Kidney-tonifying anti-aging tablet fingerprint spectrum establishment method

Abstract

The invention belongs to the technical field of quality control of traditional Chinese medicines, and particularly relates to a method for establishing a fingerprint spectrum of kidney-tonifying and anti-aging tablets. The establishment method of the fingerprint spectrum of the kidney-tonifying anti-aging tablet comprises the following steps: mixing the kidney-tonifying anti-aging tablet and a methanol aqueous solution, and carrying out ultrasonic treatment to obtain a test solution; reference substances in the mixed reference substance solution comprise ferulic acid, hyperoside, quercitrin, hesperidin, rosmarinic acid and salvianolic acid B; and respectively carrying out UHPLC detection on the test solution and the mixed reference substance solution, and introducing obtained chromatograms into fingerprint spectrum software for processing to obtain the fingerprint spectrum of the kidney-tonifying and anti-aging tablets. The pretreatment method is simple, and the characteristic components of the kidney-tonifying and anti-aging tablet are completely reserved; the ultra-high performance liquid chromatography is adopted, so that the precision is high, the reproducibility is good, and the stability is good; according to the fingerprint spectrum obtained by the invention, 15 common peaks are determined, so that the overall characteristics of the kidney-tonifying and anti-aging tablet can be reflected, and the quality of the kidney-tonifying and anti-aging tablet can be effectively monitored.

Description

Technical Field

[0001] The invention belongs to the technical field of quality control of traditional Chinese medicines, and particularly relates to a method for establishing a fingerprint spectrum of a kidney-tonifying and anti-aging tablet. Background Art

[0002] Kidney-tonifying and anti-aging tablets are composed of Poria cocos, Chuanxiong, dried orange peel, cinnamon bark, Codonopsis pilosula, vinegar tortoise shell, Acorus calamus, Salvia miltiorrhiza, Eucommia ulmoides, Cuscuta australis, Prunella vulgaris, processed Polygonum multiflorum, seaweed, kelp and Morus alba. They have the effects of harmonizing yin and yang, strengthening the body and eliminating evil, replenishing qi and lightening the body, replenishing essence and marrow, strengthening the body and brain, and prolonging life. They are often used for the prevention and treatment of various diseases in the middle-aged and elderly. Modern pharmacological studies have shown that kidney-tonifying and anti-aging tablets have the effects of anti-oxidative damage, reducing inflammatory reactions, and regulating HO-1 protein levels to maintain cellular redox balance.

[0003] At present, the research on Bushen Kangsui Tablets mainly focuses on pharmacological activity. The ingredients of Chinese medicine preparations are complex, and there is currently a lack of control methods that can fully reflect the quality of Bushen Kangsui Tablets. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method for establishing a fingerprint spectrum of Bushen Kangsui Tablets. The fingerprint spectrum established by the establishment method of the present invention can fully reflect the quality of Bushen Kangsui Tablets and can be used to evaluate and control the quality of Bushen Kangsui Tablets.

[0005] The present invention provides a method for establishing a fingerprint spectrum of a kidney-tonifying and anti-aging tablet, comprising the following steps:

[0006] The Bushenkangsui tablets and the methanol aqueous solution are mixed and ultrasonically treated to obtain a test solution;

[0007] Providing a mixed reference solution, wherein the reference substances in the mixed reference solution include ferulic acid, hyperoside, quercetin, hesperidin, rosmarinic acid and salvianolic acid B;

[0008] The test solution and the mixed reference solution are respectively subjected to UHPLC detection, the obtained test sample chromatogram is imported into the fingerprint software, the common peaks are marked, and the obtained reference solution chromatogram is used for identification to obtain the fingerprint of the Bushen Kangsui Tablets;

[0009] The conditions of the UHPLC detection include:

[0010] Chromatographic column: C18 column; mobile phase: phase A is 0.05-0.2% formic acid aqueous solution, phase B is acetonitrile; gradient elution, elution program: 0-16min, phase B volume fraction increased from 5% to 38%; flow rate: 0.2-0.4mL / min; column temperature: 25-35℃; detection wavelength: 310-330nm;

