A method for constructing a fingerprint spectrum of Danpu granules

Abstract

The application discloses a kind of danpu granules fingerprint spectrum construction methods, belong to the technical field of pharmaceutical analysis.The application constructs HPLC fingerprint spectrum for danpu granules, the atlas contains 11 sample common characteristic peaks, including gallic acid, protocatechuic aldehyde, paeoniflorin, chicory acid, osmanthus glycoside, rosemary acid, shikonin and salvianolic acid B, comprehensively covers the main active ingredient in danpu granules, can be complete, accurately present the chemical substance basis of danpu granules, provides rich, reliable information for quality evaluation.The construction method of fingerprint spectrum of the application is good in stability, can be used for the quality evaluation and quality control of multiple batches of danpu granules, provides solid technical support for the factory production process establishment of danpu granules.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for constructing a fingerprint spectrum of Danpu granules. Background Technology

[0002] Chronic prostatitis is a common and difficult-to-treat disease in urology, with a high incidence and easy recurrence, seriously affecting patients' quality of life. Danpu Granules (disclosed in patent CN102755563A), formulated with Salvia miltiorrhiza, Patrinia scabiosaefolia, raw Coix lacryma-jobi, Paeonia lactiflora, Taraxacum mongolicum, and Lycium barbarum, has the effects of promoting blood circulation, removing blood stasis, detoxifying, and relieving urinary tract infections. Clinically, it is used for chronic prostatitis of the qi stagnation and blood stasis type, and can improve symptoms such as lower abdominal and perineal pain, frequent urination, and whitish urine. Pharmacological studies have confirmed its anti-inflammatory, antibacterial, and prostate pathological damage repair effects, with definite clinical efficacy and good safety. Danpu Granules are a traditional Chinese medicine compound preparation with complex chemical components, containing flavonoids, phenolic acids, anthraquinones, polysaccharides, and other active ingredients. Its quality is significantly affected by the origin of raw materials, harvesting and processing, and production technology. However, current quality standards for traditional Chinese medicine compound preparations mostly focus on quantifying single indicator components, making it difficult to comprehensively reflect the overall quality and efficacy correlation, thus restricting the standardization, industrialization, and internationalization of the preparation.

[0003] Fingerprint mapping of traditional Chinese medicine (TCM) is an internationally recognized core technology for overall quality control. Through chromatographic / spectral separation and detection, it obtains a spectrum characterizing chemical features, comprehensively reflecting the types and quantities of components, identifying authenticity, evaluating batch consistency, and linking pharmacodynamic material basis, thus providing a scientific means for TCM quality control. However, current research on Danpu granules focuses on efficacy, clinical applications, and preparation processes, with no reports on fingerprint mapping and overall quality control methods. The lack of a standard spectrum that comprehensively characterizes its intrinsic quality makes it difficult to effectively control batch-to-batch quality differences, affecting the stability of clinical efficacy and medication safety. Therefore, establishing a dedicated fingerprint spectrum for Danpu granules and clarifying the attribution of characteristic peaks and quality control parameters is of significant practical importance for improving quality standards, ensuring formulation stability, and promoting modern applications. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a fingerprint spectrum of Danpu granules to solve the problems existing in the prior art. This invention constructs an HPLC fingerprint spectrum for Danpu granules, which contains 11 common characteristic peaks shared by the samples, including gallic acid, protocatechuic aldehyde, paeoniflorin, chicoric acid, luteolin, rosmarinic acid, shikonin, and salvianolic acid B. This comprehensively covers the main active ingredients in Danpu granules, and can completely and accurately present the chemical composition of Danpu granules, providing rich and reliable information for quality evaluation.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for constructing a fingerprint spectrum of Danpu particles, comprising the following steps: (1) Prepare the test solution and the reference solution; The test solution is prepared by taking Danpu granules, adding methanol aqueous solution, and then ultrasonically treating it to obtain the test solution. (2) Perform HPLC chromatographic analysis on the test solution and the reference solution to obtain the fingerprint spectrum of Danpu granules composed of common characteristic peaks of the samples; The HPLC chromatographic analysis conditions were as follows: octadecylsilane-bonded silica gel was used as the stationary phase; 0.05% phosphoric acid aqueous solution was used as mobile phase A; and acetonitrile was used as mobile phase B. The elution program was as follows: 0-12 min, 2%-6% B; 12-25 min, 6%-12% B; 25-35 min, 12%-15% B; 35-45 min, 15%-20% B; 45-47 min, 20%-21% B; 47-57 min, 21%-22% B; 57-65 min, 22%-23% B; and 65-75 min, 23%-39% B.

