Establishment method of fingerprint spectrum of folium artemisiae argyi and radix aconiti carmichaeli uterus warming pill and fingerprint spectrum thereof

Abstract

The invention provides a method for establishing a fingerprint spectrum of a Yifu uterus-warming pill and the fingerprint spectrum thereof, and the method comprises the following steps: preparation of a test solution: taking Yifu uterus-warming pill powder, adding a methanol solution, weighing, carrying out ultrasonic extraction, cooling, weighing again, complementing the lost weight with the methanol solution, uniformly shaking, filtering with a microporous filter membrane, and taking a subsequent filtrate to obtain the test solution; obtaining a test solution; preparation of reference substance solutions: respectively taking ferulic acid and paeoniflorin reference substances, and adding a methanol solution to dissolve the ferulic acid and paeoniflorin reference substances to obtain the reference substance solutions; 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 software to obtain the fingerprint of the Yifu uterus-warming pill. The fingerprint spectrum of the moxa-moxibustion uterus-warming pill has 14 fingerprint peaks, the types of chemical components contained in the moxa-moxibustion uterus-warming pill can be comprehensively reflected, and the quality of the moxa-moxibustion uterus-warming pill can be comprehensively monitored.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a method for establishing a fingerprint spectrum of Aifu Nuangong Pills and a fingerprint spectrum thereof. Background Art

[0002] Aifu Nuangong Pills are a Chinese patent medicine made from Artemisia argyi (charcoal), Cyperus rotundus, Evodia rutaecarpa (processed), Cinnamon bark, Angelica sinensis, Chuanxiong, White Peony Root (fried with wine), Rehmannia glutinosa, Astragalus membranaceus (fried with honey), and Dipsacus asper. They are dark brown to black honey pills with a slight smell and a sweet taste followed by bitterness and spiciness. Aifu Nuangong Pills are warming agents that have the effects of regulating qi and replenishing blood, warming the uterus and regulating menstruation. They are used for menstrual disorders and dysmenorrhea caused by blood deficiency, qi stagnation, and coldness in the lower jiao. Symptoms include delayed menstruation, scanty menstruation, blood clots, lower abdominal pain, lower abdominal cold pain during menstruation and preference for heat, and soreness in the waist and knees.

[0003] The content test under the item "Ai Fu Nuangong Pills" in the 2020 edition of the Chinese Pharmacopoeia is to determine the content of paeoniflorin in white peony root. The measured chemical components are single, and it is difficult to effectively control the quality of Ai Fu Nuangong Pills. At present, the quality control of Ai Fu Nuangong Pills mainly uses high performance liquid chromatography to determine the content of one or several components, which cannot fully reflect the overall quality of the preparation. Traditional Chinese medicine and its preparations are complex multi-component systems. Therefore, the evaluation of their quality cannot simply determine the content of the main active ingredients, but should adopt a detection method that can provide rich component information. 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. It is mainly used to evaluate the authenticity, excellence and stability of the quality 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 Ai Fu Nuan Gong Pills and its fingerprint spectrum in view of the deficiencies of the above-mentioned prior art, which can more comprehensively reflect the types of chemical components contained in Ai Fu Nuan Gong Pills (powder) and better control the quality of Ai Fu Nuan Gong Pills.

[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 Aifu Nuangong Pills comprises the following steps:

[0007] S1. Preparation of test solution

[0008] Take the powder of Aifu Nuangong Pills, accurately add methanol solution, weigh it, then perform ultrasonic or reflux extraction, let it cool, weigh it again, make up the lost weight with methanol solution, shake well, filter with a microporous filter membrane, take the filtrate, and get the test solution;

[0009] S2. Preparation of reference solution

[0010] Take ferulic acid and paeoniflorin reference substances respectively, add methanol solution 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 Ai Fu Nuangong Pills.

[0013] Preferably, the mass fraction of the methanol solution in S1 and S2 is 50%-100%.

[0014] Preferably, the ratio of the Ai Fu Nuan Gong Wan powder to the methanol solution in S1 is (1.0-3.0 g): (25-50 mL).

[0015] Preferably, the power of the ultrasonic extraction in S1 is 200-300 W, the frequency is 50-100 kHz, and the time is 40-60 min.

[0016] Preferably, the concentrations of ferulic acid and paeoniflorin in the reference solution in S2 are 0.16 mg / mL and 0.50 mg / 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 in S3 are: the chromatographic column is Waters Symmetry C 18 , 250mm×4.6mm, 5μm; mobile phase: A-0.1% phosphoric acid water, B-acetonitrile; flow rate: 1.0mL / min; column temperature: 30℃; detection wavelength: 230nm; injection volume: 10μL; gradient elution program: 0~5min, 90%A; 5~35min, 90%-80%A; 35~52min, 80%-70%A; 52~67min, 70%-40%A; 67~82min, 40%A.

