A method for constructing a characteristic spectrum of Zhenlong Xingnao Capsule

Abstract

The present invention relates to the field of medical technology, and more particularly to a method for constructing a characteristic spectrum of Zhenlong Xingnao Capsules. The method of the present invention can simultaneously detect chebulic acid, gallic acid, dehydrodiisoeugenol, dehydrocostus lactone, cinnamaldehyde, and cinnamic acid in Zhenlong Xingnao Capsules, and can increase the initial content of cinnamaldehyde and ensure its stable presence during the stability storage period. In addition, the present invention establishes an HPLC characteristic spectrum for Zhenlong Xingnao Capsules, identifies 11 characteristic peaks, and more fully demonstrates the chemical characteristics of Zhenlong Xingnao Capsules. This method can reflect the major chemical components in Zhenlong Xingnao Capsules. The overall quality of Zhenlong Xingnao Capsules can be controlled more efficiently and quickly.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for constructing a characteristic spectrum of Zhenlong Xingnao Capsule. Background Art

[0002] Zhenlong Nao Xingnao Capsules are made from a crushed blend of pearl, rhodiola rosea, saffron, cloves, nutmeg, cardamom, tsaoko, sandalwood, red sandalwood, agarwood, terminalia chebula, terminalia chebula, emblica, costus root, cinnamon bark, piper longifolia, crab, goldstone, celery, artificial bezoar, musk, jujube, lixiangdu, corydalis chinensis, short-spiked rabbit ear grass, iron powder (processed), winter mallow fruit, licorice, and black seed. It is primarily used to awaken the mind, clear heat, and dredge the meridians. It is used to treat stroke caused by phlegm and blood stasis blocking the meridians, as well as slurred speech, hemiplegia, and facial paralysis.

[0003] Currently, the Zhenlong Xingnao Capsules have a problem with the stability of cinnamaldehyde content, which may decrease and fail to meet the standard when placed. Existing methods for Zhenlong Xingnao Capsules may involve thin layer identification of Piper smilax, costus root, cinnamon bark, gallic acid, cardamom, cloves, pearl, bile acid, saffron, cinnamaldehyde, and licorice in the formula, and content determination of cinnamaldehyde, piperine, bile acid, crocin-I, and crocin-II. Neither involves the characteristic spectrum method of Zhenlong Xingnao Capsules. Since Zhenlong Xingnao Capsules contain a large variety of Chinese medicinal materials, and some of the ingredients are volatile, detection is relatively difficult. It is relatively difficult to simultaneously extract and separate the various active ingredients of Zhenlong Xingnao Capsules. In view of this, the present invention is specially proposed. Summary of the Invention

[0004] In order to make up for the deficiencies of the prior art, the present invention provides a method for constructing a characteristic spectrum of Zhenlong Xingnao Capsule, which can simultaneously detect chebulic acid, gallic acid, dehydrodiisoeugenol, dehydrocostus lactone, cinnamaldehyde and cinnamic acid in Zhenlong Xingnao Capsule.

[0005] Zhenlong Xingnao Capsule is prepared by the following method:

[0006] The raw materials of Zhenlong Xingnao Capsule are: pearl 51.9g, Tianzhuhuang 6.6g, clove 29.1g, nutmeg 26.1g, cardamom 26.1g, tsaoko 19.5g, sandalwood 26.1g, red sandalwood 64.8g, agarwood 51.9g, terminalia chebula 84.3g, hairy terminalia chebula 51.9g, emblica 64.8g, costus root 64.8g, cinnamon 51.9g, longan 26.1g, square sea 32.4g, gold stone 26.1g, fragrant celery 16.2g, artificial bezoar 6.6g, jujube 19.5g, fierce fragrant azalea 19g. .5g, Saibei Corydalis 39.0g, Short-spiked Rabbit Ear Grass 129.9g, Iron Powder (processed) 12.9g, Winter Mallow Fruit 51.9g, Licorice 39.0g, Nigella Sativa Seed 16.2g, except cinnamon, the medicinal materials are crushed at room temperature for 20 minutes using YSF-8 ultrafine grinder, and cinnamon is crushed separately by ultrafine sealing using a universal grinder, and the powder is taken according to the prescribed amount and added into a square cone mixer for mixing for 30 minutes, and the evenly mixed powder is put into capsules; a capsule sealing glue station device is installed on the capsule filling machine to seal the capsules, install blisters, and install aluminum foil composite film bags.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for constructing a characteristic spectrum of Zhenlong Xingnao Capsule comprises the following steps:

