stable, comprehensive and uniform medicinal ingredients in "Shentong Zhuyu Decoction". The fingerprint spectrum detection method of the present invention for Shentong Zhuyu Decoction and its preparation has high spectrum separation, many calibrated common peaks, strong characteristic, and can accurately, comprehensively and stably reflect the chemical quality profile of the classic prescription Shentong Zhuyu Decoction, realize the quality control of the traditional preparation and modern preparation of Shentong Zhuyu Decoction, and has the value of popularization and application.

[0020] The Shentong Zhuyu Decoction described in the present invention comes from "Medical Error Correction" written by Wang Qingren in the Qing Dynasty and is one of the 100 ancient classic prescriptions. It is composed of 1 qian of qinjiao, 2 qian of chuanxiong, 3 qian of peach kernel, 3 qian of safflower, 2 qian of liquorice, 1 qian of qianghuo, 2 qian of myrrh, 3 qian of angelica, 2 qian of aconiti lobata (fried), 1 qian of xiangfu, 3 qian of cyperus rotundus and 2 qian of earthworm (soil removed).

[0021] The material standard of the Shentong Zhuyu Decoction used in the present invention is the standard of the medicinal material of the Shentong Zhuyu Decoction prepared according to the preparation method of the Shentong Zhuyu Decoction recorded in the ancient medical book "Yi Lin Gaicuo". Except for the molding process, the rest of the preparation method is basically consistent with the record in "Yi Lin Gaicuo". The specific preparation method is:

[0022] Weigh the raw materials according to the formula of Shentong Zhuyu Decoction; add water, cover and boil twice, add 12 times the amount of water for the first time, soak for 30 minutes, boil over high heat, then boil over low heat for 30 minutes, filter while hot, add 10 times the amount of water for the second time, boil over high heat, then boil over low heat for 20 minutes, filter while hot, combine the filtrate, concentrate under reduced pressure to a concentrate volume of about 1:1 (g:ml), freeze-dry, and crush to obtain the actual dry paste powder corresponding to the material benchmark of Shentong Zhuyu Decoction.

[0023] The Shentong Zhuyu Decoction granules used in the present invention are prepared by adding pharmaceutically acceptable auxiliary materials to the actual dry paste powder corresponding to the substance standard of Shentong Zhuyu Decoction, granulating, drying and sieving to form granules.

[0024] 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.

[0025] 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

[0026] Figure 1 Fingerprint search map-1

[0027] Figure 2 Fingerprint search map-2

[0028] Figure 3 Results of the test sample extraction solvent investigation

[0029] Figure 4 HPLC comparison of drug flavors

[0030] Figure 5 HPLC comparison charts for confirmation of related components

[0031] Figure 6 HPLC comparison of test sample, reference sample (ferulic acid) and blank solvent

[0032] Figure 7 Common pattern diagram of the fingerprint spectrum of Shentong Zhuyu Decoction (R reference spectrum)

[0033] Figure 8 Reference fingerprint (reference substance confirmed chromatographic peaks: Peak 1: protocatechuic acid, Peak 4: caffeic acid, Peak 5: gentiopicroside, Peak 7: p-coumaric acid, Peak 8 (S): ferulic acid, Peak 9: neoliquiritigenin, Peak 10: liquiritigenin, Peak 11: kaempferol-3-O-rutinoside, Peak 12: ligusticolide I, Peak 13: ligusticolide H, Peak 15: isoliquiritigenin, Peak 16: neoliquiritigenin) DETAILED DESCRIPTION

[0034] Example 1 Detection of the substance standard of Shentong Zhuyu Decoction

[0035] 1) Preparation of reference solution:

[0036] Take an appropriate amount of ferulic acid reference substance, weigh accurately, and add methanol to make a solution containing 0.16 mg per 1 ml.

