Establishment method of HPLC (High Performance Liquid Chromatography) fingerprint spectrum of qi-reinforcing and blood-nourishing granules

Abstract

The invention belongs to the field of analysis of traditional Chinese medicine preparations, and particularly discloses a method for establishing an HPLC (High Performance Liquid Chromatography) fingerprint spectrum of qi-reinforcing and blood-nourishing granules, which comprises the following steps: preparing reference substance and test substance solutions, determining HPLC chromatographic conditions and establishing an HPLC standard fingerprint spectrum. The invention also discloses an HPLC standard fingerprint spectrum of the qi-reinforcing and blood-nourishing granules obtained by the method, and the fingerprint spectrum has 32 characteristic peaks. The obtained fingerprint spectrum is high in separation degree and good in peak shape, all characteristic peaks are subjected to baseline separation, the quality of the qi-reinforcing and blood-nourishing particles can be evaluated more comprehensively and accurately, and the safety and effectiveness of clinical medication are improved.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine preparation analysis and relates to a method for establishing an HPLC fingerprint of Yiqi Yangxue granules and the fingerprint thereof. Background Art

[0002] Surgery, radiotherapy, and chemotherapy are all effective means for cancer patients to combat cancer and eliminate pathogenic factors. While the goal of treatment is to eliminate cancerous lesions and cancer cells, this can compromise the body's ability to fight cancer and the function of its internal organs. The depletion of the tumor, the intervention of surgery, radiotherapy, and chemotherapy, and especially the toxic side effects of chemotherapy, often lead to imbalances in yin and yang, deficiencies in qi and blood, and impaired organ function in cancer patients.

[0003] Guided by the principle of "making the past serve the present, applying the foreign to the Chinese, and introducing the new through the old," the integration of Traditional Chinese Medicine and Western Medicine inherits and promotes traditional Chinese medicine, conducting extensive and in-depth research on the etiology, pathogenesis, and treatment principles of malignant tumors in Traditional Chinese Medicine. Guided by a holistic approach and based on syndrome differentiation and treatment, Traditional Chinese Medicine employs a method of strengthening the body's vital energy and consolidating the underlying foundation to treat these patients. By strengthening the body's vital energy and regulating yin and yang, Traditional Chinese Medicine can enhance the patient's immune function, mitigate surgical damage and the toxic side effects of chemotherapy and radiotherapy, and simultaneously intervene in cancer recurrence and metastasis, thereby alleviating patient suffering, improving quality of life, prolonging survival, and reducing mortality.

[0004] Yiqi Yangxue Granules are made from seven raw medicinal ingredients: Astragalus, Codonopsis, Ligustrum lucidum fruit, Chinese Angelica, Epimedium, Cornus officinalis, and Salvia miltiorrhiza. This formula is derived from the "Yiqi Yangxue Recipe," a representative recipe of Academician Wu Xianzhong's "Qi-Nourishing and Blood-Nourishing Method." It boasts the effects of nourishing Qi and blood, strengthening the body's defenses, and is used for cancer patients undergoing chemotherapy and radiotherapy to alleviate reactions, address weak constitutions, and provide postoperative conditioning. This recipe utilizes Astragalus and Codonopsis as the main ingredients to strengthen the spleen, replenish Qi, and strengthen the body's foundation; Ligustrum lucidum fruit and Chinese Angelica as the auxiliary ingredients to nourish Yin and blood. This combination of main and auxiliary ingredients strengthens Qi and blood, balancing Yin and Yang. Cornus officinalis and Epimedium are added to tonify the kidneys and strengthen Yang, replenishing Essence and nourishing the marrow, and thus benefiting the source of Qi and blood. Salvia miltiorrhiza serves as the guiding ingredient, promoting blood circulation, removing blood stasis, and promoting menstruation, providing nourishment without being greasy. This recipe is simple and effective, addressing Qi, blood, Yin and Yang, and the spleen and kidneys, making it particularly beneficial for patients experiencing post-illness physical weakness.

[0005] To ensure stable and controllable quality during the preparation process of Yiqi Yangxue Granules, the present invention investigated the quality standards for the preparation. Traditional Chinese medicine fingerprinting is a multi-index quality control method that comprehensively reflects the types and contents of the active ingredients in a preparation, thus enabling comprehensive, macroscopic, and integrated quality control of traditional Chinese medicine preparations.