[0009] (1) Preparation of reference solution: accurately weigh the reference substances of chebulic acid, gallic acid, dehydrodiisoeugenol, dehydrocostus lactone, cinnamaldehyde from Zhenlong Xingnao Capsules, and cinnamic acid, add methanol to prepare a solution containing 120 μg per ml, shake well, and use it as the reference solution;

[0010] (2) Preparation of test solution: Take 1 g of the contents of Zhenlong Xingnao Capsule, first add 30 ml of n-hexane-ethyl acetate solution, and ultrasonicate at 250W, 40kHz, and 25℃ for 20 minutes, filter and collect filtrate A; then add 30 ml of 60% methanol-water solution with 0.1% formic acid to the residue obtained by filtering filtrate A, and ultrasonicate in a constant temperature water bath at 300W, 40kHz, and 40℃ for 40 minutes, filter and collect filtrate B; after combining filtrates A and B, pass through C18 and HLB solid phase extraction columns in series, the extraction columns are pre-equilibrated with methanol-water, discard the first 3 ml of effluent, and use 5 ml of water, 5 ml of The impurities were eluted with 20% methanol, and the target compound was finally eluted with a gradient of 90% methanol solution containing 0.5% ammonia water. The eluate was concentrated to near dryness by nitrogen blowing, and the upper phase and lower phase (volume ratio 1.2:1) after separation of the eluted and concentrated residue in a mixed solvent system of petroleum ether-ethanol-ethyl acetate-water in a volume ratio of 12:5:3:2 were added to the residue. The mixture was dissolved and injected into a high-speed countercurrent chromatograph. The effluents at 15–25 min, 35–50 min, and 60–75 min were collected. The target fractions were combined and dried under reduced pressure at low temperature. The dried product was re-dissolved in 60% methanol solution, ultrasonically shaken for 5 minutes, filtered through a 0.22 μm filter membrane, and the filtrate was transferred to a brown sample bottle.

[0011] (3) Determination: Inject the reference solution and the test solution into a high performance liquid chromatograph under the following chromatographic conditions, inject 10 μl of each sample, and record the chromatogram;

[0012] Chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler; acetonitrile-tetrahydrofuran (volume ratio 95:5) as mobile phase A, 0.1% citrate buffer (pH 2.8±0.1) containing 0.15% ammonium phosphate and 0.1% sodium dodecylsulfonate as mobile phase B, elution according to the following gradient; column temperature, 30°C; flow rate, 1 ml / min; detection with a diode array detector at a wavelength of 210 nm.

[0013] The gradient conditions are:

[0014] From 0 to 7 min, the volume ratio of phase A:phase B changed from 2:98 to 3:97;

[0015] From 7 to 15 min, the volume ratio of phase A:phase B changed from 3:97 to 5:95;

[0016] From 15 to 25 min, the volume ratio of phase A:phase B changed from 5:95 to 13:87;

[0017] From 25 to 40 min, the volume ratio of phase A:phase B changed from 13:87 to 14:86;

[0018] From 40 to 55 min, the volume ratio of phase A:phase B changed from 14:86 to 18:82;

[0019] From 55 to 70 min, the volume ratio of phase A:phase B changed from 18:82 to 24:76;

[0020] From 70 to 92 min, the volume ratio of phase A:phase B changed from 24:76 to 52:48;

[0021] From 92 to 110 min, the volume ratio of phase A:phase B changed from 52:48 to 70:30;

[0022] From 110 to 125 min, the volume ratio of phase A:phase B changed from 70:30 to 100:0;

[0023] At 125–126 min, the volume ratio of phase A:phase B changed from 100:0 to 2:98;

[0024] 126-135 min, the volume ratio of phase A:phase B was 2:98;

[0025] (4) Generate a control characteristic spectrum: select the chromatographic peaks that exist in the chromatograms of different batches of Zhenlong Xingnao Capsules as common peaks, and use the average value calculation method to generate a control characteristic spectrum of Zhenlong Xingnao Capsules.

[0026] Preferably, the volume ratio of n-hexane to ethyl acetate in the n-hexane-ethyl acetate solution in step (2) is 3:1.

[0027] Preferably, the parameters of the high-speed countercurrent chromatography in step (2) are set as follows: rotation speed 850 r / min, flow rate 3.0 mL / min, and detection wavelength 280 nm.