[0037] 2) Preparation of test solution:

[0038] Take 1.0g of the actual dry powder of the substance benchmark of Shentong Zhuyu Decoction, weigh it accurately, put it in a 100ml stoppered conical bottle, add 25ml of water, dissolve it by ultrasonic treatment, shake it with ethyl acetate for 3 times, 50ml each time, combine the ethyl acetate liquid, evaporate to dryness, add an appropriate amount of methanol to dissolve the residue, transfer it to a 5ml volumetric bottle, add methanol to dilute to the scale, shake well, filter, and obtain;

[0039] 3) Determination of fingerprint

[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] Chromatographic column: Ultimate XB-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 ferulic acid peak should be no less than 5000.

[0043]

[0044] 4) Analyze fingerprint

[0045] There are 17 characteristic peaks in the fingerprint of the actual dry paste powder corresponding to the material benchmark of Shentong Zhuyu Decoction. The ferulic acid chromatographic peak is taken as the reference peak (S peak), and the relative retention time of the remaining characteristic peaks and the S peak is calculated. The relative retention time should be within ±7% of the specified value, and the specified value is: Peak 1 protocatechuic acid 0.349, Peak 2 0.509, Peak 3 0.607, Peak 4 caffeic acid 0.637, Peak 5 gentiopicroside 0.675, Peak 6 0.83, Peak 7 p-coumaric acid 0.880, Peak 8 ferulic acid 1.000, Peak 9 neoliquiritin 1.028, Peak 10 liquiritin 1.063, Peak 11 kaempferol-3-O-rutinoside 1.278, Peak 12 ligusticol I 1.440, Peak 13 ligusticol H 1.570, Peak 14 1.667, peak 15 isoliquiritigenin 1.727, peak 16 neoisoliquiritigenin 1.820, peak 17 2.066.

[0046] The similarity evaluation software system of Chinese medicine chromatographic fingerprint was further used to compare the fingerprint of the obtained substance benchmark of Shentong Zhuyu Decoction with the established reference fingerprint ( Figure 7) comparison, the similarity is greater than 0.90, and the method is accurate and reliable.

[0047] Example 2 Detection of Shentong Zhuyu Decoction Granules

[0048] 1) Preparation of reference solution:

[0049] Take an appropriate amount of ferulic acid reference substance, weigh accurately, and add methanol to make a solution containing 0.16 mg per 1 ml.

[0050] 2) Preparation of test solution:

[0051] Take 5.0g of Shentong Zhuyu Decoction Granules, weigh accurately, place in a 100ml stoppered conical flask, add 25ml of water, and dissolve and mix by ultrasonic treatment. Shake and extract with ethyl acetate 3 times, 50ml each time. Combine the ethyl acetate liquid, evaporate to dryness, add an appropriate amount of methanol to dissolve the residue, transfer to a 5ml volumetric flask, add methanol to dilute to the scale, shake well, filter, and obtain;

[0052] 3) Determination of fingerprint

[0053] Accurately pipette the reference solution and the test solution into the liquid chromatograph to obtain a chromatogram;

[0054] The chromatographic conditions were as follows:

[0055] Chromatographic column: Waters symmetry-C18, 4.6×250mm, 5μm; acetonitrile is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is performed as specified in the following table; column temperature is 30°C; flow rate is 1ml per minute; detection wavelength: 280nm, and the number of theoretical plates calculated based on the ferulic acid peak should be no less than 5000.

[0056]

[0057] 4) Analyze fingerprint

[0058] There are 17 characteristic peaks in the fingerprint of the actual dry paste powder corresponding to the material benchmark of Shentong Zhuyu Decoction. The ferulic acid chromatographic peak is taken as the reference peak (S peak), and the relative retention time of the remaining characteristic peaks and the S peak is calculated. The relative retention time should be within ±7% of the specified value, and the specified value is: Peak 1 protocatechuic acid 0.349, Peak 2 0.509, Peak 3 0.607, Peak 4 caffeic acid 0.637, Peak 5 gentiopicroside 0.675, Peak 6 0.83, Peak 7 p-coumaric acid 0.880, Peak 8 ferulic acid 1.000, Peak 9 neoliquiritin 1.028, Peak 10 liquiritin 1.063, Peak 11 kaempferol-3-O-rutinoside 1.278, Peak 12 ligusticol I 1.440, Peak 13 ligusticol H 1.570, Peak 14 1.667, peak 15 isoliquiritigenin 1.727, peak 16 neoisoliquiritigenin 1.820, peak 17 2.066.