[0011] The fingerprint spectrum of the kidney-tonifying and anti-aging tablets includes 15 common peaks, and the retention times are as follows: peak 1: 1.48-1.49 min, peak 2: 3.32-3.34 min, peak 3: 3.81-3.85 min, peak 4: 4.86-4.89 min, peak 5: 5.27-5.39 min, peak 6: 7.99-8.04 min, peak 7: 8.75-8.79 min, peak 8: 9.98-10.02 min, peak 9: 10.37-10. 39min, peak 10: 10.56~10.60min, peak 11: 10.68~10.73min, peak 12: 11.08~11.12min, peak 13: 11.82~11.86min, peak 14: 12.72~12.76min, peak 15: 13.41~13.45min; among them, peak 6 is ferulic acid, peak 7 is hyperoside, peak 8 is quercetin, peak 9 is hesperidin, peak 11 is rosmarinic acid, and peak 13 is salvianolic acid B.

[0012] Preferably, the volume fraction of methanol in the methanol aqueous solution is 40-60%.

[0013] Preferably, the dosage ratio of the kidney-tonifying and anti-aging tablets to the methanol aqueous solution is 0.1-0.3 g:10 mL.

[0014] Preferably, the conditions of the ultrasonic treatment include: time of 20 to 40 min, power of 250 to 350 W, and frequency of 35 to 45 kHz.

[0015] Preferably, the detection wavelength is 320 nm.

[0016] Preferably, the phase A is a formic acid aqueous solution with a volume fraction of 0.1%.

[0017] Preferably, the flow rate is 0.3 mL / min.

[0018] Preferably, the chromatographic column is an ACQUITY UPLC CORTECS C18 chromatographic column with a specification of 2.1 mm×100 mm, 1.6 μm.

[0019] Preferably, the injection volume of the UHPLC detection is 1 to 4 μL.

[0020] The present invention also provides the application of the fingerprint spectrum of Bushen Kangsui Tablets obtained by the establishment method described in the above technical scheme in the quality control of Bushen Kangsui Tablets.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The invention provides a method for establishing a fingerprint spectrum of a kidney-tonifying and anti-aging tablet, comprising the following steps: mixing the kidney-tonifying and anti-aging tablet and a methanol aqueous solution for ultrasonic treatment to obtain a test solution; providing a mixed reference solution, wherein the reference substances in the mixed reference solution include ferulic acid, hyperoside, quercetin, hesperidin, rosmarinic acid and salvianolic acid B; respectively performing UHPLC detection on the test solution and the mixed reference solution, importing the obtained test sample chromatogram into a fingerprint spectrum software, marking common peaks, and using the obtained reference substance chromatogram for identification to obtain a fingerprint spectrum of the kidney-tonifying and anti-aging tablet; the conditions of the UHPLC detection include: chromatographic column: C18 column; mobile phase: phase A is a formic acid aqueous solution with a volume fraction of 0.05-0.2%, and phase B is acetonitrile; gradient elution, elution program: 0-16min, and the volume fraction of phase B is increased from 5% to 38%; flow rate: 0.2-0.4mL / min; column temperature: 25-35℃; detection wavelength: 310-330nm.

[0023] The pretreatment method of the present invention is simple, and the characteristic components of the Bushen Kangsui Tablets are kept intact; the present invention adopts ultra-high performance liquid chromatography (UHPLC), which has high precision, good reproducibility and good stability; the fingerprint spectrum obtained by the present invention determines 15 common peaks in total, which can reflect the overall characteristics of the Bushen Kangsui Tablets and can effectively monitor the quality of the Bushen Kangsui Tablets. The present invention adopts UHPLC to detect the Bushen Kangsui Tablets to obtain the fingerprint spectrum, which reveals the material basis of the Bushen Kangsui Tablets, can be used to evaluate and control the quality, and provides a guarantee for the comprehensive and effective control of the quality of the Bushen Kangsui Tablets. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The fingerprints of 10 batches of Bushen Kangsui Tablets tested in Example 1;