[0028] Preferably, the chromatographic column in step (3) is Platisil ODS C18, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0029] Preferably, the chromatogram obtained in step (4) presents 11 characteristic peaks, wherein peak 1, peak 2, peak 3, peak 4, peak 7, and peak 9 correspond to the peaks of the reference substance of gallic acid, chebulic acid, cinnamic acid, cinnamaldehyde, dehydrocostus lactone, and dehydrodiisoeugenol, respectively; the peak corresponding to the peak of the reference substance of cinnamaldehyde is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within the range of ±10% of the specified value. The specified value is: 0.29 (peak 1), 0.67 (peak 2), 0.97 (peak 3), 1.12 (peak 5), 1.31 (peak 6), 1.39 (peak 7), 1.42 (peak 8), 1.45 (peak 9), 1.54 (peak 10), and 1.61 (peak 11).

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] (1) The method of the present invention can simultaneously detect chebulic acid, gallic acid, dehydrodiisoeugenol, dehydrocostus lactone, cinnamaldehyde, and cinnamic acid in Zhenlong Xingnao Capsules, and can increase the initial content of cinnamaldehyde and stably exist during the stability storage period; in addition, the present invention establishes an HPLC characteristic spectrum for Zhenlong Xingnao Capsules, identifies 11 characteristic peaks, and more fully demonstrates the chemical characteristics of Zhenlong Xingnao Capsules;

[0032] (2) The present invention investigated the extraction solvent and extraction method of the sample. Within the investigated range, the chromatographic method showed 11 characteristic peaks, and the relative retention time of each peak had a small change, with RDS < 2.0%;

[0033] (3) The method of the characteristic spectrum of Zhenlong Xingnao Capsule constructed in the present invention was investigated by methodology, including precision experiments, method repeatability experiments, and sample stability experiments. The RSD values ​​of the relative retention time of each peak in each experimental result were all ≤2.0%, and the RSD values ​​of the relative peak area were all ≤10.0%. This indicates that the method is good and can reflect the major chemical components of Zhenlong Xingnao Capsule. The overall quality of Zhenlong Xingnao Capsule can be controlled more efficiently and quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention will be further described below with reference to the accompanying drawings.

[0035] Figure 1 This is the chromatogram of the precision test, from bottom to top: S1~S6: precision 1~6;

[0036] Figure 2 This is the chromatogram of the repeatability test, from bottom to top: S1~S6: repeatability 1~6;

[0037] Figure 3 This is the stability test chromatogram, from bottom to top: S1~S6: stability 1~6;

[0038] Figure 4 The chromatograms of eight batches of Zhenlong Xingnao Capsules samples are as follows: S1: 01231110; S2: 01231111; S3: 01231112; S4: 01231113; S5: 01231114; S6: 01231116; S7: 01231117; S8: 01231119;

[0039] Figure 5 This is the characteristic spectrum of Zhenlong Xingnao Capsule. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the accompanying drawings.

[0041] Example 1

[0042] 1. Instruments and test drugs

[0043] 1.1 Instruments and Equipment

[0044] .

[0045] 1.2 Test materials

[0046] .

[0047] 1.3 Reference Material Information

[0048] .

[0049] 1.4 Sample Information

[0050] .

[0051] Example 2 Investigation of the method for constructing the characteristic spectrum of Zhenlong Xingnao Capsule

[0052] 2.1 Investigation of test sample preparation methods

[0053] 2.1.1 Investigation of extraction solvents for test samples

[0054] Chromatographic conditions and system suitability test

[0055] The column was packed with octadecylsilane bonded silica gel (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); mobile phase A was acetonitrile-tetrahydrofuran (95:5), and mobile phase B was 0.1% citrate buffer (pH 2.8 ± 0.1) containing 0.15% ammonium phosphate and 0.1% sodium dodecylsulfonate. Gradient elution was performed according to the table below. The column temperature was 30°C, and the detection wavelength was 210 nm. The number of theoretical plates, calculated based on the cinnamaldehyde peak, should be no less than 5000.

[0056] .

[0057] Preparation of reference solution

[0058] Take appropriate amount of chebulic acid reference substance, gallic acid reference substance, dehydrodiisoeugenol reference substance, dehydrocostus lactone reference substance, cinnamaldehyde reference substance, and cinnamic acid reference substance, accurately weigh them, add methanol to make a solution containing 120 μg per 1 ml, shake well, and use it as the reference substance solution.