[0059] The similarity evaluation software system of Chinese medicine chromatographic fingerprint was further used to compare the fingerprint of the obtained substance benchmark of Shentong Zhuyu Decoction with the established reference fingerprint ( Figure 7 ) comparison, the similarity is greater than 0.90, and the method is accurate and reliable.

[0060] The beneficial effects of the present invention are described below through test examples.

[0061] Test Example 1

[0062] 1. Instruments and test drugs

[0063] Liquid chromatograph: Shimadzu LC-20A, Agilent 1260; chromatographic columns: Ultimate XB-C18 (4.6×250mm, 5μm); Agilent Zorbax SB-C18 (4.6×250mm, 5μm); Waters symmetry-C18 (4.6×250mm, 5μm); electronic analytical balance: Sartorius Scientific Instrument (Beijing) Co., Ltd. SQP (one thousandth); Mettler-Toledo Instrument (Shanghai) Co., Ltd. MS204TS / 02 (one ten-thousandth); Mettler-Toledo Instrument (Shanghai) Co., Ltd. MS204TS (one ten-thousandth); Sartorius Scientific Instrument (Beijing) Co., Ltd. BT25S (one hundred thousandth).

[0064] Acetonitrile was chromatographically pure and produced by Tianjin Saifului Technology Co., Ltd.; phosphoric acid was chromatographically pure and produced by MREDA; methanol and ethyl acetate were analytically pure and produced by Beijing Chemical Plant; Wahaha purified water was from Hangzhou Wahaha Group Co., Ltd. Ferulic acid reference substance (batch number: 110773-201614, purity: 99.0%) was purchased from China Food and Drug Administration.

[0065] 2. Prescription and Preparation of Shentong Zhuyu Decoction

[0066] Take 3.73g of Gentiana macrophylla, 7.46g of Chuanxiong, 11.19g of peach kernel (crushed), 11.19g of safflower, 7.46g of licorice, 3.73g of Notopterygium wilfordii, 7.46g of vinegar myrrh, 11.19g of angelica sinensis, 7.46g of vinegar aconiti lobata, 3.73g of Cyperus rotundus, 11.19g of Achyranthes bidentata, and 7.46g of earthworm, cover and decoct twice, add 12 times the amount of water for the first time, soak for 30 minutes, boil over high heat and then simmer for 30 minutes, filter while hot, add 10 times the amount of water for the second time, boil over high heat and then simmer for 20 minutes, filter while hot, combine the filtrate, concentrate under reduced pressure, freeze-dry until the volume of the concentrate is about 1:1 (g:ml), and grind to obtain dry paste powder.

[0067] 3. Fingerprint Exploration

[0068] Method 1: directly shake the corresponding dry paste powder aqueous solution of the substance benchmark of Shentong Zhuyu Decoction with ethyl acetate to extract and determine the chromatogram. The method is as follows:

[0069] 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 as specified in the following table; the detection wavelength was 254 nm.

[0070] The time program is as follows:

[0071]

[0072] Preparation of test solution: Take about 1.0g of the substance corresponding to the standard of Shentong Zhuyu Decoction, weigh it accurately, put it in a 100ml stoppered conical flask, add 20ml of water to dissolve it, shake and extract with 20ml of ethyl acetate, collect the ethyl acetate solution, evaporate it to dryness, add an appropriate amount of methanol to dissolve the residue, transfer it to a 5ml volumetric flask, add methanol to dilute to the scale, shake well, filter, and obtain the solution.

[0073] Determination method: Accurately draw 10μl of the test solution, inject it into the liquid chromatograph, determine, and record the chromatogram. According to the chromatogram information, from 0 to 70 minutes, the chromatographic peaks are well separated and the peaks are sharp; from 70 to 130 minutes, the chromatographic peaks are poorly separated and the baseline is uneven. The results are shown in Figure 1 .