[0019] The present invention provides a fingerprint spectrum of Aifu Nuangong Pills established by the above method, wherein there are 14 common fingerprint peaks in the fingerprint spectrum of Aifu Nuangong Pills, and the retention time of each fingerprint peak is respectively:

[0020] Peak 1: 11.873min;

[0021] Peak 2: 19.163min;

[0022] Peak 3: paeoniflorin, 22.395min;

[0023] Peak 4: ferulic acid, 28.405 min;

[0024] Peak 5: 38.808min;

[0025] Peak 6: 41.401min;

[0026] Peak 7: 42.674min;

[0027] Peak 8: 64.522min;

[0028] Peak 9: 65.386min;

[0029] Peak 10: 67.597min;

[0030] Peak 11: 68.482min;

[0031] Peak 12: 69.33min;

[0032] Peak 13: 72.52min;

[0033] Peak 14: 76.853min.

[0034] The present invention has the following significant technical effects:

[0035] 1. The present invention provides a method for establishing a fingerprint spectrum of Aifu Nuangong Pills and its fingerprint spectrum. The test sample processing method is simple and can completely retain its effective ingredients. The chromatographic conditions are accurately controlled by high performance liquid chromatography. The established fingerprint spectrum of Aifu Nuangong Pills has 14 common fingerprint peaks, which can perform a more comprehensive quality evaluation of Aifu Nuangong Pills.

[0036] 2. The present invention establishes the fingerprint spectrum of Ai Fu Nuan Gong Pills, which can effectively characterize the quality of Ai Fu Nuan Gong Pills, is conducive to comprehensive monitoring of the quality of Ai Fu Nuan Gong Pills, makes up for the shortcomings of existing quality control technology, and makes the quality control of Ai Fu Nuan Gong Pills 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 Aifu Nuangong Pills in Example 1 of the present invention;

[0041] Figure 4 It is the matching chromatogram of 10 batches of Ai Fu Nuangong Pills test products in Example 1 of the present invention;

[0042] Figure 5 It is the HPLC spectrum of Aifu Nuangong Pills at a wavelength of 230nm in Example 2 of the present invention;

[0043] Figure 6 It is the HPLC spectrum of Aifu Nuangong Pills at a wavelength of 321 nm in Example 2 of the present invention;

[0044] Figure 7 is the HPLC spectrum under the conditions of mobile phase gradient elution program 1 in Example 2 of the present invention;

[0045] Figure 8 is the HPLC spectrum under the conditions of mobile phase gradient elution program 2 in Example 2 of the present invention;

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

[0047] Fig.10 It is the HPLC spectrum of the test sample under reflux extraction in Example 2 of the present invention. DETAILED DESCRIPTION

[0048] Example 1

[0049] This embodiment is a method for establishing a fingerprint spectrum of Aifu Nuangong Pills, which comprises the following steps:

[0050] S1. Preparation of test solution

[0051] Take 3.0g of Ai Fu Nuangong Pill powder, accurately weigh it, put it in a stoppered conical flask, accurately add 50mL of 50% methanol solution by mass, stopper it, weigh it, ultrasonically treat it (300W, 50kHz) for 40min, let it cool, weigh it again, make up the lost weight with 50% methanol solution by mass, shake it well, filter it with a 0.45μm microporous filter membrane, take the filtrate to obtain the test solution.

[0052] S2. Preparation of reference solution

[0053] Take appropriate amount of ferulic acid reference substance and paeoniflorin reference substance, weigh accurately, add 50% methanol solution by mass to make solutions with concentrations of 0.16 mg / mL and 0.50 mg / mL, respectively, to obtain reference substance solutions.

[0054] S3. High performance liquid chromatography detection

[0055] Chromatographic conditions: Waters Symmetry C 18The chromatographic column (250 mm × 4.6 mm, 5 μm) was used with 0.1% phosphoric acid water as mobile phase A and acetonitrile as mobile phase B. The flow rate was 1.0 mL / min, the column temperature was 30°C, the detection wavelength was 230 nm, the injection volume was 10 μL, and the gradient elution program was shown in Table 1:

[0056] Table 1 Mobile phase gradient elution program

[0057] Time (min) A-0.1% phosphoric acid water (%) B-Acetonitrile (%) 90 10 5 90 10 35 80 20 52 70 30 67 40 60 82 40 60

[0058] 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 two peaks from left to right are paeoniflorin and ferulic acid) and the HPLC spectrum of the test solution ( Figure 2 ).

[0059] Take Ai Fu Nuangong Pills (powder) (10 batches, each batch is processed in parallel with 2 test sample solutions), analyze according to the fingerprint method established above, import the test results into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", and generate a control fingerprint after data matching, which can be used as the fingerprint of Ai Fu Nuangong Pills (see Figure 3 ). The chromatograms of 10 batches of Aifu Nuangong Pills were evaluated for similarity, and the matching chromatograms are shown in Figure 4 ,The similarity results are shown in Table 2. The results show that the similarity of the 10 batches of Aifu Nuangong Pills tested samples is between 0.976 and 1.000.