[0059] Preparation of test solution

[0060] Take an appropriate amount of the contents of this product (batch number: 01231110), mix well, take 1g, first add 30ml of n-hexane-ethyl acetate solution with a volume ratio of 3:1, 250W, 40kHz, 25℃ low-temperature ultrasonic for 20 minutes, filter and collect filtrate A; add 30ml of 60% methanol-water solution with 0.1% formic acid to the residue, 300W, 40kHz, 40℃ constant temperature water bath ultrasonic for 40 minutes, filter and collect filtrate B; after combining filtrates A and B, pass through C18 and HLB solid phase extraction columns in series, the extraction columns are pre-equilibrated with methanol-water, discard the first 3ml of effluent, and wash with 5ml of water, 5ml Impurities were washed with 20% methanol, and the target compound was finally eluted with a gradient of 90% methanol solution containing 0.5% ammonia water. The eluate was concentrated to near dryness by nitrogen blowing, and the residue was added to a mixed solvent system of petroleum ether-ethanol-ethyl acetate-water with a volume ratio of 12:5:3:2. The upper phase and lower phase (volume ratio 1.2:1) after separation were dissolved and injected into a high-speed countercurrent chromatograph with the following parameters: speed 850 r / min, flow rate 3.0 mL / min, detection wavelength 280 nm, and effluents from 15–25 min, 35–50 min, and 60–75 min were collected. The target fractions were combined and dried under reduced pressure at low temperature. The dried product was re-dissolved in 60% methanol solution, ultrasonically shaken for 5 minutes, filtered through a 0.22 μm filter membrane, and the filtrate was transferred to a brown sample bottle.

[0061] Assay

[0062] Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, measure, and record the chromatogram.

[0063] Solvent investigations revealed that a 3:1 volume ratio of n-hexane to ethyl acetate resulted in better peak separation and a more optimal peak shape. However, a 2:2 volume ratio or a 1:3 volume ratio of n-hexane to ethyl acetate resulted in poor peak shape and missing peaks, failing to achieve the desired results. Therefore, a 3:1 volume ratio of n-hexane to ethyl acetate was selected as the extraction solvent.

[0064] Table 1 Results of relative retention time of solvent investigation

[0065] .

[0066] 2.2 Investigation of chromatographic conditions

[0067] 2.2.1 Investigation of mobile phase types

[0068] Chromatographic Conditions 1: Octadecylsilane bonded silica gel as the packing (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); acetonitrile-tetrahydrofuran (95:5) as mobile phase A, 0.1% citrate buffer (pH 2.8 ± 0.1) containing 0.15% ammonium phosphate and 0.1% sodium dodecylsulfonate as mobile phase B, gradient elution as specified in the table below; column temperature, 30°C; detection wavelength, 210 nm. The number of theoretical plates calculated based on the cinnamaldehyde peak should be no less than 5000.

[0069] .

[0070] Chromatographic Conditions 2: Octadecylsilane bonded silica gel as the packing (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); acetonitrile as mobile phase A, mobile phase B: 0.1% citrate buffer (pH 2.8 ± 0.1) containing 0.15% ammonium phosphate and 0.1% sodium dodecyl sulfate, with gradient elution as specified in the table below; column temperature, 30°C; detection wavelength, 210 nm. The number of theoretical plates, calculated based on the cinnamaldehyde peak, should be no less than 5000.

[0071] .

[0072] Chromatographic Conditions 3: Octadecylsilane bonded silica gel as the packing (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); acetonitrile-tetrahydrofuran (95:5) as mobile phase A, 0.1% citrate buffer as mobile phase B, gradient elution as specified in the table below; column temperature, 30°C; detection wavelength, 210 nm. The number of theoretical plates calculated based on the cinnamaldehyde peak should be no less than 5000.

[0073] .

[0074] The reference solution is the same as that in item “2.1.1”.