[0074] Method 2: Considering that the overall time of the chromatogram is relatively long, the time program is further adjusted. The preparation method of the test sample is the same as that of method 1, and the time program is as follows:

[0075]

[0076] The chromatographic peaks are well separated and the baseline is relatively stable. Figure 2 , tentatively designated as the fingerprint method.

[0077] IV. Investigation of test sample preparation methods

[0078] Take 1.0 g of the actual dry paste powder corresponding to the material standard of Shentong Zhuyu Decoction, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of methanol, seal it, and treat it with ultrasound (power 500 W, frequency 40 kHz) for 30 minutes. Cool it, shake it evenly, filter it, inject it into liquid chromatograph, and measure it.

[0079] Take 1.0g of the actual dry paste powder corresponding to the material benchmark of Shentong Zhuyu Decoction (3 portions in total), weigh it accurately, put it in a stoppered conical flask, accurately measure 25ml of water, dissolve it by ultrasonic treatment, add ethyl acetate and shake to extract 1 time, 2 times, and 3 times, 50ml each time, combine the ethyl acetate liquids, evaporate to dryness, dissolve the residue in methanol and transfer it to a 5ml volumetric flask, add methanol to the scale, shake well, filter, and inject into liquid chromatograph for determination.

[0080] Results Figure 3 The results show that: compared with ethyl acetate shaking extraction 1, 2, and 3 times, shaking extraction 3 times is more complete; compared with methanol ultrasonic sample, the peak response values ​​of ethyl acetate shaking extraction samples are higher, so ethyl acetate shaking extraction 3 times is selected as the preparation method of the test solution.

[0081] 5. Selection of detection wavelength

[0082] The test sample was prepared by shaking and extracting ethyl acetate three times under the above-mentioned "Test Sample Preparation Method Investigation". The test sample solution was scanned at a wavelength of 190-400nm using a PDA detector. The analysis was performed through isoabsorption spectra and 3D views. The results showed that the chromatographic peaks were in the wavelength range of 230-280nm, with a large amount of information. Wavelengths of 230nm, 240nm, 254nm, 270nm, and 280nm were selected for analysis. The results showed that at a wavelength of 254nm, the response values ​​of each chromatographic peak were also moderate, and the baseline was relatively stable.

[0083] VI. Peak attribution and reference selection

[0084] (1) Attribution of drug flavor peaks

[0085] Preparation of reference solution: Take an appropriate amount of ferulic acid reference substance, weigh it accurately, and add methanol to make a solution containing 0.16 mg per 1 ml.

[0086] Preparation of single-herb test solution: Take appropriate amount of coarse powder of each medicinal ingredient in the prescription (0.35g each of Angelica sinensis and Carthami, 0.24g each of Chuanxiong, Licorice, and Aconiti Lateralis Preparata, 0.12g each of Gentiana macrophylla and Notopterygium wilfordii, 1.0g of Myrrh), accurately weigh, put in a round-bottom flask, add 25ml of water, boil for 30 minutes, filter, cool, add ethyl acetate to the filtrate and shake for extraction 3 times, 50ml each time, combine the ethyl acetate solutions, evaporate to dryness, add appropriate amount of methanol to the residue to dissolve it, transfer to a 5ml volumetric flask, add methanol to the scale, shake well, filter, and obtain.

[0087] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and record the chromatogram.

[0088] Determine the attribution of each peak in the material reference fingerprint (see Figure 4 and Table 1).

[0089] Table 1: Table of medicinal taste

[0090]

[0091] (2) Chromatographic peak identification

[0092] The main components related to each medicinal taste were selected through literature research: peak positioning research was conducted on 12 known components (gentiopicroside, ferulic acid, licorice glycoside, caffeic acid, p-coumaric acid, protocatechuic acid, kaempferol-3-O-rutinoside, isoliquiritigenin, neoisoliquiritigenin, neoliquiritigenin, ligusticum chuanxiong lactone I, ligusticum chuanxiong lactone H) (see Figure 5 ), and ultimately a total of 12 known components were identified.

