Method for establishing kidney-tonifying and anti-aging tablet fingerprint spectrum

Abstract

The application belongs to the technical field of traditional Chinese medicine quality control, and particularly relates to a method for establishing a kidney-tonifying and anti-aging tablet fingerprint spectrum. The method for establishing the kidney-tonifying and anti-aging tablet fingerprint spectrum comprises the following steps: mixing the kidney-tonifying and anti-aging tablet with a methanol aqueous solution and performing ultrasonic treatment to obtain a test sample solution; the control samples in the mixed control sample solution comprise ferulic acid, hyperoside, quercitrin, hesperidin, rosmarinic acid and salvianolic acid B; the test sample solution and the mixed control sample solution are respectively subjected to UHPLC detection, the obtained chromatograms are introduced into fingerprint spectrum software for processing, and the kidney-tonifying and anti-aging tablet fingerprint spectrum is obtained. The pre-treatment method is simple, and the characteristic components of the kidney-tonifying and anti-aging tablet are completely reserved; the application adopts the ultra-high performance liquid chromatography method, and has high precision, good reproducibility and good stability; the obtained fingerprint spectrum determines 15 common peaks, can reflect the overall characteristics of the kidney-tonifying and anti-aging tablet, and can effectively monitor the quality of the kidney-tonifying and anti-aging tablet.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality control technology, specifically relating to a method for establishing the fingerprint spectrum of a kidney-tonifying and anti-aging tablet. Background Technology

[0002] The Kidney-Nourishing and Anti-Aging Tablets are composed of Poria cocos, Ligusticum chuanxiong, Citrus reticulata peel, Cinnamomum cassia, Codonopsis pilosula slices, vinegar-processed tortoise shell, Acorus tatarinowii, Salvia miltiorrhiza, salt-processed Eucommia ulmoides, Cuscuta chinensis, Prunella vulgaris, processed Polygonum multiflorum, Sargassum, Laminaria japonica, and Taxillus chinensis. They possess the effects of harmonizing Yin and Yang, strengthening the body and eliminating pathogens, invigorating Qi and promoting lightness of body, replenishing essence and marrow, strengthening the body and brain, and prolonging life. They are commonly used for the prevention and treatment of various diseases in middle-aged and elderly people. Modern pharmacological research shows that the Kidney-Nourishing and Anti-Aging Tablets have effects such as anti-oxidative damage, reducing inflammatory responses, and regulating HO-1 protein levels to maintain cellular redox balance.

[0003] Currently, research on kidney-tonifying and anti-aging tablets mainly focuses on pharmacological activity. Traditional Chinese medicine preparations have complex components, and there is currently a lack of comprehensive quality control methods to reflect the quality of kidney-tonifying and anti-aging tablets. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for establishing a fingerprint spectrum of kidney-tonifying and anti-aging tablets. The fingerprint spectrum established by the method of this invention can comprehensively reflect the quality of kidney-tonifying and anti-aging tablets and can be used to evaluate and control their quality.

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

[0006] The kidney-tonifying and anti-aging tablets were mixed with a methanol-water solution and subjected to ultrasonic treatment to obtain the test solution;

[0007] A mixed reference solution is provided, wherein the references 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 were separately detected by UHPLC. The obtained chromatogram of the test solution was imported into fingerprinting software to identify common peaks. The obtained chromatogram of the reference solution was used for identification to obtain the fingerprint chromatogram of the kidney-tonifying and anti-aging tablets.

[0009] The conditions for UHPLC detection include:

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

[0011] The fingerprint spectrum of the kidney-tonifying and anti-aging tablets includes 15 common peaks with retention times of: 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, and peak 9: 10.37–10.02 min. 39 min, peak 10: 10.56~10.60 min, peak 11: 10.68~10.73 min, peak 12: 11.08~11.12 min, peak 13: 11.82~11.86 min, peak 14: 12.72~12.76 min, peak 15: 13.41~13.45 min; 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-water solution is 40-60%.

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

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

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

[0016] Preferably, phase A is a 0.1% (v / v) aqueous solution of formic acid.

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

[0018] Preferably, the chromatographic column is an ACQUITYUPLC CORTECS C18 column with dimensions of 2.1 mm × 100 mm and 1.6 μm.

[0019] Preferably, the injection volume for UHPLC detection is 1–4 μL.

[0020] This invention also provides the application of the fingerprint spectrum of kidney-tonifying and anti-aging tablets obtained by the above-described technical solution in the quality control of kidney-tonifying and anti-aging tablets.

