Construction method of fingerprint of qiangshen tablet

Abstract

The application belongs to the field of traditional Chinese medicines, and provides a method for constructing a kidney-strengthening tablet fingerprint spectrum, which comprises: detecting test sample solution and reference material solution by HPLC, wherein the chromatographic conditions are as follows: using octadecylsilane bonded silica gel as a filler; using acetonitrile as mobile phase A and 0.05% formic acid water as mobile phase B, and identifying 13 chromatographic peaks, which are 5-hydroxymethyl furfural, morroniside, motherwort alkaloid hydrochloride, loganin, pinoresinol di-glucoside, paeoniflorin, salvianolic acid B, psoralen, isopsoralen, paeonol, cryptotanshinone, tanshinone I and tanshinone IIA, so that the quality of the kidney-strengthening tablet can be comprehensively and accurately characterized. Meanwhile, the detection method in the application is stable and feasible. The characteristic spectrum established by the application can comprehensively reflect the complex chemical components and relative proportions of the kidney-strengthening tablet, and can effectively characterize the quality of the medicine.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine, and particularly relates to a construction method of a kidney-strengthening tablet fingerprint spectrum. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of generally presenting the context of the application. The information disclosed in this Background section is not to be taken as an acknowledgement or any form of suggestion that this information forms prior art to the application.

[0003] With the application of new technologies and new methods of traditional Chinese medicine and the popularization of large-scale analytical instruments, the fingerprint spectrum technology, which can reflect the integrity and dynamics of the internal chemical components of traditional Chinese medicine, provides a new idea for the quality control of traditional Chinese medicine. The solid research foundation in the early stage provides a strong guarantee for the wide application and gradual improvement of fingerprint spectrum in scientific research practice. It is time to establish a quality standard system in line with the characteristics of traditional Chinese medicine and gradually transform from single index component qualitative and quantitative to overall quality control mode of fingerprint spectrum. The technology of traditional Chinese medicine fingerprint spectrum has been collected in the Chinese Pharmacopoeia 2010 edition and further strengthened in the Chinese Pharmacopoeia 2015 edition and 2020 edition. It expresses the characteristics of traditional Chinese medicine as a complex system as much as possible, more comprehensively reflects the overall identification characteristics of traditional Chinese medicine, enriches and expands the connotation of traditional Chinese medicine identification, and significantly enhances the controllability of the safety and effectiveness of traditional Chinese medicine quality. The technology of traditional Chinese medicine fingerprint spectrum adheres to the combination of inheritance and innovation, is an important embodiment of adhering to the principle of internationalization of drug standard development, perfects the national drug standard with the Chinese Pharmacopoeia as the core, speeds up the pace of internationalization of Chinese drug standards, and promotes the internationalization of traditional Chinese medicine standards.

[0004] The kidney-strengthening tablet (State Food and Drug Administration Approval Number Z21021750, production enterprise: Liaoning Shangyao Haohushi Pharmaceutical (Group) Co., Ltd.) has the effects of tonifying kidney and filling essence, benefiting qi and strengthening yang. It is used for treating edema due to kidney deficiency, waist pain, spermatorrhea, impotence, premature ejaculation, frequent nocturia, chronic nephritis and pyelonephritis that has been treated for a long time. The quality control research of the kidney-strengthening tablet in the Chinese Pharmacopoeia 2020 edition is only the research on thin-layer identification and content determination. At present, there is still a lack of a method that can comprehensively and accurately evaluate the quality of the kidney-strengthening tablet in the industry. SUMMARY

[0005] In order to solve the above problems, the application provides a method for constructing a fingerprint spectrum of Qiangshen tablets, a method for detecting the fingerprint spectrum is established based on a high-performance liquid chromatograph, octadecylsilane bonded silica gel is used as a filler, acetonitrile is used as mobile phase A, and 0.05% formic acid water is used as mobile phase B, the index components in the prescription are identified, and a total of 13 chromatographic peaks are identified, which are 5-hydroxymethyl furfural, moranin, leucocyanidol hydrochloride, loganin, pinoresinol diglucoside, paeoniflorin, salvianolic acid B, psoralen, isopsoralen, paeonol, cryptotanshinone, tanshinone I, and tanshinone IIA. The characteristic spectrum established by the application can comprehensively reflect the complex chemical components and relative proportions of Qiangshen tablets, and effectively represent the quality of the medicine.