[0093] (3) Selection of reference objects

[0094] Among them, the ferulic acid chromatographic peak has a moderate retention time and a high response value, achieving baseline separation, so ferulic acid is selected as the reference peak and marked as S peak.

[0095] VII. Preliminary determination of the method

[0096] 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 254 nm; the theoretical plate number calculated based on the ferulic acid peak should be no less than 3000.

[0097]

[0098] Preparation of reference solution: Take an appropriate amount of ferulic acid reference substance, weigh it accurately, and add methanol to make a solution containing 0.16 mg per 1 ml.

[0099] Preparation of the test solution: Take 1.0g of the dry powder corresponding to the substance standard of Shentong Zhuyu Decoction, weigh it accurately, add 25ml of water, dissolve it by ultrasonic treatment, extract it with ethyl acetate by shaking for 3 times, 50ml each time, combine the ethyl acetate liquid, evaporate to dryness, add appropriate amount of methanol to dissolve the residue, transfer it to a 5ml volumetric flask, dilute it to the scale with methanol, shake it well, filter it, and obtain the solution.

[0100] The determination method is to accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, determine, and record the chromatogram.

[0101] 8. Methodological Investigation

[0102] ①Specificity test

[0103] In order to investigate whether the blank solvent has any interference, the test solution, reference solution and blank solvent were injected into the liquid chromatograph and the chromatogram was recorded. The blank solvent had no interference and the specificity was good. At the same time, the integrity of the test product was also investigated. There was no obvious chromatographic peak within the extended 30 minutes, and the integrity was good. Figure 6 .

[0104] The time program is as follows:

[0105]

[0106] ②Instrument precision test

[0107] Take 1.0 g of the actual dry powder corresponding to the material standard of Shentong Zhuyu Decoction, weigh it accurately, and operate according to the preparation and determination method of the test solution under the "Preliminary Determination of the Method", inject the sample continuously for 6 times, and the relative retention time results are shown in Table 2.

[0108] Table 2 Relative retention time results of instrument precision test

[0109]

[0110] The results showed that the RSD of the relative retention time of the fingerprint was less than 2%, the similarity was above 0.90, and the instrument precision was good.

[0111] ③Stability test

[0112] Take 1.0 g of the actual dry powder corresponding to the substance standard of Shentong Zhuyu Decoction, weigh it accurately, and operate according to the preparation and determination method of the test solution under the "Preliminary Determination of the Method". After 0, 2, 4, 8, 12, and 24 hours after preparation, the samples are injected and measured. The relative retention time results are shown in Table 3.

[0113] Table 3 Relative retention time results of fingerprint stability test

[0114]

[0115]

[0116] The results showed that the relative retention time RSD was less than 2%, the similarities were all above 0.90, and the test solution had good stability within 24 hours.

[0117] ④Repeatability test

[0118] Take 1.0g of the actual dry powder of the substance standard of Shentong Zhuyu Decoction (6 portions in total), weigh it accurately, and operate according to the preparation and determination method of the test solution under the "Preliminary Determination of the Method". The relative retention time results are shown in Table 4.

[0119] Table 4 Relative retention time results of fingerprint repeatability test

[0120]

[0121] The results showed that the relative retention time RSD was less than 2% and the similarities were all above 0.90, indicating that the method had good repeatability.

[0122] ⑤Durability test

[0123] The durability of three different flow rates, three different column temperatures, three different acid concentrations, three different chromatographic columns, and two different instruments for the chromatographic conditions was investigated, and the relative retention time and similarity of each characteristic peak to the S peak were calculated. The results of the relative retention time determination are shown in Tables 5 to 9.