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

[0022] This 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 with a methanol-water solution and subjecting it to ultrasonic treatment to obtain a test solution; providing a mixed reference solution, wherein the reference standards in the mixed reference solution include ferulic acid, hyperoside, quercetin, hesperidin, rosmarinic acid, and salvianolic acid B; performing UHPLC detection on the test solution and the mixed reference solution respectively, importing the obtained chromatogram of the test solution into fingerprinting software, identifying common peaks, and using the obtained reference chromatogram for identification to obtain the fingerprint spectrum of the kidney-tonifying and anti-aging tablet; the UHPLC detection conditions include: chromatographic column: C18 column; mobile phase: phase A is a 0.05-0.2% (v / v) formic acid aqueous solution, phase B is acetonitrile; gradient elution, elution program: 0-16 min, phase B volume fraction increases from 5% to 38%; flow rate: 0.2-0.4 mL / min; column temperature: 25-35℃; detection wavelength: 310-330 nm.

[0023] The pretreatment method of this invention is simple and retains the characteristic components of the kidney-tonifying and anti-aging tablets completely. This invention employs ultra-high performance liquid chromatography (UHPLC), which offers high precision, good reproducibility, and good stability. The fingerprint spectrum obtained by this invention identifies 15 common peaks, reflecting the overall characteristics of the kidney-tonifying and anti-aging tablets and effectively monitoring their quality. This invention uses UHPLC to detect the fingerprint spectrum of kidney-tonifying and anti-aging tablets, revealing their material basis and enabling quality evaluation and control, thus providing a guarantee for comprehensive and effective quality control of kidney-tonifying and anti-aging tablets. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The fingerprint chromatograms of 10 batches of kidney-tonifying and anti-aging tablets in Example 1;

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

[0027] Figure 3 Chromatograms of different extraction solvents used in Example 3;

[0028] Figure 4 The chromatograms are for different detection wavelengths in Example 4. Detailed Implementation

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

[0030] The kidney-tonifying and anti-aging tablets were mixed with a methanol-water solution and subjected to ultrasonic treatment to obtain the test solution;

[0031] A mixed reference solution is provided, wherein the references 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 were separately detected by UHPLC. The obtained chromatogram of the test solution was imported into fingerprinting software to identify common peaks. The obtained chromatogram of the reference solution was used for identification to obtain the fingerprint chromatogram of the kidney-tonifying and anti-aging tablets.

[0033] The conditions for UHPLC detection include:

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

[0035] The fingerprint spectrum of the kidney-tonifying and anti-aging tablets includes 15 common peaks with retention times of: 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, and peak 9: 10.37–10.02 min. 39 min, peak 10: 10.56~10.60 min, peak 11: 10.68~10.73 min, peak 12: 11.08~11.12 min, peak 13: 11.82~11.86 min, peak 14: 12.72~12.76 min, peak 15: 13.41~13.45 min; 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] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0037] This invention involves mixing kidney-tonifying and anti-aging tablets with a methanol-water solution and then subjecting the mixture to ultrasonic treatment to obtain a test solution.

[0038] In this 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 powder form, and this invention does not impose any special requirements on the pulverization method or particle size of the powder.

[0039] In this invention, the volume fraction of methanol in the methanol-water solution is preferably 40-60%, more preferably 50%. The methanol volume fraction specified in this invention allows for the full extraction of the effective components from the kidney-tonifying and anti-aging tablets, resulting in symmetrical chromatographic peaks and a large number of peaks.

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

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

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

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

[0044] This invention performs UHPLC detection on the test sample solution and the mixed reference solution respectively, imports the obtained chromatogram of the test sample into fingerprinting software, identifies common peaks, and uses the obtained reference chromatogram for identification to obtain the fingerprint spectrum of the kidney-tonifying and anti-aging tablets;

[0045] The conditions for UHPLC detection include:

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

[0047] In this invention, the chromatographic column is preferably an ACQUITYUPLC CORTECS C18 chromatographic column, with a preferred specification of 2.1 mm × 100 mm and 1.6 μm.

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

[0049] In this invention, the flow rate is preferably 0.3 mL / min. The gradient elution procedure described in this invention effectively separates the components in the kidney-tonifying and anti-aging tablets, and the total analysis time is short.

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

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

[0052] In this invention, the detection wavelength is preferably 320 nm. The detection wavelength described in this invention provides a high response to the various components in the kidney-tonifying and anti-aging tablets.