[0006] Furthermore, the Danpu granules are prepared from Salvia miltiorrhiza, dandelion, Paeonia lactiflora, Patrinia scabiosaefolia, Coix lacryma-jobi, and Lycium barbarum.

[0007] Furthermore, the preparation method of the test solution is as follows: accurately weigh 0.20 g of Danpu granules, add 20 mL of 70% methanol aqueous solution, and sonicate at 200 W and 53 kHz for 30 minutes. After weighing and filtration, the test solution is obtained.

[0008] Furthermore, the HPLC chromatographic analysis was performed at a flow rate of 0.4 mL / min, a detection wavelength of 220 nm, a column temperature of 35 °C, and an injection volume of 10 μL.

[0009] Furthermore, the reference solution comprises gallic acid solution, protocatechuic aldehyde solution, paeoniflorin solution, chicoric acid solution, luteolin solution, rosmarinic acid solution, lithospermic acid solution, and salvianolic acid B solution prepared using 70% methanol aqueous solution as solvent.

[0010] Furthermore, the fingerprint spectrum of the Danpu granules contains 11 common characteristic peaks of the samples, of which peak 1 is gallic acid, peak 4 is protocatechuic aldehyde, peak 5 is paeoniflorin, peak 6 is chicoric acid, peak 7 is luteolin, peak 9 is rosmarinic acid, peak 10 is shikonin, and peak 11 is salvianolic acid B.

[0011] This invention also provides the application of the fingerprint spectrum of Danpu granules constructed by the above method in the quality control of the Danpu granules production process, the evaluation of finished product quality, the batch stability analysis and the identification of authenticity.

[0012] The present invention discloses the following technical effects: This invention constructs an HPLC fingerprint for Danpu granules, containing 11 common characteristic peaks, including gallic acid, protocatechuic aldehyde, paeoniflorin, chicoric acid, luteolin, rosmarinic acid, shikonin, and salvianolic acid B. This comprehensively covers the main active ingredients in Danpu granules, accurately and completely presenting their chemical composition and providing rich and reliable information for quality evaluation. The fingerprints constructed using this method for multiple batches of Danpu granules all showed a similarity greater than 0.9, indicating good stability and applicability for quality evaluation and control of multiple batches of Danpu granules. This provides solid technical support for establishing a factory-scale production process for Danpu granules. Attached Figure Description

[0013] 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.

[0014] Figure 1 The images show the BPI chromatogram (A), HPLC chromatogram (B), and HPLC chromatogram (C) of Danpu granules in negative ion mode. In A and B, peak 1 is gallic acid, peak 4 is protocatechuic aldehyde, peak 5 is paeoniflorin, peak 6 is chicoric acid, peak 7 is luteolin, peak 9 is rosmarinic acid, peak 10 is shikonin, peak 11 is salvianolic acid B, and peaks 2, 3, and 8 are unknown. In C, peak 1 is rosmarinic acid, peak 2 is gallic acid, peak 3 is paeoniflorin, peak 4 is protocatechuic aldehyde, peak 5 is salvianolic acid B, peak 6 is luteolin, peak 7 is shikonin, and peak 8 is chicoric acid. Figure 2 The fingerprint chromatograms (S1-S9) and control chromatograms (R) of 9 batches of Danpu granules are shown. Detailed Implementation