[0124] Table 5 Relative retention time results at different column temperatures

[0125]

[0126] Table 6 Relative retention time results of different acidity tests

[0127]

[0128]

[0129] Table 7 Relative retention time results of different chromatographic column tests

[0130]

[0131] Table 8 Relative retention time results of different flow rate tests

[0132]

[0133] Table 9 Relative retention time results of different instrument tests

[0134]

[0135]

[0136] The results showed that the relative retention time RSD of this method was less than 5% under different column temperatures, different acidities, different chromatographic columns, different flow rates and different instrument conditions. There are 17 characteristic peaks in the chromatograms, peak 1 is protocatechuic acid, peak 4 is caffeic acid, peak 5 is gentiopicroside, peak 7 is p-coumaric acid, peak 8 is ferulic acid, peak 9 is neoliquiritigenin, peak 10 is liquiritigenin, peak 11 is kaempferol-3-O-rutinoside, peak 12 is ligusticolide I, peak 13 is ligusticolide H, peak 15 isoliquiritigenin, peak 16 is neoliquiritigenin, and the peak corresponding to the ferulic acid 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 2 0.509, peak 3 0.607, peak 6 0.83, peak 141.667, peak 17 2.066; the common peaks in the chromatograms are sharp and symmetrical with good separation, and the similarity is not less than 0.90. This indicates that the method has good durability at different column temperatures, different acidities, different chromatographic columns, different flow rates, and different instruments.

[0137] IX. Determination of fingerprint detection method

[0138] 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 254 nm; the theoretical plate number calculated based on the ferulic acid peak should be no less than 3000.

[0139]

[0140] Preparation of reference solution: Take an appropriate amount of ferulic acid reference substance, weigh it accurately, and add methanol to make a solution containing 0.16 mg per 1 ml.

[0141] Preparation of test solution: Take 1.0g of the dry powder corresponding to the substance standard of Shentong Zhuyu Decoction, weigh it accurately, put it in a stoppered conical flask, add 25ml of water, dissolve it by ultrasonic treatment, extract it with ethyl acetate by shaking for 3 times, 50ml each time, combine the ethyl acetate solutions, evaporate to dryness, add an appropriate amount of methanol to dissolve the residue, transfer it to a 5ml volumetric flask, dilute it to the scale with methanol, shake it well, filter it, and obtain the solution.

[0142] The determination method is to accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, determine, and record the chromatogram.

[0143] 10. Establishment of Fingerprint

[0144] ① Common peak calibration

[0145] Fifteen batches (30 copies) of test samples were determined by the method under the fingerprint spectrum, and the obtained fingerprints were analyzed. The chromatographic peaks with good stability and appropriate response values ​​in the fingerprints of 15 batches (30 copies) of samples were selected as common peaks. A total of 17 common peaks were calibrated. The ferulic acid chromatographic peak was used as the reference peak (S peak), and the relative retention time of the remaining characteristic peaks and the S peak was calculated. The relative retention time should be within ±7% of the specified value, and the specified value is: Peak 1 protocatechuic acid 0.349, Peak 2 0.509, Peak 3 0.607, Peak 4 caffeic acid 0.637, Peak 5 gentiopicroside 0.675, Peak 6 0.83, peak 7 p-coumaric acid 0.880, peak 8 ferulic acid 1.000, peak 9 neoliquiritigenin 1.028, peak 10 liquiritigenin 1.063, peak 11 kaempferol-3-O-rutinoside 1.278, peak 12 ligusticolide I 1.440, peak 13 ligusticolide H 1.570, peak 14 1.667, peak 15 isoliquiritigenin 1.727, peak 16 neoisoliquiritigenin 1.820, peak 17 2.066.

[0146] ② Establishment of reference map

[0147] 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 the Shentong Zhuyu Decoction material benchmark dry paste powder to form a common pattern diagram. The common pattern diagram is shown in Figure 7 , and establish a reference map (see Figure 8 ).

[0148] ③ Calculation of similarity

[0149] The similarity evaluation software system of Chinese medicine chromatographic fingerprint was used to calculate the similarity of 15 batches (30 copies) of material reference dry paste powder and the reference fingerprint based on the common peaks. The similarity calculated by fingerprint was the lowest at 0.961, and all were greater than 0.90.