[0006] In order to achieve the above-mentioned purpose, the application adopts the following technical solutions:

[0007] In a first aspect, the application provides a method for constructing a fingerprint spectrum of Qiangshen tablets, comprising:

[0008] The Qiangshen tablets are removed from the coating, finely ground, and the powder is taken, added to a methanol aqueous solution, extracted, filtered, and the filtrate is collected to obtain a test sample solution;

[0009] 5-hydroxymethyl furfural, moranin, loganin, pinoresinol diglucoside, paeoniflorin, psoralen, isopsoralen, paeonol, cryptotanshinone, tanshinone I, and tanshinone IIA are taken and methanol is added to prepare a solution; leucocyanidol hydrochloride reference substance is taken and an ethanol aqueous solution is added to prepare a leucocyanidol hydrochloride-containing solution; and salvianolic acid B reference substance is taken and methanol water is added to prepare a salvianolic acid B-containing solution; the above solutions are mixed to obtain a reference solution;

[0010] The test sample solution and the reference solution are detected by HPLC, and the chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as a filler; acetonitrile is used as mobile phase A, and 0.05% formic acid water is used as mobile phase B, and the gradient elution program is as follows:

[0011] 0-5min, A phase: 5%-11%;

[0012] 5-15min, A phase: 11%-12%;

[0013] 15-22min, A phase: 12%-16%;

[0014] 22-25min, A phase: 16%-18%;

[0015] 25-30min, A phase: 18%-20%;

[0016] 30-32min, A phase: 20%-23%;

[0017] 32-40min, A phase: 23%-25%;

[0018] 40-45 min, phase A: 25-34%;

[0019] 45-50 min, phase A: 34-49%;

[0020] 50-52 min, phase A: 49-59%;

[0021] 52-60 min, phase A: 59-79%;

[0022] 60-65 min, phase A: 79-85%;

[0023] 65-70 min, phase A: 85%;

[0024] The detection wavelength is 200-300 nm.

[0025] The application constructs a research method for the fingerprint spectrum of Qiangshen tablets, and provides a whole quality control method for the fingerprint spectrum of Qiangshen tablets, which can effectively control the quality of Qiangshen tablets.

[0026] In some embodiments, the volume concentration of the methanol aqueous solution is 70-75%.

[0027] In some embodiments, the extraction is ultrasonic extraction, and the ultrasonic time is 30-40 min.

[0028] In some embodiments, the specific steps for preparing the reference solution include: accurately weighing psoralen and isopsoralen reference substances, and adding methanol to prepare a solution containing 0.4 mg of psoralen and 0.1 mg of isopsoralen per 1 mL, thereby obtaining the solution.

[0029] In the reference solution, the concentration of psoralen is 0.4-0.5 mg / mL, and the concentration of isopsoralen is 0.1-0.15 mg / mL.

[0030] In some embodiments, the concentration of each component in the reference solution is 0.2-0.5 mg / mL.

[0031] In some embodiments, the concentration of the test solution is 40-50 mg / mL.

[0032] In some embodiments, the flow rate of the HPLC is 1-1.5 mL / min, and the injection amount is 10-15 muL.

[0033] In some embodiments, the column temperature of the HPLC is 30-40 DEG C.

[0034] In some embodiments, 13 chromatographic peaks are identified as 5-hydroxymethylfurfural, monoglucoside, leonurine hydrochloride, loganin, pinoresinol diglucoside, paeoniflorin, tanshinone B, psoralen, isopsoralen, paeonol, cryptotanshinone, tanshinone I, and tanshinone IIA.

[0035] In a second aspect, the present invention provides a fingerprint spectrum of kidney-strengthening tablets constructed by the above-described method.

[0036] A third aspect of the present invention provides the application of the above-mentioned fingerprint spectrum of Qiangshen tablets in the quality control of Qiangshen tablets.

[0037] Beneficial effects of the present invention

[0038] (1) This invention conducted HPLC characteristic chromatographic experiments on Qiangshen tablets, established a quality detection method for the characteristic chromatograms, and performed methodological verification tests such as precision and repeatability on the established method. The results show that the detection method in this invention is stable and feasible. The characteristic chromatograms established by this invention can comprehensively reflect the complex chemical components and their relative proportions of Qiangshen tablets, and can effectively characterize the quality of the drug;

[0039] (2) This invention achieves effective separation of numerous components in Qiangshen tablets, with a large number of characteristic peaks, high peak areas, and good separation between characteristic peaks. A total of 13 chromatographic peaks were identified, namely 5-hydroxymethylfurfural, monoglucoside, leonurine hydrochloride, loganin, pinoresinol diglucoside, paeoniflorin, tanshinone B, psoralen, isopsoralen, paeonol, cryptotanshinone, tanshinone I, and tanshinone IIA, which can comprehensively and accurately characterize the quality of Qiangshen tablets.