[0026] Figure 2 is the chromatogram of the reference substance solution in Example 1;

[0027] Figure 3 3 is the chromatogram of different extraction solvents in Example 3;

[0028] Figure 4 It is the chromatogram of different detection wavelengths in Example 4. DETAILED DESCRIPTION

[0029] The present invention provides a method for establishing a fingerprint spectrum of a kidney-tonifying and anti-aging tablet, comprising the following steps:

[0030] The Bushenkangsui tablets and the methanol aqueous solution are mixed and ultrasonically treated to obtain a test solution;

[0031] Providing a mixed reference solution, wherein the reference substances in the mixed reference solution include ferulic acid, hyperoside, quercetin, hesperidin, rosmarinic acid and salvianolic acid B;

[0032] The test solution and the mixed reference solution are respectively subjected to UHPLC detection, the obtained test sample chromatogram is imported into the fingerprint software, the common peaks are marked, and the obtained reference solution chromatogram is used for identification to obtain the fingerprint of the Bushen Kangsui Tablets;

[0033] The conditions of the UHPLC detection include:

[0034] Chromatographic column: C18 column; mobile phase: phase A is 0.05-0.2% formic acid aqueous solution, phase B is acetonitrile; gradient elution, elution program: 0-16min, phase B volume fraction increased from 5% to 38%; flow rate: 0.2-0.4mL / min; column temperature: 25-35℃; detection wavelength: 310-330nm;

[0035] The fingerprint spectrum of the kidney-tonifying and anti-aging tablets includes 15 common peaks, and the retention times are as follows: peak 1: 1.48-1.49 min, peak 2: 3.32-3.34 min, peak 3: 3.81-3.85 min, peak 4: 4.86-4.89 min, peak 5: 5.27-5.39 min, peak 6: 7.99-8.04 min, peak 7: 8.75-8.79 min, peak 8: 9.98-10.02 min, peak 9: 10.37-10. 39min, peak 10: 10.56~10.60min, peak 11: 10.68~10.73min, peak 12: 11.08~11.12min, peak 13: 11.82~11.86min, peak 14: 12.72~12.76min, peak 15: 13.41~13.45min; among them, peak 6 is ferulic acid, peak 7 is hyperoside, peak 8 is quercetin, peak 9 is hesperidin, peak 11 is rosmarinic acid, and peak 13 is salvianolic acid B.

[0036] In the present invention, unless otherwise specified, the materials and equipment used are commercially available products in the art.

[0037] The invention mixes the kidney-tonifying and anti-aging tablets with a methanol aqueous solution and performs ultrasonic treatment to obtain a test solution.

[0038] In the present invention, the kidney-tonifying and anti-aging tablets are sourced from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine. The kidney-tonifying and anti-aging tablets are preferably used in the form of powders, and the present invention has no special requirements on the pulverization method and particle size of the powders.

[0039] In the present invention, the volume fraction of methanol in the methanol aqueous solution is preferably 40-60%, more preferably 50%. The volume fraction of methanol in the present invention can fully extract the effective ingredients in the Bushen Kangsui Tablets, and the obtained chromatographic peaks are symmetrical and have many peaks.

[0040] In the present invention, the dosage ratio of the kidney-tonifying and anti-aging tablets to the methanol aqueous solution is preferably 0.1-0.3 g:10 mL, more preferably 0.2 g:10 mL.

[0041] In the present invention, the ultrasonic treatment time is preferably 20 to 40 min, more preferably 30 min, the power is preferably 250 to 350 W, more preferably 300 W, and the frequency is preferably 35 to 45 kHz, more preferably 40 kHz.

[0042] The invention provides a mixed reference substance solution, wherein the reference substances in the mixed reference substance solution include ferulic acid, hyperoside, quercetin, hesperidin, rosmarinic acid and salvianolic acid B.

[0043] In the present invention, the concentrations of ferulic acid, hyperoside, quercetin, hesperidin, rosmarinic acid and salvianolic acid B in the mixed reference solution are preferably independently 40 to 60 μg / mL, more preferably 50 μg / mL.