[0006] The present invention discloses a method for establishing an HPLC fingerprint of Yiqi Yangxue Granules, and an HPLC standard fingerprint of Yiqi Yangxue Granules produced using the method. When similarity is used for evaluation, the standard fingerprint can be used to comprehensively evaluate and control the quality of Yiqi Yangxue Granules, thereby ensuring the stability of product quality and the safety and effectiveness of clinical medication.

[0007] At present, there are no reports on the use of fingerprints to control the quality of Yiqi Yangxue Granules. Therefore, there is an urgent need to provide a method for establishing the fingerprint of Yiqi Yangxue Granules to ensure product quality, provide a reliable basis for the authenticity identification and intrinsic quality control of Yiqi Yangxue Granules, and provide a reliable analytical method and technical means to ensure the stability of product quality and the safety and effectiveness of clinical use. Summary of the Invention

[0008] The purpose of the present invention is to address the deficiencies in the existing quality control methods for Yiqi Yangxue granules and provide a method for establishing an HPLC fingerprint of Yiqi Yangxue granules. According to the method, Yiqi Yangxue granules are prepared into a test solution, and subjected to HPLC separation and detection to obtain an HPLC standard fingerprint of Yiqi Yangxue granules, thereby achieving all-round and multi-faceted detection, improving its quality control system, and further providing a reliable basis for the authenticity identification and intrinsic quality of Yiqi Yangxue granules.

[0009] The present invention provides a method for establishing an HPLC fingerprint of Yiqi Yangxue Granules, and the specific technical scheme is as follows:

[0010] 1) Preparation of reference and test solutions

[0011] Weigh appropriate amounts of morroniside, loganin, ferulic acid, calycosin, privet glycoside, codonopsis pilosula, salvianolic acid B, icariin, and baohuoside I, and dissolve them in methanol to prepare the reference solution.

[0012] Accurately weigh an appropriate amount of the test sample, add methanol, ultrasonically treat, cool, make up the weight of methanol lost, centrifuge, filter, and take the filtrate to obtain the test sample solution.

[0013] 2) Determination of HPLC chromatographic conditions

[0014] The chromatographic column is Perkin Elmer Brownlee Validated AQ C 18 The column was used; mobile phase A was acetonitrile, and mobile phase B was formic acid solution; gradient elution was used; the detection wavelength was multi-wavelength; the flow rate was 1.0 mL / min; the column temperature was 35°C; and the injection volume was 10 μL.

[0015] 3) Fingerprint preparation

[0016] The reference solution and the test solution were analyzed and compared according to the chromatographic conditions in step 2) to obtain the standard fingerprint of Yiqi Yangxue Granule consisting of the common characteristic peaks of the samples.

[0017] Preferably, the Qi-Yin Yang Xue granules are Qi-Yin Yang Xue granules.

[0018] Preferably, the test solution in step 1) is prepared as follows: accurately measure 5 g of Yiqi Yangxue Granules, place them in a 50 mL conical flask, accurately add 20 mL of methanol and weigh them, ultrasonically treat, cool, make up the weight of methanol lost, centrifuge, filter, and take the filtrate to obtain the test solution.

[0019] Preferably, the specification of the chromatographic column in step 2) is 4.6×250 mm, and the particle size is 5 μm.

[0020] Preferably, in step 2), the mobile phase B is a 0.1% formic acid solution.

[0021] Preferably, the gradient elution conditions in step 2) are:

[0022] Preferably, the detection wavelength conditions in step 2) are:

[0023] Another aspect of the present invention is to provide a fingerprint of Yiqi Yangxue Granules. Ten batches of prepared Yiqi Yangxue Granule test solutions were analyzed and compared, separated and detected by HPLC, and analyzed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software recommended by the State Pharmacopoeia Commission to obtain a standard fingerprint of Yiqi Yangxue Granules composed of common characteristic peaks of the samples.

[0024] By matching the fingerprints of 10 batches of Yiqi Yangxue Granules, 32 common characteristic peaks were obtained. Taking peak 19 (teruncle glycoside) as the reference peak, peaks 6, 8, 11, 13, 16, 17, 18, 19, 20, 22, 23, 25, 27, 29, and 32 were identified, a total of 15 main characteristic peaks of Yiqi Yangxue Granules, and their relative retention times were 0.20, 0.25, 0.37, 0.46, 0.62, 0.95, 1.00, 1.10, 1.37, 1.52, 1.63, 1.74, 1.81, 1.85, and 2.07, respectively.