[0053] In this invention, the fingerprinting software is a "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System," preferably version 130723, published by the National Pharmacopoeia Commission in 2012. The process of importing the fingerprinting software includes: setting reference spectra and time window width, and generating chromatograms of multiple batches of kidney-tonifying and anti-aging tablet samples using multi-point calibration; the preferred time window width is 0.1 min.

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

[0055] This invention also provides the application of the fingerprint spectrum of kidney-tonifying and anti-aging tablets obtained by the above-described technical solution in the quality control of kidney-tonifying and anti-aging tablets.

[0056] 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 will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

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

[0058] 1. Instruments: Agilent 1290 ultra-high performance liquid chromatograph (Agilent Technologies, USA); Agilent MassHunter analysis software (Agilent Technologies, USA); Milli-Q IQ 7005 ultrapure water preparation system (Millipore); 5424R high-speed centrifuge (Eppendorf, Germany); AS 60 / 220.R2 0.0001 g balance (Radiwag, Poland); G3KT 18273 vortex mixer (Thermo Fisher Scientific).

[0059] 2. Materials: Methanol and acetonitrile (chromatographic grade) were purchased from Fisher Scientific, USA; formic acid (chromatographic grade) was purchased from ROE, USA; ultrapure water was prepared using a Milli-Q ultrapure water system. Standard reference standards: 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 Desite Biotechnology Co., Ltd., all with a purity greater than 98%. The kidney-tonifying and anti-aging tablets were from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine; drug batch numbers are shown in Table 1.

[0060] Table 1. List of drug batch numbers for kidney-tonifying and anti-aging tablets

[0061] 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% 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] Accurately weigh ferulic acid, hyperoside, quercetin, hesperidin, rosmarinic acid, and salvianolic acid B reference standards, dissolve them in 50% (v / v) methanol aqueous solution to prepare standard reference solutions with a concentration of 50 μg / mL, and store them in a refrigerator at 4℃ for later use.

[0067] 3. Preparation of the test solution

[0068] Accurately weigh 200.0 mg of the kidney-tonifying and anti-aging tablet powder, place it in a 10 mL volumetric flask, add 50% methanol aqueous solution to dilute to the mark, sonicate for 30 min (300 W, 40 kHz), cool, make up the weight loss, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0069] 4. Establishment and similarity evaluation of UHPLC fingerprint of kidney-tonifying and anti-aging tablets

[0070] Ten batches of kidney-tonifying and anti-aging tablets, each weighing 200.0 mg, were prepared according to procedure 3. The samples were then injected and analyzed, yielding chromatograms for all ten batches. The chromatogram data were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2012.130723)," with the reference chromatogram set to S1 and a time window width of 0.1 min. After multi-point correction, chromatograms for all ten batches were generated. (See attached image.) Figure 1 A total of 15 common peaks were identified. Six chromatographic peaks were identified by comparing the chromatogram of the reference solution: peak 6 (ferulic acid), peak 7 (hyperoside), peak 8 (quercetin), peak 9 (hesperidin), peak 11 (rosmarinic acid), and peak 13 (tanshinone B). The chromatogram of the reference solution is shown below. Figure 2 The similarity evaluation results are shown in Table 2. The similarity of the medicinal materials of the 10 batches of kidney-tonifying and anti-aging tablets is greater than 0.977, indicating that the quality of the 10 batches of kidney-tonifying and anti-aging tablets is relatively stable, and the established chromatographic conditions and analytical methods are suitable for the quality evaluation study of kidney-tonifying and anti-aging tablets.

[0071] Table 210 Similarity Evaluation Results of Kidney-Tonifying and Anti-Aging Tablets Samples

[0072] 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 kidney-tonifying and anti-aging tablets (S1), accurately weigh 200.0 mg, prepare the test solution according to the method in Example 1, and inject the sample 6 times consecutively. The relative standard deviation (RSD) of the peak area of ​​each common peak was less than 4.13%, and the RSD of the retention time was less than 0.28%, indicating that the instrument precision was good. The results are shown in Tables 3 and 4.