[0075] Preparation of test solution

[0076] Take an appropriate amount of the contents of this product, i.e., the test sample (batch number: 01231110), mix well, take 1 g, first add 30 ml of n-hexane-ethyl acetate solution with a volume ratio of 3:1, and sonicate at 250 W, 40 kHz, and 25 ° C for 20 minutes, filter and collect filtrate A; add 30 ml of 60% methanol-water solution with 0.1% formic acid to the residue, and sonicate in a constant temperature water bath at 300 W, 40 kHz, and 40 ° C for 40 minutes, filter and collect filtrate B; the A and B filtrates are combined and passed through a C18 and HLB solid phase extraction column in series. The extraction column is pre-equilibrated with methanol-water, and the first 3 ml of the effluent is discarded, and 5 ml of water, 5 ml of Impurities were washed with 20% methanol, and the target compound was finally eluted with a gradient of 90% methanol solution containing 0.5% ammonia water. The eluate was concentrated to near dryness by nitrogen blowing, and the residue was added to a mixed solvent system of petroleum ether-ethanol-ethyl acetate-water with a volume ratio of 12:5:3:2. The upper phase and lower phase (volume ratio 1.2:1) after separation were dissolved and injected into a high-speed countercurrent chromatograph with the following parameters: speed 850 r / min, flow rate 3.0 mL / min, detection wavelength 280 nm, and effluents from 15–25 min, 35–50 min, and 60–75 min were collected. The target fractions were combined and dried under reduced pressure at low temperature. The dried product was re-dissolved in 60% methanol solution, ultrasonically shaken for 5 minutes, filtered through a 0.22 μm filter membrane, and the filtrate was transferred to a brown sample bottle.

[0077] Assay

[0078] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0079] The results showed that when acetonitrile-tetrahydrofuran (95:5) was used as mobile phase A and 0.1% citric acid buffer (pH 2.8±0.1) containing 0.15% ammonium phosphate and 0.1% sodium dodecyl sulfate was used as mobile phase B for elution, the test sample presented a total of 11 characteristic peaks in the chromatogram, with good peak separation and excellent peak shape. When acetonitrile was used as mobile phase A and 0.1% citric acid buffer (pH 2.8±0.1) containing 0.15% ammonium phosphate and 0.1% sodium dodecyl sulfate was used as mobile phase B, or when acetonitrile-tetrahydrofuran (95:5) was used as mobile phase A and 0.1% citric acid buffer (pH 2.8±0.1) was used as mobile phase B, all peaks were missing, and the corresponding effect was not achieved. Therefore, acetonitrile-tetrahydrofuran (95:5) was selected as mobile phase A, and 0.1% citric acid buffer (pH 2.8 ± 0.1) containing 0.15% ammonium phosphate and 0.1% sodium dodecyl sulfate was selected as mobile phase B.

[0080] Table 2 Relative retention time results of mobile phase acid types

[0081] .

[0082] Example 3 Methodological Verification of the Characteristic Spectrum of Zhenlong Xingnao Capsule

[0083] 3.1 Feature Graph Method

[0084] Chromatographic conditions and system suitability test

[0085] The column was packed with octadecylsilane bonded silica gel (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); mobile phase A was acetonitrile-tetrahydrofuran (95:5), and mobile phase B was 0.1% citrate buffer (pH 2.8 ± 0.1) containing 0.15% ammonium phosphate and 0.1% sodium dodecylsulfonate. Gradient elution was performed according to the table below. The column temperature was 30°C, and the detection wavelength was 210 nm. The number of theoretical plates, calculated based on the cinnamaldehyde peak, should be no less than 5000.

[0086] .

[0087] Preparation of reference solution

[0088] Take appropriate amount of chebulic acid reference substance, gallic acid reference substance, dehydrodiisoeugenol reference substance, dehydrocostus lactone reference substance, cinnamaldehyde reference substance, and cinnamic acid reference substance, accurately weigh them, add methanol to make a solution containing 120 μg per 1 ml, shake well, and use it as the reference substance solution.

[0089] Preparation of test solution

[0090] Take an appropriate amount of the contents of this product (batch number: 01231110), mix well, take 1g, first add 30ml of n-hexane-ethyl acetate solution with a volume ratio of 3:1, 250W, 40kHz, 25℃ low-temperature ultrasonic for 20 minutes, filter and collect filtrate A; add 30ml of 60% methanol-water solution with 0.1% formic acid to the residue, 300W, 40kHz, 40℃ constant temperature water bath ultrasonic for 40 minutes, filter and collect filtrate B; after combining filtrates A and B, pass through C18 and HLB solid phase extraction columns in series, the extraction columns are pre-equilibrated with methanol-water, discard the first 3ml of effluent, and wash with 5ml of water, 5ml Impurities were washed with 20% methanol, and the target compound was finally eluted with a gradient of 90% methanol solution containing 0.5% ammonia water. The eluate was concentrated to near dryness by nitrogen blowing, and the residue was added to a mixed solvent system of petroleum ether-ethanol-ethyl acetate-water with a volume ratio of 12:5:3:2. The upper phase and lower phase (volume ratio 1.2:1) after separation were dissolved and injected into a high-speed countercurrent chromatograph with the following parameters: speed 850 r / min, flow rate 3.0 mL / min, detection wavelength 280 nm, and effluents from 15–25 min, 35–50 min, and 60–75 min were collected. The target fractions were combined and dried under reduced pressure at low temperature. The dried product was re-dissolved in 60% methanol solution, ultrasonically shaken for 5 minutes, filtered through a 0.22 μm filter membrane, and the filtrate was transferred to a brown sample bottle.