[0015] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0016] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0017] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0018] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0019] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0021] Example 1 1. Instruments and Materials Instruments: Waters Acquity UPLC / Xevo G2-S Q-TOF LC-MS system (Waters Corporation, USA), Shimadzu LC-20 AT high performance liquid chromatograph (Shimadzu Corporation, Japan); Waters ACQUITY UPLC BEH C18 column (2.1×100 mm, 1.7 μm), Kromasil 100-5-C18 column (4.6×250 mm, 5 μm); FW100 high-speed universal grinder (Tianjin Tester Instrument Co., Ltd.); ultrasonic cleaner (model: SK10GT, frequency: 53kHz, Shanghai Kedao Ultrasonic Instrument Co., Ltd.); ME104 Mettler 0.001 g electronic balance [Mettler-Toledo Instruments (Shanghai) Co., Ltd.]; MS105DU Mettler 0.001 g electronic balance [Mettler-Toledo Instruments (Shanghai) Co., Ltd.].

[0022] Materials: The Danpu Granules formula consists of the following components by weight: 30 parts Salvia miltiorrhiza, 15 parts Taraxacum mongolicum, 15 parts Paeonia lactiflora, 30 parts Patrinia scabiosaefolia, 30 parts Coix lacryma-jobi, and 15 parts Lycium barbarum. The sources of the Danpu Granules samples used in this example are shown in Table 1. The preparation processes for each sample are as follows: Salvia miltiorrhiza was extracted twice with 70% ethanol under reflux, each time with 8 times the volume of the extract and for 1 hour each time. The extracts were filtered, the filtrates were combined, the ethanol was recovered, and the extract was concentrated to a clear extract with a relative density of 1.05-1.10 (60℃). The dregs were then decocted three times with water, each time with 8 times the volume of the extract and for 1 hour each time. The extracts were filtered, the filtrates were combined, and the extract was concentrated to a clear extract with a relative density of 1.05-1.10 (60℃). The extract was then combined with the above-mentioned clear extract and concentrated to a clear extract with a relative density of 1.15-1.25 (60℃). The extracts were dried, pulverized, and a suitable amount of dextrin was added. The mixture was then mixed, granulated, and dried to obtain the final product.

[0023] Table 1. Information on Danpu Granules Samples 2. Methods and Results 2.1 Preparation of test solution Accurately weigh 0.20 g of Danpu granules powder (batch number 2408001) (pass through a No. 4 sieve), add 20 mL of 70% methanol, accurately weigh the mass, sonicate (200 W, 53 kHz) for 30 min, let stand to room temperature, add the weight, and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0024] 2.2 Preparation of the reference solution Accurately weigh appropriate amounts of gallic acid, protocatechuic aldehyde, paeoniflorin, chicoric acid, luteolin, rosmarinic acid, lithospermic acid, and salvianolic acid B, and prepare a single reference stock solution with a mass concentration of 0.1 mg / mL using 70% methanol.

[0025] 2.3 Qualitative Analysis using UPLC-Q-TOF-MS / MS 2.3.1 Chromatographic conditions Waters ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm); mobile phase: 0.1% formic acid water (A) - acetonitrile (B), gradient elution (0–2.4 min, 2%–6% B; 2.4–3.6 min, 6%–8% B; 3.6–5.6 min, 8%–12% B; 5.6–8 min, 12%–17% B; 8–10 min, 17%–22% B; 10–14 min, 22%–24% B), flow rate: 0.4 mL / min, column temperature: 35 °C, injection volume: 2 μL.