[0025] After positioning the retention time of the reference substances and analyzing the chromatographic peaks, peak 13 was identified as morroniside, peak 16 as loganin, peak 17 as ferulic acid, peak 18 as calycoside, peak 20 as codonopsis pilosula, peak 23 as salvianolic acid B, peak 29 as icariin, and peak 32 as baohuoside I.

[0026] Among them, Peak 13 and Peak 16 are mainly components from Cornus officinalis; Peak 17 is a component from Angelica sinensis; Peak 18 is a component from Astragalus membranaceus; Peak 19 is a component from Ligustrum lucidum fruit; Peak 20 is a component from Codonopsis pilosula; Peak 23 is a component from Salvia miltiorrhiza; Peaks 29 and 32 are components from Epimedium.

[0027] The third aspect of the present invention provides the application of the fingerprint spectrum in the quality evaluation of Yiqi Yangxue Granules.

[0028] Compared with the prior art, the technical solution of the present invention has achieved the following beneficial technical effects:

[0029] 1. The present invention provides for the first time a method for establishing an HPLC fingerprint of Yiqi Yangxue Granules. Based on the composition characteristics of the Yiqi Yangxue Granules prescription, the optimal mobile phase composition and its gradient elution procedure, flow rate, and detection wavelength are optimized. The resulting fingerprint has high resolution and good peak shape, and all characteristic peaks are baseline-separated, enabling a more comprehensive and accurate evaluation of the quality of Yiqi Yangxue Granules.

[0030] 2. The detection method of the present invention uses terglucosidoside as the reference peak and determines that 32 common peaks are the main characteristic peaks constituting the standard fingerprint of Yiqi Yangxue Granules, making the composition of the fingerprint more abundant and comprehensive. Nine chemical components, including morroniside, loganin, ferulic acid, calycosin, privet glycoside, codonopsis pilosula, salvianolic acid B, icariin, and baohuoside I, are identified from the common peaks and classified as components, further clarifying the chemical substance basis of Yiqi Yangxue Granules.

[0031] 3. The application of the fingerprint spectrum of the present invention in the quality evaluation of Yiqi Yangxue Granules can make a more comprehensive evaluation of the quality of Yiqi Yangxue Granules by comparing the presence or absence of common peaks in the obtained standard fingerprint spectrum, more effectively ensure the quality of the finished product, and overcome the problem that the existing technology has a single detection indicator and cannot reflect the insufficient intrinsic quality of Yiqi Yangxue Granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is an overlay of HPLC fingerprints of 10 batches of Yiqi Yangxue Granules;

[0033] Figure 2 This is the HPLC standard fingerprint of Yiqi Yangxue Granule (1-32 are 32 common characteristic peaks), among which peak 13 is morroniside, peak 16 is loganin, peak 17 is ferulic acid, peak 18 is calycoside, peak 19 is privet glycoside, peak 20 is codonopsis pilosula, peak 23 is salvianolic acid B, peak 29 is icariin, and peak 32 is baohuoside I;

[0034] Figure 3 HPLC fingerprint of the reference substance. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, obvious modifications and improvements made without departing from the concept of the present invention also fall within the scope of protection of the present invention.

[0036] Example 1 Establishment of the HPLC Standard Fingerprint of Yiqi Yangxue Granules

[0037] 1. Instruments and reagents

[0038] 1.1 Instruments

[0039] Agilent 1260 high performance liquid chromatograph (USA), OpenLab CDS, ChemStation Edition chromatography workstation.

[0040] 1.2 Drug testing

[0041] Yiqi Yangxue granules were provided by Lunan Houpu Pharmaceutical Co., Ltd., and the sample batch numbers are shown in Table 1. Methanol and acetonitrile were of chromatographic grade, water was ultrapure water, and the remaining reagents were of analytical grade.