[0076] Table 3. Peak area precision results of fingerprint spectrum for Kidney-tonifying and Anti-aging Tablets (n=6)

[0077] 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) and precision results of the fingerprint spectrum of the Kidney-Tonifying and Anti-Aging Tablets (n=6)

[0079]

[0080]

[0081] 2. Repeatability experiment

[0082] Take 200.0 mg of the kidney-tonifying and anti-aging tablet sample (S1) and prepare 6 test solutions in parallel according to the method in Example 1. Inject the samples according to the chromatographic conditions in Example 1. The peak area RSD of each common peak was less than 9.93% and the retention time RSD was 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 the kidney-tonifying and anti-aging tablets (n=6)

[0084] 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) and repeatability results of fingerprint chromatograms of Kidney-tonifying and Anti-aging Tablets (n=6)

[0086]

[0087]

[0088] 3. Stability test

[0089] Take 200.0 mg of the kidney-tonifying and anti-aging tablet sample (S1), prepare the test solution according to the method in Example 1, and analyze it at 0, 2, 6, 8, 12 and 24 h according to the chromatographic conditions in Example 1. The peak area RSD of each common peak was less than 9.83% and the retention time RSD was 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 spectrum of the Kidney-Tonifying and Anti-Aging Tablets (n=6)

[0091] 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) and stability results of the fingerprint spectrum of the Kidney-Tonifying and Anti-Aging Tablets (n=6)

[0093] 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] Extraction was performed using different extraction solvents: 30% (v / v) methanol-water solution, 50% (v / v) methanol-water solution, and 70% (v / v) methanol-water solution. The elution program was 0–30 min, 5%–100% B, with other conditions the same as in Example 1.

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

[0097] Example 4

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

[0099] Figure 4 The chromatograms for different detection wavelengths (sample S1) show that at a detection wavelength of 320 nm, the obtained chromatographic peaks have good shapes and stable baselines. Therefore, this invention determines 320 nm as the optimal detection wavelength.

[0100] This invention constructed a UHPLC fingerprint chromatogram for a kidney-tonifying and anti-aging tablet. Ten batches of samples from different origins were analyzed using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software (Version 2012.130723)". The results showed that 15 common peaks were identified in the established fingerprint chromatogram. By comparing with reference standards, six peaks were identified: ferulic acid, hyperoside, quercetin, hesperidin, rosmarinic acid, and salvianolic acid B. The similarity of all ten batches of samples was above 0.977, indicating that the quality of the ten batches of samples was relatively stable and their chemical composition characteristics were basically consistent. The established fingerprint chromatogram provides relatively comprehensive information for the identification of the chemical components of the kidney-tonifying and anti-aging tablet and can serve as a reference for quality evaluation research of this tablet.

[0101] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for establishing the fingerprint spectrum of a kidney-tonifying and anti-aging tablet, characterized in that, Includes the following steps: The kidney-tonifying and anti-aging tablets were mixed with a methanol-water solution and subjected to ultrasonic treatment to obtain the test solution; A mixed reference solution is provided, wherein the references 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 were separately detected by UHPLC. The obtained chromatogram of the test solution was imported into fingerprinting software to identify common peaks. The obtained chromatogram of the reference solution was used for identification to obtain the fingerprint chromatogram of the kidney-tonifying and anti-aging tablets. The conditions for UHPLC detection include: Chromatographic column: C18 column; mobile phase: phase A is 0.05-0.2% (v / v) formic acid aqueous solution, phase B is acetonitrile; gradient elution, elution program: 0-16 min, phase B volume fraction increases from 5% to 38%; flow rate: 0.2-0.4 mL / min; column temperature: 25-35℃; detection wavelength: 310-330 nm; The fingerprint spectrum of the kidney-tonifying and anti-aging tablets includes 15 common peaks with retention times of: 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, and peak 9: 10.37~10.02 min. 39 min, peak 10: 10.56~10.60 min, peak 11: 10.68~10.73 min, peak 12: 11.08~11.12 min, peak 13: 11.82~11.86 min, peak 14: 12.72~12.76 min, peak 15: 13.41~13.45 min; 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; The volume fraction of methanol in the methanol-water solution is 40-60%. The ratio of the kidney-tonifying and anti-aging tablets to the methanol aqueous solution is 0.1~0.3g:10mL; The conditions for ultrasonic treatment include: time of 20-40 min, power of 250-350 W, and frequency of 35-45 kHz; The chromatographic column was an ACQUITY UPLC CORTECS C18 column with dimensions of 2.1 mm × 100 mm and a diameter of 1.6 μm.

2. The method for establishing according to claim 1, characterized in that, The detection wavelength is 320nm.

3. The method for establishing according to claim 1, characterized in that, Phase A is a 0.1% (v / v) aqueous solution of formic acid.

4. The method for establishing according to claim 1 or 3, characterized in that, The flow rate is 0.3 mL / min.

5. The method for establishing according to claim 1, characterized in that, The injection volume for UHPLC detection is 1~4 μL.

6. The application of the fingerprint spectrum of the kidney-tonifying and anti-aging tablets obtained by the method described in any one of claims 1 to 5 in the quality control of the kidney-tonifying and anti-aging tablets.

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