[0026] 2.3.2 Mass Spectrometry Conditions Electrospray ionization (ESI) was used in negative ion mode. The capillary voltage was 2.0 kV, the cone voltage was 40 V, the compensation voltage was 80 V, the ion source temperature was 120℃, the desolvation temperature was 500℃, the desolvation nitrogen flow rate was 400 L / h, the cone gas flow rate was 50 L / h, and bar graphs were acquired in MSE mode. The mass scan range was 100-1200 m / z, the scan interval was 0.2 s, the transmission collision energy was 6 eV for low-energy scans and 25-55 eV for high-energy scans. Leucine enkephalin at a mass concentration of 1.0 ng / L was selected for real-time mass correction at a flow rate of 10 μL / min. Data acquisition and analysis were performed using MassLynx 4.1 software.

[0027] 2.4 HPLC fingerprint of Danpu granules Chromatographic conditions: Kromasil 100-5-C18 column (4.6×250mm, 5μm); mobile phase: 0.05% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient elution (0-12min, 2%-6%B; 12-25min, 6%-12%B; 25-35min, 12%-15%B; 35-45min, 15%-20%B; 45-47min, 20%-21%B; 47-57min, 21%-22%B; 57-65min, 22%-23%B; 65-75min, 23%-39%B), flow rate: 0.4mL / min, detection wavelength: 220nm, column temperature: 35℃, injection volume: 10μL.

[0028] 2.5 Results The test solution and reference solution were loaded and tested according to methods 2.3 and 2.4, and the chromatographic peaks of the test solution were identified based on the chromatographic peaks of the reference solution. The results are as follows: Figure 1 As shown in the figure, A is the BPI chromatogram of Danpu granules in negative ion mode, B is the HPLC chromatogram of Danpu granules, and C is the HPLC chromatogram of Danpu granules and eight reference solutions. Based on the HPLC chromatograms of the reference solutions, eight peaks were identified: peak 1 is gallic acid, peak 4 is protocatechuic aldehyde, peak 5 is paeoniflorin, peak 6 is chicoric acid, peak 7 is luteolin, peak 9 is rosmarinic acid, peak 10 is shikonin, peak 11 is salvianolic acid B, and peaks 2, 3, and 8 are unknown.

[0029] 3. Methodological Examination 3.1 Precision Test A sample of Danpu granules from the same batch (batch number: 2408001) was taken, and a test solution was prepared according to the method described in section "2.1". The sample was injected six times consecutively under the chromatographic conditions described in section "2.4", and the chromatograms were recorded. The RSD of each chromatographic peak was calculated using peak number 11 (tanshinone B) as the reference peak. The results are shown in Tables 2, 3, and 4. The RSD of the relative peak area of ​​each chromatographic peak was less than 4.2%, and the RSD of the relative retention time was less than 0.43%. The similarity of the chromatograms was >0.999, indicating that the method of this invention has good precision.

[0030] Table 2. Relative peak area data of the precision of the fingerprint spectrum of Danpu particles Table 3. Relative retention time data of the precision of Danpu particle fingerprint spectrum Table 4. Similarity matching results of fingerprint spectrum precision of Danpu particles 3.2 Repeatability Test Six test solutions were prepared in parallel using the same batch of Danpu granules (batch number: 2408001) according to the method described in section "2.1". These solutions were then injected sequentially under the chromatographic conditions described in section "2.4", and the chromatograms were recorded. The RSD of each chromatographic peak was calculated using peak 11 (tanshinone B) as the reference peak. The results are shown in Tables 5, 6, and 7. The RSD of the relative peak area of ​​each chromatographic peak was less than 4.8%, and the RSD of the relative retention time was less than 0.7%, indicating that the method of this invention has good repeatability.

[0031] Table 5. Relative peak area data for repeatability of Danpu particle fingerprint spectrum Table 6. Relative retention time data for repeatability of Danpu particle fingerprint spectra Table 7. Similarity matching results of repeatability fingerprint spectra of Danpu particles 3.3 Stability Test A sample of Danpu granules from the same batch (batch number: 2408001) was taken and a test solution was prepared according to the method in section "2.1". The solution was injected and analyzed at 0, 2, 4, 8, 12, 16, and 24 hours under the chromatographic conditions in section "2.4". Chromatograms were recorded, and the RSD of each peak was calculated using peak 11 (tanshinone B) as the reference peak. The results are shown in Tables 8, 9, and 10. The RSD of the relative peak area of ​​each peak was less than 3.8%, and the RSD of the relative retention time was less than 0.9%. The similarity of the chromatograms was >0.999, indicating that the test solution had good stability within 24 hours.