[0042] Table 1 Batch number of Yiqi Yangxue granule test samples

[0043] 2 Methods and Results

[0044] 2.1 Chromatographic conditions

[0045] The chromatographic column is Perkin Elmer Brownlee Validated AQ C 18 (4.6×250 mm, 5 μm) column; mobile phase A was acetonitrile, mobile phase B was 0.1% formic acid solution, gradient elution was used; detection wavelength was multi-wavelength; flow rate was 1.0 mL / min; column temperature was 35°C; injection volume was 10 μL.

[0046] Perform gradient elution and wavelength detection according to the following two tables:

[0047] Multi-wavelength detection table:

[0048] 2.2 Preparation of reference and test solutions

[0049] Accurately weigh appropriate amounts of morroniside, loganin, ferulic acid, calycosin, privet glycoside, codonopsis pilosula, salvianolic acid B, icariin, and baohuoside I reference substances and dilute them with methanol to prepare reference substance solutions containing 348.235 µg, 162.941 µg, 38.824 µg, 31.765 µg, 634.706 µg, 81.961 µg, 595.294 µg, 112.941 µg, and 18.529 µg of the above reference substances per 1 mL.

[0050] Accurately weigh 5 g of Yiqi Yangxue Granules and place them in a 50 mL conical flask. Accurately add 20 mL of methanol and weigh the weight. Ultrasonicate, cool, make up the weight of methanol, centrifuge, filter, and take the filtrate to obtain the test solution.

[0051] 2.3 Fingerprint establishment

[0052] Take 10 batches of Yiqi Yangxue Granule samples, prepare them into test solution according to the method under "2.2", and perform the test analysis according to the method under "2.1". The chromatogram overlay of 10 batches of samples is obtained (see Appendix). Figure 1 The HPLC fingerprints of 10 batches of Yiqi Yangxue Granules were imported into the “Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System” (2012 Edition) for chromatographic peak matching and fitted to generate standard fingerprints (see Appendix). Figure 2 The obtained standard fingerprint has 32 common characteristic peaks. Peak 19 (teruncle glycoside) was used as the reference peak to calculate the ratios of the relative retention times and relative peak areas of the 15 main characteristic peaks. See Table 2 and Table 3 for details.

[0053] Table 2 Relative retention time ratios of the main characteristic peaks of 10 batches of Yiqi Yangxue Granules

[0054] Table 3 Relative peak areas of the main characteristic peaks of 10 batches of Yiqi Yangxue Granules (%)

[0055] 2.4 Similarity Evaluation

[0056] The chromatograms of 10 batches of Yiqi Yangxue Granules were compared with the standard fingerprint. The similarity calculation results were: 0.996, 0.998, 0.998, 0.999, 0.995, 0.997, 0.996, 0.997, 0.995, and 0.997. The similarities were all above 0.990, indicating good quality stability and uniformity among the sample batches.

[0057] 2.5 Attribution and identification of shared peaks

[0058] Take the reference solution and analyze it according to the chromatographic conditions under "2.1". The results are as follows: Figure 3 As shown. Based on retention time positioning and chromatographic peak analysis of reference substances, peak 13 was identified as morroniside, peak 16 as loganin, peak 17 as ferulic acid, peak 18 as calycoside, peak 19 as terunoside, peak 20 as codonopsis pilosula, peak 23 as salvianolic acid B, peak 29 as icariin, and peak 32 as baohuoside I. Peaks 13 and 16 are primarily components of Cornus officinalis; peak 17 is a component of Angelica sinensis; peak 18 is a component of Astragalus membranaceus; peak 19 is a component of Ligustrum lucidum fruit; peak 20 is a component of Codonopsis pilosula; peak 23 is a component of Salvia miltiorrhiza; and peaks 29 and 32 are components of Epimedium.

[0059] 2.6 Methodological Review

[0060] 2.6.1 Precision test

[0061] Take the sample (batch number: 250115-1), prepare the test solution according to the method under 2.2, and inject the sample 6 times continuously. Taking peak 19 as the reference peak, the RSD values ​​of the relative retention time and relative peak area of ​​common peaks 1-32 were calculated to be less than 3%. At the same time, the similarity of each chromatographic fingerprint calculated by the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software was greater than 0.99, indicating that the instrument has good stability and precision.