[0091] Assay

[0092] Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, measure, and record the chromatogram.

[0093] 3.2 Precision

[0094] Take 1 g of the contents of Zhenlong Xingnao Capsules and prepare the test solution according to the test solution preparation method under "3.1". Inject 6 times according to the chromatographic conditions under "3.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0095] Table 3 Precision relative retention time results

[0096] .

[0097] Table 4 Precision relative peak area results

[0098] .

[0099] The results showed that the relative retention time RSD of each chromatographic peak was less than 2.0%, and the relative peak area RSD was less than 10.0%, indicating good precision. The chromatogram is attached. Figure 1 .

[0100] 3.3 Repeatability

[0101] Take 1 g of the contents of Zhenlong Xingnao Capsules (batch number: 01231110), and make 6 portions. Prepare the test solution according to the test solution preparation method under "3.1". Inject the sample according to the chromatographic conditions under "3.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0102] Table 5 Repeatability relative retention time results

[0103] .

[0104] Table 6 Repeatability relative peak area results

[0105] .

[0106] The results showed that the relative retention time RSD of each chromatographic peak was less than 2.0%, and the relative peak area RSD was less than 10.0%, with good repeatability. The chromatogram is attached. Figure 2 .

[0107] 3.4 Stability

[0108] Take 1 g of the contents of Zhenlong Xingnao Capsules (batch number: 01231110) and prepare the test solution according to the test solution preparation method under "3.1". Inject the sample at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h according to the chromatographic conditions under "3.1". Record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0109] Table 7 Stability relative retention time results

[0110] .

[0111] Table 8 Stability relative peak area results

[0112] .

[0113] The results showed that the relative retention time RSD of each chromatographic peak was less than 2.0%, and the relative peak area RSD was less than 10.0%, indicating that the solution was stable. The chromatogram is attached. Figure 3 .

[0114] In summary, the method demonstrated good precision in the precision test, with the relative retention time RSDs for each chromatographic peak less than 2.0% and the relative peak area RSDs less than 10.0%. In the repeatability test, the relative retention time RSDs for each chromatographic peak less than 2.0% and the relative peak area RSDs less than 10.0%, indicating good repeatability. In the stability test, the relative retention time RSDs for each chromatographic peak less than 2.0% and the relative peak area RSDs less than 10.0%, indicating good solution stability. This method has been methodologically validated and the results are reliable and accurate.

[0115] Example 4 Construction of the Control Characteristic Spectrum of Zhenlong Xingnao Capsule

[0116] Chromatographic conditions and system suitability test

[0117] The column was packed with octadecylsilane bonded silica gel (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); mobile phase A was acetonitrile-tetrahydrofuran (95:5), and mobile phase B was 0.1% citrate buffer (pH 2.8 ± 0.1) containing 0.15% ammonium phosphate and 0.1% sodium dodecylsulfonate. Gradient elution was performed according to the table below. The column temperature was 30°C, and the detection wavelength was 210 nm. The number of theoretical plates, calculated based on the cinnamaldehyde peak, should be no less than 5000.

[0118] .

[0119] Preparation of reference solution

[0120] Take appropriate amount of chebulic acid reference substance, gallic acid reference substance, dehydrodiisoeugenol reference substance, dehydrocostus lactone reference substance, cinnamaldehyde reference substance, and cinnamic acid reference substance, accurately weigh them, add methanol to make a solution containing 120 μg per 1 ml, shake well, and use it as the reference substance solution.