[0044] The present invention performs UHPLC detection on the test solution and the mixed reference solution respectively, imports the obtained test sample chromatogram into fingerprint software, identifies the common peaks, and uses the obtained reference sample chromatogram for identification to obtain the fingerprint of the Bushen Kangsui Tablets;

[0045] The conditions of the UHPLC detection include:

[0046] Chromatographic column: C18 column; mobile phase: phase A is a formic acid aqueous solution with a volume fraction of 0.05-0.2%, and phase B is acetonitrile; gradient elution, elution program: 0-16 min, the volume fraction of phase B increases from 5% to 38%; flow rate: 0.2-0.4 mL / min; column temperature: 25-35°C; detection wavelength: 310-330 nm.

[0047] In the present invention, the chromatographic column is preferably an ACQUITY UPLC CORTECS C18 chromatographic column, and the specification is preferably 2.1 mm×100 mm, 1.6 μm.

[0048] In the present invention, the phase A is preferably a formic acid aqueous solution with a volume fraction of 0.1%.

[0049] In the present invention, the flow rate is preferably 0.3 mL / min. The gradient elution procedure of the present invention can effectively separate the components in the Bushen Kangsui Tablets, and the total analysis time is short.

[0050] In the present invention, the column temperature is preferably 30°C.

[0051] In the present invention, the injection volume of the UHPLC detection is preferably 1 to 4 μL, more preferably 2 μL.

[0052] In the present invention, the detection wavelength is preferably 320 nm. At the detection wavelength of the present invention, the response of each component in the Kidney-Nourishing and Anti-Aging Tablet is high.

[0053] In the present invention, the fingerprint software is a "Chinese medicine chromatographic fingerprint similarity evaluation system", and the "Chinese medicine chromatographic fingerprint similarity evaluation system" is preferably version 130723, State Pharmacopoeia Committee, 2012. After the fingerprint software is imported, the following steps are included: setting a reference spectrum and a time window width, and using multi-point correction to generate multiple batches of Bushen Kangsui Tablet sample chromatograms; the time window width is preferably 0.1 min.

[0054] The fingerprint spectrum established by the present invention has high similarity. The method for establishing the fingerprint spectrum of the kidney-tonifying and anti-aging tablets provided by the present invention has good precision, good repeatability, good stability, and the similarity of 10 batches of samples is all above 0.977.

[0055] The present invention also provides the application of the fingerprint spectrum of Bushen Kangsui Tablets obtained by the establishment method described in the above technical scheme in the quality control of Bushen Kangsui Tablets.

[0056] In order to further illustrate the present invention, the method for establishing the fingerprint spectrum of the kidney-tonifying and anti-aging tablets provided by the present invention is described in detail below in combination with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present invention.

[0057] In the embodiments or comparative examples of the present invention, the instruments and materials used include:

[0058] 1. Instruments: Agilent 1290 ultra-high performance liquid chromatograph (Agilent, USA); Agilent MassHunter analysis software (Agilent, USA); Milli-Q IQ 7005 ultrapure water preparation instrument (Millipore); 5424R high-speed centrifuge (Eppendorf, Germany); AS 60 / 220.R2 1 / 100000 balance (Radwag, Poland); G3KT 18273 vortex mixer (Thermo Fisher Scientific).

[0059] 2. Materials: Methanol and acetonitrile (chromatographic grade) were purchased from Fisher, USA; formic acid (chromatographic grade) was purchased from ROE, USA; ultrapure water was prepared by Milli-Q ultrapure water instrument. Standard reference substances: ferulic acid (batch number: DSTDF008101), hyperoside (batch number: DSTDJ002304), quercetin (batch number: DST191012-006), hesperidin (batch number: DSTDC003803), rosmarinic acid (batch number: DST231123-027), and salvianolic acid B (batch number: DSTDD000902) were purchased from Chengdu Desit Biotechnology Co., Ltd., with a purity of more than 98%. Kidney-tonifying and anti-aging tablets were obtained from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine. The drug batch number is shown in Table 1:

[0060] Table 1 Drug batch number list of Bushen Kangsui Tablets

[0061] serial number batch number serial number batch number S1 KD0681201 S6 KH0681210 S2 KD0681202 S7 KH0681211 S3 KH0681207 S8 KH0681212 S4 KH0681208 S9 KI0681213 S5 KH0681209 S10 KI0681214

[0062] Example 1

[0063] 1. Chromatographic conditions

[0064] Chromatographic column: ACQUITY UPLC CORTECS C18 column (2.1 mm×100 mm, 1.6 μm); mobile phase: 0.1% by volume formic acid aqueous solution (A)-acetonitrile (B); gradient elution, elution program: 0-16 min, 5%-38% B; flow rate: 0.3 mL / min; column temperature: 30°C; injection volume: 2 μL; detection wavelength: 320 nm.

[0065] 2. Preparation of reference solution

[0066] Take ferulic acid, hyperoside, quercetin, hesperidin, rosmarinic acid and salvianolic acid B reference substances, accurately weigh them, add 50% by volume methanol aqueous solution to dissolve them, and prepare standard reference substance solutions with a concentration of 50 μg / mL. Store them in a refrigerator at 4°C for later use.

[0067] 3. Preparation of test solution

[0068] Accurately weigh 200.0 mg of Bushen Kangsui Tablets powder and place it in a 10 mL volumetric flask. Add 50% by volume methanol aqueous solution to the scale line. Ultrasonicate for 30 min (300 W, 40 kHz). Cool, make up for weight loss, shake well, and filter through a 0.22 μm microporous filter to obtain the test solution.

[0069] 4. Establishment and similarity evaluation of UHPLC fingerprint of Bushenkangshuai tablets

[0070] Weigh 200.0 mg of each of 10 batches of Bushen Kangsui Tablets samples, prepare the test solution according to 3, and inject and analyze to obtain 10 batches of sample chromatograms. Import the chromatogram data into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.130723 Version)", set the reference spectrum to S1, the time window width to 0.1 min, and generate 10 batches of sample chromatograms after multi-point correction. Figure 1 A total of 15 common peaks were calibrated. By comparing the chromatogram of the reference solution, 6 chromatographic peaks were identified, namely, peak 6 ferulic acid, peak 7 hyperoside, peak 8 quercetin, peak 9 hesperidin, peak 11 rosmarinic acid and peak 13 salvianolic acid B. The chromatogram of the reference solution is shown in Figure 2 The similarity evaluation results are shown in Table 2. The similarities of the medicinal materials of the 10 batches of Bushen Kangsui Tablets are all greater than 0.977, indicating that the quality of the 10 batches of Bushen Kangsui Tablets samples is relatively stable, and the established chromatographic conditions and analytical methods are suitable for the quality evaluation study of Bushen Kangsui Tablets.

[0071] Table 2 Similarity evaluation results of 10 batches of Bushen Kangsui Tablets samples

[0072] serial number Similarity serial number Similarity S1 0.977 S6 0.991 S2 0.979 S7 0.998 S3 0.997 S8 0.996 S4 0.995 S9 0.999 S5 0.997 S10 0.998

[0073] Example 2 Methodological Investigation

[0074] 1. Precision test

[0075] Take a sample of Bushen Kangsui Tablets (S1), accurately weigh 200.0 mg, prepare the test solution according to the method of Example 1, and inject it continuously 6 times. The relative standard deviation (RSD) of the peak area of ​​each common peak is calculated to be less than 4.13%, and the RSD of the retention time is less than 0.28%, indicating that the instrument has good precision. The results are shown in Tables 3 and 4.

