Establishment method of fingerprint spectrum of spicy rhinitis capsule and fingerprint spectrum of spicy rhinitis capsule

Abstract

The invention provides a method for establishing a fingerprint spectrum of a Xinjiang rhinitis capsule and the fingerprint spectrum thereof, which comprises the following steps: preparation of a test solution: taking Xinjiang rhinitis capsule powder, adding a methanol solution, weighing, carrying out ultrasonic extraction, cooling, weighing again, complementing the lost mass with the methanol solution, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the test solution; preparing a reference substance solution, namely respectively taking naringin, neohesperidin, baicalin and magnolin reference substances, and adding methanol for dissolving to obtain the reference substance solution; and performing high performance liquid chromatography detection: precisely sucking the test solution and the reference substance solution, respectively injecting the test solution and the reference substance solution into a high performance liquid chromatograph, measuring and recording a chromatogram, and processing the chromatogram by using fingerprint spectrum software to obtain the fingerprint spectrum of the Xinjiang rhinitis capsule. The fingerprint spectrum of the Xinjiang rhinitis capsule has 15 common fingerprint peaks, chemical components contained in the Xinjiang rhinitis capsule can be comprehensively reflected, and more comprehensive quality evaluation and monitoring on the Xinjiang rhinitis capsule are facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for establishing a fingerprint spectrum of Xinfang rhinitis capsule and a fingerprint spectrum thereof. Background Art

[0002] Xinfang Rhinitis Capsules are capsules made from 15 medicinal materials including magnolia, bupleurum, mint, fructus aurantii (fried), vitex rotundi (fried), angelica dahurica, ligusticum chuanxiong, chrysanthemum, saposhnikovia divaricata, gentian, scutellaria baicalensis, platycodon, schizonepeta spicate, asarum, and buffalo horn concentrated powder. The content is gray-yellow powder with a slightly spicy and fishy taste. It can dispel wind, clear away heat and detoxify, and promote lung function and open the orifices. It is used for chronic rhinitis and sinusitis.

[0003] At present, the quality control of Xinfang Biyan Capsules is mainly carried out by high performance liquid chromatography to determine the content of one or several ingredients, which cannot fully reflect the overall quality of the preparation. Moreover, the content determination method of Xinfang Biyan Capsules is not included in the departmental standard of Xinfang Biyan Capsules, making it difficult to effectively control the quality of Xinfang Biyan Capsules. Traditional Chinese medicine and its preparations are multi-component complex systems. Therefore, their quality cannot be evaluated by simply determining the content of the main active ingredients, but a detection method that can provide rich component information should be used. Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method. It is based on the systematic study of the chemical components of traditional Chinese medicine and is mainly used to evaluate the authenticity, quality and stability of traditional Chinese medicines and traditional Chinese medicine preparations. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for establishing a fingerprint spectrum of Xinfang rhinitis capsules and its fingerprint spectrum in view of the deficiencies of the above-mentioned prior art, which will be able to more comprehensively reflect the types of chemical components contained in Xinfang rhinitis capsules and better control the quality of Xinfang rhinitis capsules.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A method for establishing a fingerprint spectrum of Xinfang Biyan Capsule comprises the following steps:

[0007] S1. Preparation of test solution

[0008] Take the powder of Xinfang Biyan Capsule, accurately add methanol solution, weigh, soak, and then perform ultrasonic extraction. After completion, cool to room temperature, weigh again, and make up the lost mass with methanol solution, shake well, filter with a microporous filter membrane, and take the filtrate to obtain the test solution;

[0009] S2. Preparation of reference solution

[0010] Take naringin, neohesperidin, baicalin and magnolin reference substances respectively, add anhydrous methanol to dissolve, and obtain reference substance solutions;

[0011] S3. High performance liquid chromatography detection

[0012] Accurately pipette the test solution and the reference solution into high performance liquid chromatograph respectively, measure and record the chromatogram, and process the chromatogram with fingerprint software to obtain the fingerprint of Xinfang Biyan Capsule.

[0013] Preferably, the mass fraction of the methanol solution in S1 is 80%.