[0121] Preparation of test solution

[0122] Take an appropriate amount of the contents of this product, mix well, take 1g, first add 30ml of n-hexane-ethyl acetate solution with a volume ratio of 3:1, 250W, 40kHz, 25℃ low-temperature ultrasonic for 20 minutes, filter and collect filtrate A; add 30ml of 60% methanol-water solution with 0.1% formic acid to the residue, 300W, 40kHz, 40℃ constant temperature water bath ultrasonic for 40 minutes, filter and collect filtrate B; after combining filtrates A and B, pass through C18 and HLB solid phase extraction columns in series, the extraction columns are pre-equilibrated with methanol-water, discard the first 3ml of effluent, and use 5ml of water, 5ml Impurities were washed with 20% methanol, and the target compound was finally eluted with a gradient of 90% methanol solution containing 0.5% ammonia water. The eluate was concentrated to near dryness by nitrogen blowing, and the residue was added to a mixed solvent system of petroleum ether-ethanol-ethyl acetate-water with a volume ratio of 12:5:3:2. The upper phase and lower phase (volume ratio 1.2:1) after separation were dissolved and injected into a high-speed countercurrent chromatograph with the following parameters: speed 850 r / min, flow rate 3.0 mL / min, detection wavelength 280 nm, and effluents from 15–25 min, 35–50 min, and 60–75 min were collected. The target fractions were combined and dried under reduced pressure at low temperature. The dried product was re-dissolved in 60% methanol solution, ultrasonically shaken for 5 minutes, filtered through a 0.22 μm filter membrane, and the filtrate was transferred to a brown sample bottle.

[0123] Preparation of negative solution

[0124] Take a blank, add 30 ml of 60% methanol, and ultrasonically treat for 30 minutes (power 250 W, frequency 40 kHz), shake well, filter, and take the filtrate to obtain the product.

[0125] Assay

[0126] Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, measure, and record the chromatogram.

[0127] The chromatograms of the test samples of 8 batches of Zhenlong Xingnao Capsules were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" and the chromatographic peaks present in the chromatograms of different batches of Zhenlong Xingnao Capsules were selected as common peaks; the average value calculation method was used to generate a reference characteristic spectrum, and the relative retention time and relative peak area of ​​each common peak were calculated. Figure 4 、 5 .

[0128] Table 9 Relative retention time results of eight batches of Zhenlong Xingnao Capsule samples

[0129] .

[0130] Table 10 Relative peak area results of eight batches of Zhenlong Xingnao Capsule samples

[0131] .

[0132] The chromatograms of the eight batches of Zhenlong Xingnao Capsule samples all showed 11 common peaks, the RSDs of the relative retention times of the 11 common peaks were all less than 2.0%, and the RSDs of the relative peak areas were all less than 10.0%.

[0133] The chromatogram of the test sample shows 11 characteristic peaks, of which Peaks 1, 2, 3, 4, 7, and 9 should correspond to the peaks of the reference substance of gallic acid, chebulic acid, cinnamic acid, cinnamaldehyde, dehydrocostus lactone, and dehydrodiisoeugenol, respectively. The peak corresponding to the peak of the reference substance of cinnamaldehyde is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be within ±10% of the specified values. The specified values ​​are: 0.29 (Peak 1), 0.67 (Peak 2), 0.97 (Peak 3), 1.12 (Peak 5), 1.31 (Peak 6), 1.39 (Peak 7), 1.42 (Peak 8), 1.45 (Peak 9), 1.54 (Peak 10), and 1.61 (Peak 11).