[0040] (3) The preparation method of the present invention is simple, practical and easy to promote. Attached Figure Description

[0041] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0042] Figure 1 According to the fingerprint spectrum, the following substances were identified: 1. 5-hydroxymethylfurfural, 2. monoglucoside, 3. leonurine hydrochloride, 4. loganin, 5. pinoresinol diglucoside, 6. paeoniflorin, 7. salvianolic acid B, 8. psoralen, 9. isopsoralen, 10. paeonol, 11. cryptotanshinone, 12. tanshinone I, and 13. tanshinone IIA.

[0043] Figure 2 HPLC 3D image;

[0044] Figure 3 Comparison of HPLC results at different detection wavelengths;

[0045] Figure 4 Comparison of HPLC data with different mobile phases;

[0046] Figure 5 HPLC comparison chart of different extraction methods;

[0047] Figure 6 Comparison of HPLC results for different extraction solvents;

[0048] Figure 7 Comparison of HPLC data for different ultrasonic times;

[0049] Figure 8 Comparison of HPLC results with different sample volumes;

[0050] Figure 9 Comparison of HPLC at different column temperatures;

[0051] Figure 10 Comparison of HPLC flow rates at different flow rates;

[0052] Figure 11 Comparison chart of HPLC instruments from different brands;

[0053] Figure 12 chromatograms after matching characteristic HPLC chromatograms of continuous injection;

[0054] Figure 13 HPLC characteristic chromatograms after matching at different injection times;

[0055] Figure 14 The HPLC characteristic chromatograms of different test samples share a common pattern. Detailed Implementation

[0056] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0057] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0058] In the following examples, the instruments used were: an Agilent 1260Ⅱ high-performance liquid chromatograph and an MS105DV electronic analytical balance.

[0059] Reagents: Acetonitrile (chromatographic grade, Sigma-Aldrich Shanghai Trading Co., Ltd., batch number: 34851); Formic acid (chromatographic grade, batch number: A2304429).

[0060] Qiang Shen Pian (National Drug Approval Number Z21021750, Manufacturer: Liaoning Shangyao Hao Hushi Pharmaceutical (Group) Co., Ltd.)

[0061] Example 1: Preparation of the reference solution

[0062] Accurately weigh 5-hydroxymethylfurfural, mononoside, loganin, pinoresinol diglucoside, paeoniflorin, psoralen, isopsoralen, paeonol, cryptotanshinone, tanshinone I, and tanshinone II. A Appropriate amounts are added to methanol to prepare a solution containing, per 1 ml: 0.3 mg of 5-hydroxymethylfurfural, 0.3 mg of monoglucoside, 0.3 mg of loganin, 0.1 mg of pinoresinol diglucoside, 0.2 mg of psoralen, 0.4 mg of isopsoralen, 0.1 mg of paeonol, 0.2 mg of cryptotanshinone, 0.2 mg of tanshinone I, and 0.2 mg of tanshinone II. A To prepare a solution containing 0.2 mg of leonurine hydrochloride reference standard, accurately weigh an appropriate amount of leonurine hydrochloride reference standard and add 70% ethanol to prepare a solution containing 0.3 mg of leonurine hydrochloride per ml. To prepare a solution containing 0.5 mg of salvianolic acid B reference standard, accurately weigh an appropriate amount of salvianolic acid B reference standard and add 80% methanol to prepare a solution containing 0.5 mg of salvianolic acid B per ml.

[0063] Example 2: Preparation of the test solution

[0064] Take about 1.0g of Qiangshen tablet powder, grind it into a fine powder, weigh it accurately, add 25ml of 75% methanol solution accurately, weigh it, sonicate for 30 minutes, take it out and let it cool, weigh it again, make up the weight loss with methanol, shake well, filter it, and you will get the product.

[0065] Example 3 Selection of Detection Wavelength

[0066] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A and 0.05% formic acid water was used as mobile phase B, with gradient elution performed according to the table below; flow rate was 1 mL / min; column temperature was 30℃; and detection wavelength was 230 nm.

[0067] The gradient elution procedure is shown in Table 1:

[0068] Table 1 Gradient elution program

[0069]

[0070] Accurately pipette 10 μL of the reference solution and the test solution into the liquid chromatograph, respectively, and measure and record the chromatogram after 70 minutes. Compare with the fingerprint chromatogram as shown below. Figure 1 As shown.