[0014] Preferably, the ratio of the Xinfang Rhinitis Capsule powder to the methanol solution in S1 is 1.0 g:25 mL.

[0015] Preferably, the power of the ultrasonic extraction in S1 is 200-300 W, the frequency is 80-100 kHz, and the time is 30-50 min.

[0016] Preferably, the concentrations of naringin, neohesperidin, baicalin and magnolin in the reference solution in S2 are 174.2 μg / mL, 96.9 μg / mL, 23.8 μg / mL and 100.1 μg / mL, respectively.

[0017] Preferably, the amount of the test solution in S3 is 10 μL, and the amount of the reference solution is 10 μL.

[0018] Preferably, the chromatographic conditions described in S3 are: octadecylsilane bonded silica gel as filler; mobile phase A-methanol, B-water, flow rate of 1.0 mL / min, column temperature of 30°C, detection wavelength of 230 nm, injection volume of 10 μL; gradient elution program: 0-18 min, 36% A; 18-20 min, 36%-45% A; 20-30 min, 45% A; 30-33 min, 45%-55% A; 33-65 min, 55% A; 65-67 min, 55%-36% A.

[0019] The present invention also provides a fingerprint spectrum of Xinfang Biyan Capsule established by the above method, wherein there are 15 common fingerprint peaks in the fingerprint spectrum of Xinfang Biyan Capsule, and the retention time of each fingerprint peak is respectively:

[0020] Peak 1: 6.425min;

[0021] Peak 2: 6.996min;

[0022] Peak 3: 12.229min;

[0023] Peak 4: 13.441min;

[0024] Peak 5: 15.919min;

[0025] Peak 6: naringin, 22.834 min;

[0026] Peak 7: 23.76min;

[0027] Peak 8: neohesperidin, 25.051min;

[0028] Peak 9: 37.127min;

[0029] Peak 10: 38.703min;

[0030] Peak 11: baicalin, 42.082min;

[0031] Peak 12: 47.749min;

[0032] Peak 13: magnolin, 48.924 min;

[0033] Peak 14: 50.178min;

[0034] Peak 15: 53.882min.

[0035] The present invention has the following significant technical effects:

[0036] The invention provides a method for establishing a fingerprint spectrum of Xinfang rhinitis capsule and the fingerprint spectrum thereof. The test sample processing method is simple, and the effective ingredients can be completely retained. The chromatographic conditions are accurately controlled by high performance liquid chromatography. The established fingerprint spectrum of Xinfang rhinitis capsule has 15 common fingerprint peaks, which can effectively characterize the Xinfang rhinitis capsule, is conducive to more comprehensive quality evaluation and monitoring of the Xinfang rhinitis capsule, makes up for the shortcomings of the existing quality control technology, and makes the quality control of the Xinfang rhinitis capsule more complete and scientific.

[0037] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the HPLC spectrum of the reference substance solution of Example 1 of the present invention;

[0039] Figure 2 is the HPLC spectrum of the test solution of Example 1 of the present invention;

[0040] Figure 3 It is the fingerprint spectrum of Xinfang Biyan Capsule of Example 1 of the present invention;

[0041] Figure 4 It is the matching chromatogram of 5 batches of Xinfang Biyan Capsule powder test sample of Example 1 of the present invention;

[0042] Figure 5is the HPLC spectrum of Xinfang Biyan Capsule at a wavelength of 205 nm in Example 2 of the present invention;

[0043] Figure 6 is the HPLC spectrum of Xinfang Biyan Capsule at a wavelength of 230 nm in Example 2 of the present invention;

[0044] Figure 7 is the HPLC spectrum of Xinfang Biyan Capsule at a wavelength of 270 nm in Example 2 of the present invention;

[0045] Figure 8 This is the HPLC spectrum under 20% methanol ultrasonic extraction in Example 2 of the present invention;

[0046] Fig. 9 This is the HPLC spectrum under 50% methanol ultrasonic extraction in Example 2 of the present invention;

[0047] Fig.10 This is the HPLC spectrum under 80% methanol ultrasonic extraction in Example 2 of the present invention;

[0048] Fig.11 This is the HPLC spectrum under 100% methanol ultrasonic extraction in Example 2 of the present invention;