Claims

1. A method for constructing a characteristic spectrum of Zhenlong Xingnao Capsule, characterized in that: The method can simultaneously detect chebulic acid, gallic acid, dehydrodiisoeugenol, dehydrocostus lactone, cinnamaldehyde, and cinnamic acid in Zhenlong Xingnao Capsules; and comprises the following steps: (1) Preparation of reference solution: accurately weigh the reference substances of chebulic acid, gallic acid, dehydrodiisoeugenol, dehydrocostus lactone, cinnamaldehyde, and cinnamic acid, add methanol to prepare a solution containing 120 μg per 1 mL, shake well, and use it as the reference solution; (2) Preparation of test solution: Take 1 g of the contents of Zhenlong Xingnao Capsule, add 30 mL of n-hexane-ethyl acetate solution, and ultrasonicate at 250W, 40kHz, and 25℃ for 20 minutes, filter and collect filtrate A; then add 30 mL of 60% methanol-water solution with 0.1% formic acid to the residue obtained by filtering filtrate A, and ultrasonicate in a constant temperature water bath at 300W, 40kHz, and 40℃ for 40 minutes, filter and collect filtrate B; after combining filtrates A and B, pass through a series of C18 and HL B solid-phase extraction column, the extraction column was pre-equilibrated with methanol-water, the first 3 mL of effluent was discarded, impurities were eluted with 5 mL of water and 5 mL of 20% methanol in sequence, and finally the target compound was eluted with a gradient of 90% methanol solution containing 0.5% ammonia water; the eluate was concentrated to near dryness by nitrogen blowdown, and a mixed solvent system of petroleum ether-ethanol-ethyl acetate-water in a volume ratio of 12:5:3:2 was added to the residue after elution and concentration to near dryness. The upper and lower phases after separation were dissolved and injected into a high-speed countercurrent chromatography, and the effluents at 15–25 min, 35–50 min, and 60–75 min were collected; the target fractions were combined and dried under reduced pressure at low temperature; the dried material was re-dissolved in 60% methanol solution, ultrasonically shaken for 5 minutes, filtered through a 0.22 μm filter membrane, and the filtrate was transferred to a brown sample bottle. (3) Determination: Inject the reference solution and the test solution into a high performance liquid chromatograph under the following chromatographic conditions, inject 10 μL of each sample, and record the chromatogram; Chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler; acetonitrile-tetrahydrofuran (95:5, volume ratio) as mobile phase A; 0.1% citrate buffer containing 0.15% ammonium phosphate and 0.1% sodium dodecyl sulfate as mobile phase B, pH 2.8±0.1, elution according to the following gradient; column temperature, 30°C; flow rate, 1 ml / min; detection with a diode array detector at a wavelength of 210 nm. The gradient conditions are: From 0 to 7 min, the volume ratio of phase A:phase B changed from 2:98 to 3:97; From 7 to 15 min, the volume ratio of phase A:phase B changed from 3:97 to 5:95; From 15 to 25 min, the volume ratio of phase A:phase B changed from 5:95 to 13:87; From 25 to 40 min, the volume ratio of phase A:phase B changed from 13:87 to 14:86; From 40 to 55 min, the volume ratio of phase A:phase B changed from 14:86 to 18:82; From 55 to 70 min, the volume ratio of phase A:phase B changed from 18:82 to 24:76; From 70 to 92 min, the volume ratio of phase A:phase B changed from 24:76 to 52:48; From 92 to 110 min, the volume ratio of phase A:phase B changed from 52:48 to 70:30; From 110 to 125 min, the volume ratio of phase A:phase B changed from 70:30 to 100:0; At 125–126 min, the volume ratio of phase A:phase B changed from 100:0 to 2:98; 126-135 min, the volume ratio of phase A:phase B was 2:98; (4) Generate a control characteristic spectrum: select the chromatographic peaks that exist in the chromatograms of different batches of Zhenlong Xingnao Capsules as common peaks, and use the average value calculation method to generate a control characteristic spectrum of Zhenlong Xingnao Capsules.

2. The method for constructing a characteristic spectrum of Zhenlong Xingnao Capsule according to claim 1, characterized in that: The volume ratio of n-hexane to ethyl acetate in the n-hexane-ethyl acetate solution in step (2) is 3:

1.

3. The method for constructing a characteristic spectrum of Zhenlong Xingnao Capsule according to claim 1, characterized in that: In step (2), the parameters of the high-speed countercurrent chromatography were set as follows: rotation speed 850 r / min, flow rate 3.0 mL / min, and detection wavelength 280 nm.

4. The method for constructing a characteristic spectrum of Zhenlong Xingnao Capsule according to claim 1, characterized in that: In step (2), the volume ratio of the upper phase to the lower phase after the petroleum ether-ethanol-ethyl acetate-water mixed solvent system is separated into layers is 1.2:

1.

5. The method for constructing a characteristic spectrum of Zhenlong Xingnao Capsule according to claim 1, characterized in that: The chromatographic column in step (3) is Platisil ODS C18, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

6. The method for constructing a characteristic spectrum of Zhenlong Xingnao Capsule according to claim 1, characterized in that: There were 11 characteristic peaks in the chromatogram, among which peak 1, peak 2, peak 3, peak 4, peak 7 and peak 9 corresponded to the peaks of gallic acid, chebulic acid, cinnamic acid, cinnamaldehyde, dehydrocostus lactone and dehydrodiisoeugenol reference substance, respectively; the peak corresponding to the peak of cinnamaldehyde reference substance was designated as S peak, and the relative retention times of each characteristic peak and S peak were calculated, and the relative retention times were all within ±10% of the specified values; the specified values ​​were: peak 1 was 0.29, peak 2 was 0.67, peak 3 was 0.97, peak 5 was 1.12, peak 6 was 1.31, peak 7 was 1.39, peak 8 was 1.42, peak 9 was 1.45, peak 10 was 1.54, and peak 11 was 1.61.

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