[0060] Table 2 Similarity of 10 batches of Aifu Nuangong Pills tested

[0061]

[0062]

[0063]

[0064] Paeoniflorin was selected as a 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 10 batches of samples, chromatographic peaks with good stability, strong absorption and obvious characteristics were selected as common peaks. After matching, 14 common fingerprint peaks were calibrated ( Figure 3 ), where peak 3 is the paeoniflorin peak and peak 4 is the ferulic acid peak. The relative retention time and peak area of ​​each fingerprint peak were calculated. The results are shown in Table 3.

[0065] Table 3 Relative retention time and relative peak area of ​​each fingerprint peak

[0066]

[0067] Example 2

[0068] This embodiment is a selection of different experimental conditions.

[0069] 1. Different detection wavelengths

[0070] The fingerprints of HPLC at wavelengths of 230 nm and 321 nm were analyzed. Figure 5 and Figure 6 It can be seen from the spectrum that for the same test solution, at a detection wavelength of 230nm, there are more chromatographic peaks in the spectrum, the peak area is larger, and the peak separation is better. Therefore, the detection wavelength is set to 230nm in the experiment.

[0071] 2. Different mobile phase conditions

[0072] The fingerprints of the samples under different mobile phase conditions in Table 1, Table 4 and Table 5 were compared. Figure 3 , Figure 7 and Figure 8 ,It can be seen from the spectrum: the chromatogram under the mobile phase conditions in Table 1 has good peak separation and can meet the requirements of the fingerprint spectrum, so the mobile phase conditions in Table 1 were finally selected.

[0073] Table 4 Mobile phase gradient elution program 1

[0074] Time (min) A-0.1% phosphoric acid water (%) B-Acetonitrile (%) 90 10 5 90 10 35 80 20 52 70 30 67 50 50 82 40 60

[0075] Table 5 Mobile phase gradient elution program 2

[0076]

[0077]

[0078] 3. Different test sample processing methods

[0079] The fingerprints of the test solution prepared by ultrasonic and reflux extraction methods were analyzed. Fig. 9 and Fig.10 It can be seen from the spectrum that the chromatogram of the test solution prepared by ultrasonic treatment method has more chromatographic peaks, larger peak area and better peak separation. Therefore, the experiment selected ultrasonic treatment as the preparation method of the test solution.

[0080] Aiming at the problem that the existing technology for the quality control method of Aifu Nuangong Pills only determines one or several components by high performance liquid chromatography, which cannot fully reflect the overall quality of the preparation, the present invention adopts high performance liquid chromatography to establish a fingerprint spectrum of Aifu Nuangong Pills, which has 14 common fingerprint peaks and can effectively characterize Aifu Nuangong Pills, which is conducive to more comprehensive quality evaluation and monitoring of Aifu Nuangong Pills, making up for the shortcomings of the existing quality control technology and making the quality control of Aifu Nuangong Pills more complete and scientific.

[0081] 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 Aifu Nuangong Pills, characterized in that: The following steps are involved: S1. Preparation of test solution Take the powder of Aifu Nuangong Pills, accurately add methanol solution, weigh it, then perform ultrasonic or reflux extraction, let it cool, weigh it again, make up the lost weight with methanol solution, shake well, filter with a microporous filter membrane, take the filtrate, and get the test solution; S2. Preparation of reference solution Take ferulic acid and paeoniflorin reference substances respectively, add methanol solution 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 Ai Fu Nuangong Pills.

2. The method according to claim 1, characterized in that The mass fraction of the methanol solution in S1 and S2 is 50%-100%.

3. The method according to claim 1, characterized in that The ratio of the Ai Fu Nuan Gong Wan powder to the methanol solution in S1 is (1.0-3.0 g): (25-50 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 50-100 kHz, and a time of 40-60 min.

5. The method according to claim 1, characterized in that The concentrations of ferulic acid and paeoniflorin in the reference solution described in S2 were 0.16 mg / mL and 0.50 mg / 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: the chromatographic column is Waters Symmetry C 18 , 250mm×4.6mm, 5μm; mobile phase: A-0.1% phosphoric acid water, B-acetonitrile; flow rate: 1.0mL / min; column temperature: 30℃; detection wavelength: 230nm; injection volume: 10μL; gradient elution program: 0~5min, 90%A; 5~35min, 90%-80%A; 35~52min, 80%-70%A; 52~67min, 70%-40%A; 67~82min, 40%A.

8. A fingerprint spectrum of Aifu Nuangong Pills established by the method according to any one of claims 1 to 7, characterized in that: There are 14 common fingerprint peaks in the fingerprint spectrum of Aifu Nuangong Pills, and the retention times of each fingerprint peak are: Peak 1: 11.873min; Peak 2: 19.163min; Peak 3: paeoniflorin, 22.395min; Peak 4: ferulic acid, 28.405 min; Peak 5: 38.808min; Peak 6: 41.401min; Peak 7: 42.674min; Peak 8: 64.522min; Peak 9: 65.386min; Peak 10: 67.597min; Peak 11: 68.482min; Peak 12: 69.33min; Peak 13: 72.52min; Peak 14: 76.853min.

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