[0049] Fig.12 HPLC spectrum under ultrasonic extraction of ethyl acetate in Example 2 of the present invention;

[0050] Fig.13 is the HPLC spectrum under the conditions of mobile phase gradient elution program 1 in Example 2 of the present invention;

[0051] Fig.14 is the HPLC spectrum under the conditions of mobile phase gradient elution program 2 in Example 2 of the present invention;

[0052] Fig.15 It is the HPLC spectrum under the conditions of mobile phase gradient elution program 3 in Example 2 of the present invention. DETAILED DESCRIPTION

[0053] Example 1

[0054] This embodiment is a method for establishing a fingerprint spectrum of Xinfang Biyan Capsule, which comprises the following steps:

[0055] S1. Preparation of test solution

[0056] Take about 1.0g of Xinfang Biyan Capsule powder sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 80% methanol solution by mass, weigh it, soak it for 30 minutes, and then ultrasonically treat it for 30 minutes at a power of 300W and a frequency of 100kHz. Let it cool to room temperature, weigh it again, and make up the lost mass with 80% methanol solution by mass. Shake it well, filter it with a 0.45μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0057] S2. Preparation of reference solution

[0058] Naringin, neohesperidin, baicalin and magnoliaside reference substances were taken respectively, accurately weighed, dissolved in anhydrous methanol, and prepared into mixed solutions with concentrations of 174.2 μg / mL, 96.9 μg / mL, 23.8 μg / mL and 100.1 μg / mL, respectively, to obtain reference substance solutions.

[0059] S3. High performance liquid chromatography detection

[0060] The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as the filler; the mobile phase is A-methanol, B-water, the flow rate is 1.0 mL / min, the column temperature is 30°C, the detection wavelength is 230 nm, and the injection volume is 10 μL; the gradient elution program is shown in Table 1:

[0061] Table 1 Mobile phase gradient elution program

[0062] Time (minutes) A-Methanol (%) B-Water (%) 0 36 64 18 36 64 20 45 55 30 45 55 33 55 45 65 55 45 67 36 64

[0063] Accurately pipette 10 μL of the reference solution and the test solution, respectively, and inject them into the high performance liquid chromatograph, measure, and record the chromatogram to obtain the HPLC spectrum of the reference solution ( Figure 1 , the chromatographic peaks of the four spectra in the figure are: blue: naringin, yellow: neohesperidin, green: baicalin, brown: magnolin) and the HPLC spectrum of the test solution ( Figure 2 ).

[0064] Take Xinfang Biyan Capsule powder (5 batches, each batch is processed in parallel with 2 test sample solutions), analyze it according to the fingerprint method established above, import the test results into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", generate a control fingerprint after data matching, and use it as the fingerprint of Xinfang Biyan Capsule (see Figure 3 ). The chromatograms of 5 batches of Xinfang Biyan Capsule powder samples were evaluated for similarity, and the matching chromatograms are shown in Figure 4 The similarity results are shown in Table 2. The results showed that the similarity of the five batches of Xinfang Biyan Capsule powder samples ranged from 0.976 to 1.000.

[0065] Table 2 Similarity of 5 batches of Xinfang Biyan Capsule powder samples

[0066]

[0067] Magnoliopsin was selected as the reference, and its retention time was taken as 1 to calculate the relative retention time of other fingerprint peaks. The relative retention time of other fingerprint peaks should be relatively fixed. According to the test results of 5 batches of Xinfang rhinitis capsule powder samples, the chromatographic peaks with good stability, strong absorption and obvious characteristics were selected as common peaks. After matching, 15 common fingerprint peaks were calibrated ( Figure 3 ), where peak 6 is naringin, peak 8 is neohesperidin, peak 11 is baicalin, and peak 13 is magnolin. The relative retention time and peak area of ​​each fingerprint peak were calculated. The results are shown in Table 3.

[0068] Table 3 Relative retention time and relative peak area of ​​each fingerprint peak

[0069]

[0070] Example 2

[0071] This embodiment is a selection of different experimental conditions.