[0076] Table 3 Peak area precision results of the fingerprint of Bushen Kangsui Tablets (n=6)

[0077] Peak 1 2 3 4 5 6 average value RSD(%) 1 18.97 18.69 18.11 17.96 18.15 17.94 18.30 2.32 2 19.41 19.70 19.46 19.52 20.91 20.04 19.84 2.89 3 33.92 33.98 34.00 33.96 35.62 34.44 34.32 1.94 4 22.76 23.14 23.16 23.22 24.89 23.80 23.50 3.23 5 16.57 16.82 16.96 16.91 18.32 17.48 17.18 3.70 6 39.72 39.80 39.87 40.08 42.73 40.99 40.53 2.89 7 148.91 149.03 148.92 145.25 148.62 148.72 148.24 0.99 8 8.59 8.65 8.71 8.71 8.95 8.81 8.74 1.46 9 30.14 29.88 29.93 29.85 31.76 30.22 30.29 2.42 10 25.96 27.33 27.59 27.49 29.46 28.15 27.66 4.13 11 95.15 96.07 96.33 96.21 98.55 98.41 96.79 1.42 12 21.78 21.72 21.72 21.60 23.25 21.64 21.95 2.92 13 160.42 160.96 161.09 160.88 163.54 164.10 161.83 0.97 14 58.48 58.59 58.39 57.94 60.71 57.94 58.67 1.77 15 34.70 37.29 37.19 36.94 36.88 36.77 36.63 2.63

[0078] Table 4 Retention time (min) precision results of the fingerprint of Bushen Kangsui Tablets (n=6)

[0079]

[0080]

[0081] 2. Repeatability experiment

[0082] Take the Bushen Kangsui Tablets sample (S1), accurately weigh 200.0 mg, prepare 6 test solutions in parallel according to the method of Example 1, and inject according to the chromatographic conditions of Example 1. The peak area RSD of each common peak is calculated to be less than 9.93%, and the retention time RSD is less than 0.31%, indicating that the method has good repeatability. The results are shown in Tables 5 and 6.

[0083] Table 5 Peak area repeatability results of the fingerprint spectrum of Bushenkangsui Tablets (n=6)

[0084] Peak 1 2 3 4 5 6 average value RSD(%) 1 17.78 18.73 17.43 18.79 17.85 18.01 18.10 3.01 2 20.51 20.48 20.98 22.79 22.67 21.73 21.53 4.81 3 35.87 34.52 35.51 35.72 35.90 38.43 35.99 3.61 4 25.06 28.56 24.56 25.20 25.06 26.82 25.87 5.90 5 18.54 17.85 18.50 18.23 19.61 19.11 18.64 3.37 6 43.32 42.23 44.06 43.28 47.04 45.93 44.31 4.11 7 156.61 151.86 165.64 159.67 173.63 173.57 163.49 5.51 8 9.38 10.52 9.23 9.35 9.29 9.84 9.60 5.22 9 30.07 34.56 31.35 34.75 35.27 39.80 34.30 9.93 10 28.59 28.10 30.99 30.28 32.99 32.98 30.65 6.82 11 103.15 100.12 105.70 103.68 112.41 110.90 105.99 4.49 12 23.06 26.43 22.54 24.31 23.32 25.41 24.18 6.20 13 173.02 196.36 179.84 174.86 193.74 190.71 184.75 5.47 14 58.27 50.08 52.52 55.67 52.55 54.23 53.89 5.29 15 36.67 36.91 37.41 38.54 37.23 41.20 37.99 4.47

[0085] Table 6 Retention time (min) repeatability results of the fingerprint spectrum of Bushenkangsui tablets (n=6)

[0086]

[0087]

[0088] 3. Stability test

[0089] Take the Bushen Kangsui Tablets sample (S1), accurately weigh 200.0 mg, prepare the test solution according to the method of Example 1, and analyze the samples at 0, 2, 6, 8, 12 and 24 h according to the chromatographic conditions of Example 1. The peak area RSD of each common peak is calculated to be less than 9.83%, and the RSD of the retention time is less than 0.79%, indicating that the sample has good stability within 24 h. The results are shown in Tables 7 and 8.