[0150] In summary, the pharmaceutical composition containing "Shen Tong Zhu Yu Tang" of the present invention contains stable, comprehensive and uniform active ingredients in "Shen Tong Zhu Yu Tang". The fingerprint spectrum detection method of Shen Tong Zhu Yu Tang and its preparation of the present invention has high spectral separation, many calibrated common peaks, strong characteristic, and can accurately, comprehensively and stably reflect the chemical quality profile of the classic prescription Shen Tong Zhu Yu Tang, realize the quality control of traditional preparations and modern preparations of Shen Tong Zhu Yu Tang, and has the value of promotion and application.

Claims

1. A HPLC fingerprint detection method for Shentong Zhuyu Decoction, characterized in that: It includes the following steps: a. Preparation of reference solution: Take ferulic acid reference substance, dissolve it in methanol, and use it as reference solution; b. Preparation of test solution: Take the sample to be tested, dissolve it in water, extract it with ethyl acetate, evaporate the extract to dryness, dissolve the residue in methanol, 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; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B for gradient elution; wavelength 230-280nm; gradient elution program is as follows: There are 17 characteristic peaks in the fingerprint, of which 12 characteristic peaks are: peak 1 protocatechuic acid, peak 4 caffeic acid, peak 5 gentiopicroside, peak 7 p-coumaric acid, peak 8 ferulic acid, peak 9 neoliquiritin, peak 10 liquiritin, peak 11 kaempferol-3-O-rutinoside, peak 12 ligusticolide I, peak 13 ligusticolide H, peak 15 isoliquiritigenin, and peak 16 neoisoliquiritigenin.

2. The HPLC fingerprint detection method according to claim 1, characterized in that: The 12 characteristic peaks are calculated with the ferulic acid chromatographic peak as the reference peak, and the relative retention time of each characteristic peak should be within ±7% of the specified value, and the specified value is: peak 1 protocatechuic acid 0.349, peak 4 caffeic acid 0.637, peak 5 gentiopicroside 0.675, peak 7 p-coumaric acid 0.880, peak 8 ferulic acid 1.000, peak 9 neoliquiritigenin 1.028, peak 10 liquiritigenin 1.063, peak 11 kaempferol-3-O-rutinoside 1.278, peak 12 ligusticum lactone I 1.440, peak 13 ligusticum lactone H 1.570, peak 15 isoliquiritigenin 1.727, peak 16 neoisoliquiritigenin 1.

820.

3. The HPLC fingerprint detection method according to claim 1, characterized in that: For the other five characteristic peaks in the fingerprint, the ferulic acid chromatographic peak was used as the reference peak to calculate the relative retention time of each characteristic peak and the reference peak. The relative retention time should be within ±7% of the specified value, and the specified value is: peak 2 0.509, peak 3 0.607, peak 6 0.83, peak 141.667, peak 17 2.

066.

4. The HPLC fingerprint detection method according to claim 1, characterized in that: In step a), the reference solution has a concentration of 0.1-0.3 mg ferulic acid per 1 ml.

5. The HPLC fingerprint detection method according to claim 1, characterized in that: In step b), the mass volume ratio of the sample to be tested to water is 0.5-5 g:25 mL.

6. The HPLC fingerprint detection method according to claim 1, characterized in that: The extraction in step b) is performed by adding ethyl acetate and shaking 1 to 3 times, each time adding 1 to 3 times the volume of water, and combining the ethyl acetate solutions.

7. The HPLC fingerprint detection method according to claim 1, characterized in that: In step b), the amount of methanol added to the residue is 1 / 6 to 1 / 2 of the amount of water added to the sample to be tested, v / v, ml / ml.

8. The HPLC fingerprint detection method according to claim 1, characterized in that: In step c), the wavelength is 254 nm, and the number of theoretical plates calculated based on ferulic acid should be no less than 3000.

9. The HPLC fingerprint detection method according to claim 1, characterized in that: Step c) The octadecylsilane bonded silica gel chromatographic column is any one of Ultimate XB-C18, 4.6×250 mm, 5 μm; Agilent Zorbax SB-C18, 4.6×250 mm, 5 μm; and Waters symmetry-C18, 4.6×250 mm, 5 μm.

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