[0062] 2.6.2 Stability test

[0063] Take a sample (batch number: 250115-1) and prepare the test solution according to the method under 2.2. The samples were injected at 0, 2, 5, 8, 12, 16, and 24 hours respectively. Peak 19 was used as the reference peak. The RSD values ​​of the relative retention time and relative peak area of ​​common peaks 1-32 were calculated to be less than 3%. At the same time, the similarity of each chromatographic fingerprint calculated by similarity evaluation software was greater than 0.99, indicating that the test solution had good stability within 24 hours.

[0064] 2.6.3 Repeatability test

[0065] Take 6 samples from the same batch (batch number: 250115-1), accurately measure them, prepare the test solution according to the method under 2.2, and inject them separately. Taking peak 19 as the reference peak, the RSD values ​​of the relative retention time and relative peak area of ​​common peaks 1-32 are calculated to be less than 3%, and the similarity of each chromatographic fingerprint calculated by similarity evaluation software is greater than 0.99, indicating that the method has good repeatability.

Claims

1. A method for establishing an HPLC fingerprint of Yiqi Yangxue Granules, characterized in that: The following steps are involved: 1) Preparation of reference and test solutions Weigh appropriate amounts of morroniside, loganin, ferulic acid, calycosin, privet glycoside, codonopsis pilosula, salvianolic acid B, icariin, and baohuoside I, and dissolve them in methanol to prepare the reference solution. Accurately weigh an appropriate amount of Yiqi Yangxue Granule test sample, add methanol, ultrasonicate, cool, make up the weight of methanol lost, centrifuge, filter, and take the filtrate to obtain the test sample solution; 2) Determination of HPLC chromatographic conditions The chromatographic column is Perkin Elmer Brownlee Validated AQ C 18 The column was 1.5-μm elution column; mobile phase A was acetonitrile, and mobile phase B was formic acid solution; gradient elution was used; the detection wavelength was multi-wavelength; the flow rate was 1.0 mL / min; the column temperature was 35 °C; and the injection volume was 10 μL. 3) Fingerprint preparation The reference solution and the test solution were analyzed and compared according to the chromatographic conditions in step 2) to obtain a standard fingerprint of Yiqi Yangxue Granule consisting of the common characteristic peaks of the samples.

2. The method according to claim 1, characterized in that Step 1) The test solution is prepared as follows: accurately measure 5 g of Yiqi Yangxue Granules, place in a 50 mL conical flask, accurately add 20 mL of methanol and weigh, ultrasonicate, cool, make up the weight of methanol lost, centrifuge, filter, and take the filtrate to obtain the test solution.

3. The method according to claim 1, characterized in that The specifications of the chromatographic column in step 2) are 4.6×250 mm and the particle size is 5 μm.

4. The method according to claim 1, wherein In step 2), mobile phase B is 0.1% formic acid solution.

5. The method according to claim 1, wherein Step 2) Gradient elution conditions are:

6. The method according to claim 1, characterized in that Step 2) Detection wavelength conditions are:

7. A fingerprint prepared by the method according to any one of claims 1 to 6, characterized in that: By matching the fingerprints of 10 batches of Yiqi Yangxue Granules, 32 common characteristic peaks were obtained. Taking the peak 19, the special privet glycoside peak, as the reference peak, peaks 6, 8, 11, 13, 16, 17, 18, 19, 20, 22, 23, 25, 27, 29, and 32, a total of 15 main characteristic peaks of Yiqi Yangxue Granules were identified, and their relative retention times were 0.20, 0.25, 0.37, 0.46, 0.62, 0.95, 1.00, 1.10, 1.37, 1.52, 1.63, 1.74, 1.81, 1.85, and 2.07, respectively.

8. The fingerprint according to claim 7, characterized in that: Peak 13 is morroniside, peak 16 is loganin, peak 17 is ferulic acid, peak 18 is calycoside, peak 19 is privet glycoside, peak 20 is codonopsis pilosula, peak 23 is salvianolic acid B, peak 29 is icariin, and peak 32 is baohuoside I.

9. The fingerprint according to claim 7, characterized in that: Peaks 13 and 16 are mainly components from Cornus officinalis; Peak 17 is a component from Angelica sinensis; Peak 18 is a component from Astragalus membranaceus; Peak 19 is a component from Ligustrum lucidum fruit; Peak 20 is a component from Codonopsis pilosula; Peak 23 is a component from Salvia miltiorrhiza; Peaks 29 and 32 are components from Epimedium.

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