[0072] 1. Detection wavelength

[0073] The detection wavelengths were set to 205nm, 230nm and 270nm respectively, and the fingerprints at different detection wavelengths were analyzed experimentally. The results are shown in Figure 5 , 6 and 7. It can be seen from the spectrum that for the same sample solution, at a detection wavelength of 230 nm, the separation of the chromatographic peaks in the spectrum is better, so the detection wavelength is set at 230 nm in the experiment.

[0074] 2. Test solution treatment method

[0075] In the experiment, the sample solution was processed by ultrasonic extraction, and the fingerprints of five different solvents, including 20% ​​methanol, 50% methanol, 80% methanol, 100% methanol (anhydrous methanol) and ethyl acetate, were investigated. The results are shown in Table 1. Figure 8-12 It can be seen from the spectrum that: at a detection wavelength of 230nm, when 80% methanol is used as the ultrasonic extraction solvent, the content of the chromatographic peak of the obtained test solution is relatively high. Through comparative analysis, the experiment finally determined to use the ultrasonic treatment method and use 80% methanol as the ultrasonic extraction solvent to prepare the test solution.

[0076] 3. Selection of mobile phase conditions

[0077] The fingerprints of the samples under different mobile phase conditions in Table 1, Table 4, Table 5 and Table 6 were compared. Figure 3 and Figure 13-15It can be seen from the spectrum that: under the mobile phase elution conditions in Table 4-6 (mobile phase gradient elution program 1-3), the separation of the chromatographic peaks is poor, and some chromatographic peaks are not even separated, which does not meet the requirements of the fingerprint spectrum. Under the mobile phase conditions in Table 1, the separation of the chromatographic peaks is better and can meet the requirements of the fingerprint spectrum, so the mobile phase conditions in Table 1 were finally selected.

[0078] Table 4 Mobile phase gradient elution program 1

[0079] Time (minutes) A-Methanol (%) B-Water (%) 0 36 64 18 36 64 20 56 44 30 56 44 33 72 28 49 72 28 50 36 64 53 36 64

[0080] Table 5 Mobile phase gradient elution program 2

[0081] Time (minutes) A-Methanol (%) B-Water (%) 0 36 64 18 36 64 20 45 55 30 45 55 33 60 40 49 60 40 50 36 64 53 36 64

[0082] Table 6 Mobile phase gradient elution program 3

[0083] Time (minutes) A-Methanol (%) B-Water (%) 0 36 64 18 36 64 20 45 55 30 45 55 33 60 40 59 60 40 60 36 64 63 36 64

[0084] Example 3

[0085] This example is a methodological study on the establishment or detection of the fingerprint of Xinfang Rhinitis Capsules.

[0086] 1. Precision test

[0087] Take one portion of the unsterilized Xinfang Biyan Capsule (powder) test solution and repeat the injection 6 times under the above chromatographic conditions. Taking magnolia oleoresin as the reference peak, calculate the relative retention time and relative peak area ratio of each common peak, and calculate the RSD value. The results show that the relative retention time RSD of the specified 15 common peaks is less than 1%, and the relative peak area RSD is less than 3%. Import the data file into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for similarity evaluation. The results show that the similarity of the fingerprints of the test samples is greater than 0.98, indicating that the instrument has good precision and meets the requirements of fingerprint research technology.

[0088] 2. Repeatability test

[0089] Six unsterilized Xinfang Biyan Capsules (powder) test solutions of the same batch were taken and injected separately under the above chromatographic conditions. Taking magnolia oleoresin as the reference peak, the relative retention time and relative peak area ratio of each common peak were calculated, and the RSD value was calculated. The results showed that the relative retention time RSD of the specified 15 common peaks was less than 1%, and the relative peak area RSD was less than 3%. The data file was imported into the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" for similarity evaluation. The results showed that the similarity of the fingerprints of each test sample was greater than 0.98, indicating good repeatability and meeting the requirements of fingerprint research technology.