[0071] A photodiode array ultraviolet-visible detector was used to scan the sample at wavelengths of 190–800 nm. Figure 2 , Figure 3As shown, analysis using 3D views and isoabsorption spectroscopy revealed that the chromatographic peaks were within the wavelength range of 200 nm to 300 nm, providing a large amount of information. Analysis was performed at wavelengths of 203 nm, 220 nm, 230 nm, and 270 nm. The results showed that the chromatographic peaks measured at 230 nm were more numerous, evenly distributed, and exhibited better separation; therefore, 230 nm was selected as the detection wavelength.

[0072] Example 4: Investigation of the mobile phase ratio

[0073] The chromatographic conditions were the same as in Example 3, except that two mobile phase systems were selected: an acetonitrile-water gradient system and an acetonitrile-0.05% formic acid-water gradient system. The acetonitrile-0.05% formic acid-water gradient system was found to be optimal, exhibiting good peak resolution and moderate retention times. Figure 4 As shown, the acetonitrile-0.05% formic acid aqueous gradient system was therefore selected as the mobile phase.

[0074] Example 5: Examination of Extraction Methods

[0075] The chromatographic conditions were the same as in Example 3. Three extraction methods were selected for the preparation of the test sample: shaking, ultrasonication, and reflux extraction. The results showed that the effective components extracted by shaking and ultrasonication were similar, and the peak areas were not significantly different. Therefore, ultrasonication was chosen for extraction. Figure 5 As shown.

[0076] Example 6: Investigation of the extraction solvent

[0077] The chromatographic conditions were the same as in Example 3. Six solvents were selected for preparing the test sample: 25% methanol, 50% methanol, 75% methanol, methanol, ethanol, and aqueous solution. The results showed that the 75% methanol solution extracted the most active ingredients and had the largest peak area; therefore, 75% methanol solution was chosen as the extraction solvent. Figure 6 As shown.

[0078] Example 7: Investigation of Ultrasound Time

[0079] The chromatographic conditions were the same as in Example 3. When preparing the test sample, three different ultrasonic times were selected: 15 min, 30 min, and 60 min. The results showed that the peak area of ​​most active components extracted by ultrasonication at 30 min was the largest; therefore, an ultrasonic time of 30 min was chosen. Figure 7 As shown.

[0080] Example 8: Examination of Sampling Quantity

[0081] The chromatographic conditions were the same as in Example 3. When preparing the test sample, two different sample amounts were selected: 0.5 g and 1.0 g. The results showed that 1.0 g resulted in more complete extraction and a more uniform overall distribution of the chromatogram; therefore, 1.0 g was chosen as the sample amount. Figure 8 As shown.

[0082] Example 9: Investigation of column temperature

[0083] The chromatographic conditions were the same as in Example 3. Column temperature had a certain impact on the separation, so the column temperature was adjusted; a column temperature of 30°C was found to be suitable. Figure 9 As shown.

[0084] Example 10: Investigation of Flow Rate

[0085] The chromatographic conditions were the same as in Example 3. The flow rate had a certain impact on the separation; therefore, the flow rate was adjusted, and a flow rate of 1.0 mL / min was deemed suitable. Figure 10 As shown.

[0086] Example 11 Instrumental Examination

[0087] The determination was performed using high performance liquid chromatographs from different brands, and the number of characteristic peaks showed no significant difference.

[0088] Example 12 Precision Test

[0089] A sample of the kidney-strengthening tablets was prepared according to the method for preparing the test solution. The sample was injected six times consecutively, and the fingerprint chromatogram was detected. The similarity calculation results are shown in Table 2 and... Figure 12 The results showed that the similarity of each chromatographic peak met the requirements of fingerprint chromatograms. Time window width: 0.10; reference chromatogram generation method: mean.

[0090] Table 2. Similarity calculation results for consecutive injections

[0091]

[0092]

[0093] Example 13 Stability Test

[0094] Samples of Qiangshen tablets were prepared according to the preparation method of the test solution. Fingerprint chromatograms were detected at 0, 3, 6, 9, 12, and 24 hours. Similarity calculation results are shown in Table 3. Figure 13 The results showed that the similarity of each chromatographic peak met the requirements of fingerprint chromatograms. Time window width: 0.10; reference chromatogram generation method: mean.