[0090] Table 7 Peak area stability results of the fingerprint of Bushen Kangsui Tablets (n=6)

[0091] Peak 0h 2h 6h 8h 12h 24h average value RSD(%) 1 18.97 17.96 17.83 18.79 18.22 18.21 18.33 2.47 2 19.41 19.52 19.85 22.79 23.13 20.32 20.84 8.05 3 33.92 33.96 34.29 35.72 37.13 35.43 35.07 3.60 4 22.76 23.22 23.71 25.20 27.68 24.44 24.50 7.27 5 16.57 16.91 17.48 18.23 16.47 17.96 17.27 4.26 6 39.72 40.08 40.72 43.28 46.98 42.52 42.22 6.44 7 148.91 145.25 147.79 159.67 176.26 155.92 155.63 7.36 8 8.59 8.71 8.92 9.35 10.17 8.99 9.12 6.33 9 30.14 29.85 28.35 34.75 34.92 30.79 31.47 8.68 10 25.96 27.49 27.00 30.28 32.87 30.59 29.03 9.07 11 95.15 96.21 97.26 103.68 114.51 110.53 102.89 7.90 12 21.78 21.60 21.84 24.31 25.97 23.34 23.14 7.56 13 160.42 160.88 163.00 174.86 192.76 172.20 170.69 7.26 14 58.48 57.94 56.85 55.67 57.54 44.23 55.12 9.83 15 34.70 36.94 36.43 38.54 37.07 36.68 36.73 3.36

[0092] Table 8 Retention time (min) stability results of the fingerprint spectrum of Bushen Kangsui Tablets (n=6)

[0093] Peak 0h 2h 6h 8h 12h 24h average value RSD(%) 1 1.49 1.49 1.48 1.49 1.49 1.49 1.49 0.18 2 3.34 3.33 3.31 3.33 3.32 3.33 3.33 0.38 3 3.85 3.83 3.81 3.82 3.83 3.84 3.83 0.33 4 4.89 4.87 4.86 4.87 4.88 4.89 4.88 0.23 5 5.39 5.36 5.34 5.36 5.27 5.37 5.35 0.79 6 8.04 8.01 7.99 8.01 8.01 8.02 8.01 0.18 7 8.79 8.77 8.75 8.77 8.77 8.77 8.77 0.16 8 10.02 10.00 9.99 9.99 10.00 10.01 10.00 0.12 9 10.39 10.38 10.37 10.38 10.39 10.39 10.38 0.08 10 10.60 10.58 10.56 10.58 10.58 10.59 10.58 0.12 11 10.73 10.71 10.68 10.70 10.71 10.72 10.71 0.14 12 11.12 11.10 11.08 11.09 11.10 11.11 11.10 0.12 13 11.86 11.84 11.82 11.84 11.84 11.84 11.84 0.10 14 12.76 12.74 12.73 12.75 12.74 12.75 12.74 0.08 15 13.45 13.43 13.41 13.42 13.43 13.43 13.43 0.09

[0094] Example 3

[0095] Different extraction solvents were used for extraction, namely 30% methanol aqueous solution, 50% methanol aqueous solution and 70% methanol aqueous solution. The elution procedure was 0-30 min, 5%-100% B, and the other conditions were the same as in Example 1.

[0096] Figure 3The chromatograms of different extraction solvents (S1 sample) show that when the methanol aqueous solution with a volume fraction of 50% is used as the extraction solvent, the sample chromatographic peak has a good peak shape, a large number of peaks, and a high peak response. Therefore, the present invention determines that the methanol aqueous solution with a volume fraction of 50% is the optimal extraction solvent.

[0097] Example 4

[0098] Different detection wavelengths were used for detection, namely 210 nm, 254 nm, 280 nm, 320 nm and 360 nm, and the elution procedure was 0 to 30 min, 5% to 100% B, and the other conditions were the same as in Example 1.

[0099] Figure 4 The chromatograms of different detection wavelengths (S1 sample) show that at a detection wavelength of 320 nm, the obtained chromatographic peak has a good peak shape and a stable baseline. Therefore, the present invention determines that 320 nm is the optimal detection wavelength.