[0090] 3. Stability test

[0091] Take the unsterilized Xinfang Biyan Capsule (powder) sample and prepare it into the test solution. Under the above chromatographic conditions, inject the sample at 0, 6, 12, and 24 hours respectively, record the fingerprint, take magnolia oleoresin as the reference peak, calculate the relative retention time and relative peak area ratio of each common peak, and calculate the RSD value. The results show that the relative retention time RSD of the specified 15 common peaks is less than 1%, and the relative peak area RSD is less than 3%. The data file is imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for similarity evaluation. The results show that the similarity of the fingerprints of each test sample is greater than 0.98, indicating that the samples have good stability within 24 hours and meet the requirements of fingerprint research technology.

[0092] In view of the problem that the current existing technology for the quality control method of Xinfang Rhinitis Capsules only uses high performance liquid chromatography to determine one or several ingredients, which cannot fully reflect the overall quality of the preparation, the present invention adopts high performance liquid chromatography to establish a fingerprint spectrum of Xinfang Rhinitis Capsules, which has 15 common fingerprint peaks and can effectively characterize Xinfang Rhinitis Capsules, is conducive to more comprehensive quality evaluation and monitoring of Xinfang Rhinitis Capsules, makes up for the shortcomings of the existing quality control technology, and makes the quality control of Xinfang Rhinitis Capsules more complete and scientific.

[0093] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for establishing a fingerprint spectrum of Xinfang Biyan Capsule, characterized in that: The following steps are involved: S1. Preparation of test solution Take the powder of Xinfang Biyan Capsule, accurately add methanol solution, weigh, soak, and then perform ultrasonic extraction. After completion, cool to room temperature, weigh again, and make up the lost mass with methanol solution, shake well, filter with a microporous filter membrane, and take the filtrate to obtain the test solution; S2. Preparation of reference solution Take naringin, neohesperidin, baicalin and magnolin reference substances respectively, add anhydrous methanol to dissolve, and obtain reference substance solutions; S3. High performance liquid chromatography detection Accurately pipette the test solution and the reference solution into high performance liquid chromatograph respectively, measure and record the chromatogram, and process the chromatogram with fingerprint software to obtain the fingerprint of Xinfang Biyan Capsule.

2. The method according to claim 1, characterized in that The mass fraction of the methanol solution in S1 is 80%.

3. The method according to claim 1, characterized in that The ratio of the Xinfang Rhinitis Capsule powder to the methanol solution in S1 is 1.0 g:25 mL.

4. The method according to claim 1, characterized in that: The ultrasonic extraction in S1 has a power of 200-300 W, a frequency of 80-100 kHz, and a time of 30-50 min.

5. The method according to claim 1, characterized in that The concentrations of naringin, neohesperidin, baicalin and magnolin in the reference solution described in S2 were 174.2 μg / mL, 96.9 μg / mL, 23.8 μg / mL and 100.1 μg / mL, respectively.

6. The method according to claim 1, characterized in that The amount of the test solution described in S3 is 10 μL, and the amount of the reference solution is 10 μL.

7. The method according to claim 1, characterized in that The chromatographic conditions described in S3 are: octadecylsilane bonded silica gel as filler; mobile phase is A-methanol, B-water, flow rate is 1.0mL / min, column temperature is 30°C, detection wavelength is 230nm, injection volume is 10μL; gradient elution program is: 0-18min, 36%A; 18-20min, 36%-45%A; 20-30min, 45%A; 30-33min, 45%-55%A; 33-65min, 55%A; 65-67min, 55%-36%A.

8. A fingerprint spectrum of Xinfang Biyan Capsule established by the method according to any one of claims 1 to 7, characterized in that: There are 15 common fingerprint peaks in the fingerprint spectrum of Xinfang Biyan Capsule, and the retention time of each fingerprint peak is: Peak 1: 6.425min; Peak 2: 6.996min; Peak 3: 12.229min; Peak 4: 13.441min; Peak 5: 15.919min; Peak 6: naringin, 22.834 min; Peak 7: 23.76min; Peak 8: neohesperidin, 25.051min; Peak 9: 37.127min; Peak 10: 38.703min; Peak 11: baicalin, 42.082min; Peak 12: 47.749min; Peak 13: magnolin, 48.924 min; Peak 14: 50.178min; Peak 15: 53.882min.

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