[0095] Table 2. Similarity calculation results at different detection times

[0096]

[0097] Example 14 Repeatability Test

[0098] Six test samples were prepared from the Qiangshen tablets according to the preparation method of the test solution. The fingerprint spectra were analyzed, and the similarity calculation results are shown in Table 4. Figure 14The results showed that the relative retention times and peak similarity of each chromatographic peak met the requirements for fingerprint chromatograms. Time window width: 0.10; reference chromatogram generation method: mean.

[0099] Table 4. Calculation results of similarity between different test samples

[0100]

[0101] Therefore, the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the reference sample should show the same chromatographic peaks at the corresponding retention times, and the similarity between the two should not be less than 0.90. This indicates that the quality of the test sample is qualified.

[0102] This invention achieves effective separation of numerous components in Qiang Shen tablets, resulting in a large number of characteristic peaks with high peak areas and good separation between them. A total of 13 chromatographic peaks were identified, namely 5-hydroxymethylfurfural, monosodium glutamate, leonurine hydrochloride, loganin, pinoresinol diglucoside, paeoniflorin, salvianolic acid B, psoralen, isopsoralen, paeonol, cryptotanshinone, tanshinone I, and tanshinone IIA. This allows for comprehensive and accurate characterization of the quality of Qiang Shen tablets. Furthermore, the detection method in this invention is stable and feasible. The characteristic chromatograms established by this invention comprehensively reflect the complex chemical composition and relative proportions of Qiang Shen tablets, effectively characterizing the drug's quality.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of a kidney-strengthening tablet, characterized in that, include: Remove the coating from the kidney-strengthening tablets, grind them into a fine powder, add methanol-water solution to extract, filter, collect the filtrate to obtain the test solution; Prepare a solution by adding methanol to 5-hydroxymethylfurfural, mononoside, loganin, pinoresinol diglucoside, paeoniflorin, psoralen, isopsoralen, paeonol, cryptotanshinone, tanshinone I, and tanshinone IIA; prepare a solution containing leonurine hydrochloride reference standard by adding aqueous ethanol; prepare a solution containing salvianolic acid B reference standard by adding methanol and water; mix the above solutions to obtain the reference solution. The test solution and reference solution were analyzed by HPLC. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel as the stationary phase; acetonitrile as mobile phase A; 0.05% formic acid aqueous solution as mobile phase B; and gradient elution program as follows: 0~5min, Phase A: 5%~11%; 5~15 min, phase A: 11%~12%; 15~22 min, phase A: 12%~16%; 22~25 min, phase A: 16%~18%; 25~30 min, phase A: 18%~20%; 30~32 min, phase A: 20%~23%; 32~40 min, phase A: 23%~25%; 40~45 min, phase A: 25%~34%; 45~50 min, phase A: 34%~49%; 50~52 min, phase A: 49%~59%; 52~60 min, phase A: 59%~79%; 60~65 min, phase A: 79%~85%; 65~70 min, phase A: 85%; The detection wavelength is 200m~300nm.

2. The method for constructing the fingerprint spectrum of the kidney-strengthening tablets as described in claim 1, characterized in that, The volume concentration of the methanol aqueous solution is 70%~75%.

3. The method for constructing the fingerprint spectrum of the kidney-strengthening tablets as described in claim 1, characterized in that, The extraction was performed by ultrasonic extraction, with an ultrasonic time of 30 min to 40 min.

4. The method for constructing the fingerprint spectrum of the kidney-strengthening tablets as described in claim 1, characterized in that, In the reference solution, the concentration of each component is 0.2~0.5 mg / mL.

5. The method for constructing the fingerprint spectrum of the kidney-strengthening tablets as described in claim 1, characterized in that, The concentration of the test solution is 40~50 mg / mL.

6. The method for constructing the fingerprint spectrum of the kidney-strengthening tablets as described in claim 1, characterized in that, The HPLC flow rate is 1~1.5 mL / min, and the injection volume is 10~15 μL.

7. The method for constructing the fingerprint spectrum of the kidney-strengthening tablets as described in claim 1, characterized in that, The column temperature of the HPLC is 30–40 °C.

8. The method for constructing the fingerprint spectrum of the kidney-strengthening tablets as described in claim 1, characterized in that, Thirteen chromatographic peaks were identified as 5-hydroxymethylfurfural, monoglucoside, leonurine hydrochloride, loganin, pinoresinol diglucoside, paeoniflorin, tanshinone B, psoralen, isopsoralen, paeonol, cryptotanshinone, tanshinone I, and tanshinone IIA.

9. The application of the fingerprint spectrum of Qiangshen tablets constructed by the method of any one of claims 1-8 in the quality control of Qiangshen tablets.

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