[0100] The present invention constructs the UHPLC fingerprint of Bushen Kangsui Tablets, and analyzes 10 batches of samples from different origins by using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software (2012.130723 Version)". The results show that 15 common peaks are calibrated in the established fingerprint, and 6 of them are identified by comparing with the reference substance, namely ferulic acid, hyperoside, quercetin, hesperidin, rosmarinic acid, and salvianolic acid B. The similarities of the 10 batches of samples are all above 0.977, indicating that the quality of the 10 batches of samples is relatively stable and the chemical composition characteristics are basically the same. The established fingerprint provides relatively comprehensive information for the identification of the chemical components of Bushen Kangsui Tablets, and can provide a reference for the quality evaluation research of Bushen Kangsui Tablets.

[0101] Although the above-mentioned embodiments have made a detailed description of the present invention, they are only some embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for establishing a fingerprint spectrum of a kidney-tonifying and anti-aging tablet, characterized in that: The following steps are involved: The Bushenkangsui tablets and the methanol aqueous solution are mixed and ultrasonically treated to obtain a test solution; Providing a mixed reference solution, wherein the reference substances in the mixed reference solution include ferulic acid, hyperoside, quercetin, hesperidin, rosmarinic acid and salvianolic acid B; The test solution and the mixed reference solution are respectively subjected to UHPLC detection, the obtained test sample chromatogram is imported into the fingerprint software, the common peaks are marked, and the obtained reference solution chromatogram is used for identification to obtain the fingerprint of the Bushen Kangsui Tablets; The conditions of the UHPLC detection include: Chromatographic column: C18 column; Mobile phase: Phase A is a 0.05-0.2% formic acid aqueous solution, and phase B is acetonitrile; gradient elution, elution program: 0-16min, the volume fraction of phase B increases from 5% to 38%; flow rate: 0.2-0.4mL / min; column temperature: 25-35℃; detection wavelength: 310-330nm; The fingerprint spectrum of the kidney-tonifying and anti-aging tablets includes 15 common peaks, and the retention times are as follows: peak 1: 1.48-1.49 min, peak 2: 3.32-3.34 min, peak 3: 3.81-3.85 min, peak 4: 4.86-4.89 min, peak 5: 5.27-5.39 min, peak 6: 7.99-8.04 min, peak 7: 8.75-8.79 min, peak 8: 9.98-10.02 min, peak 9: 10.37-10. 39min, peak 10: 10.56~10.60min, peak 11: 10.68~10.73min, peak 12: 11.08~11.12min, peak 13: 11.82~11.86min, peak 14: 12.72~12.76min, peak 15: 13.41~13.45min; among them, peak 6 is ferulic acid, peak 7 is hyperoside, peak 8 is quercetin, peak 9 is hesperidin, peak 11 is rosmarinic acid, and peak 13 is salvianolic acid B.

2. The establishment method according to claim 1, characterized in that: The volume fraction of methanol in the methanol aqueous solution is 40-60%.

3. The establishment method according to claim 1 or 2, characterized in that: The dosage ratio of the kidney-tonifying and anti-aging tablets to the methanol aqueous solution is 0.1-0.3 g:10 mL.

4. The establishment method according to claim 3, characterized in that: The conditions of the ultrasonic treatment include: time of 20 to 40 minutes, power of 250 to 350 W, and frequency of 35 to 45 kHz.

5. The establishment method according to claim 1, characterized in that: The detection wavelength is 320 nm.

6. The establishment method according to claim 1, characterized in that: The phase A is a formic acid aqueous solution with a volume fraction of 0.1%.

7. The establishment method according to claim 1 or 6, characterized in that: The flow rate was 0.3 mL / min.

8. The establishment method according to claim 1, characterized in that: The chromatographic column is an ACQUITY UPLC CORTECS C18 chromatographic column with a specification of 2.1 mm×100 mm, 1.6 μm.

9. The establishment method according to claim 1, characterized in that: The injection volume of the UHPLC detection is 1 to 4 μL.

10. Application of the fingerprint spectrum of Bushen Kangsui Tablets obtained by the establishment method according to any one of claims 1 to 9 in the quality control of Bushen Kangsui Tablets.

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