[0032] Table 8. Relative peak area data of stability in the fingerprint spectrum of Danpu particles Table 9. Relative retention time data of the stability of Danpu particle fingerprint spectrum Table 10. Similarity matching results of the fingerprint spectrum stability spectrum of Danpu particles 4. Fingerprint pattern establishment and similarity evaluation Nine batches of Danpu granules samples were collected, and test solutions were prepared according to the method described in section "2.1". The samples were then injected sequentially under the chromatographic conditions described in section "2.4", and the chromatograms were recorded. The chromatograms were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". The mean method was selected, the time window was set to 0.1, multi-point correction was performed, and Mark peak matching was used to generate overlay chromatograms and reference chromatograms (R). See [link to relevant documentation]. Figure 2 The similarity evaluation results are shown in Table 11. The similarity between the nine batches of samples and the control spectrum was 0.990-0.997, indicating that the sample quality was stable and the differences between batches were small.

[0033] Table 11 Similarity evaluation results of 9 batches of Danpu granule samples The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of Danpu particles, characterized in that, Includes the following steps: (1) Prepare the test solution and the reference solution; The test solution is prepared by taking Danpu granules, adding methanol aqueous solution, and then ultrasonically treating it to obtain the test solution. (2) Perform HPLC chromatographic analysis on the test solution and the reference solution to obtain the fingerprint spectrum of Danpu granules composed of common characteristic peaks of the samples; The HPLC chromatographic analysis conditions were as follows: octadecylsilane-bonded silica gel was used as the stationary phase; 0.05% phosphoric acid aqueous solution was used as mobile phase A; and acetonitrile was used as mobile phase B. The elution program was as follows: 0-12 min, 2%-6% B; 12-25 min, 6%-12% B; 25-35 min, 12%-15% B; 35-45 min, 15%-20% B; 45-47 min, 20%-21% B; 47-57 min, 21%-22% B; 57-65 min, 22%-23% B; and 65-75 min, 23%-39% B.

2. The construction method according to claim 1, characterized in that, The Danpu granules are prepared from Danshen, Dandelion, Red Peony Root, Patrinia scabiosaefolia, Coix Seed, and Goji Berry.

3. The construction method according to claim 1, characterized in that, The test solution is prepared as follows: 0.20 g of Danpu granules are accurately weighed, 20 mL of 70% methanol aqueous solution is added, and the mixture is ultrasonically treated at 200 W and 53 kHz for 30 minutes. After weighing and filtration, the test solution is obtained.

4. The construction method according to claim 1, characterized in that, The HPLC chromatographic analysis was performed at a flow rate of 0.4 mL / min, a detection wavelength of 220 nm, a column temperature of 35 °C, and an injection volume of 10 μL.

5. The construction method according to claim 1, characterized in that, The reference solutions include gallic acid solution, protocatechuic aldehyde solution, paeoniflorin solution, chicoric acid solution, luteolin solution, rosmarinic acid solution, lithospermic acid solution, and salvianolic acid B solution, all prepared using 70% methanol aqueous solution as solvent.

6. The construction method according to claim 1, characterized in that, The fingerprint spectrum of the Danpu granules contains 11 characteristic peaks shared by the samples. Among them, peak 1 is gallic acid, peak 4 is protocatechuic aldehyde, peak 5 is paeoniflorin, peak 6 is chicoric acid, peak 7 is luteolin, peak 9 is rosmarinic acid, peak 10 is shikonin, and peak 11 is salvianolic acid B.

7. The application of the fingerprint spectrum of Danpu granules constructed by the construction method according to any one of claims 1-6 in the quality control of Danpu granules production process, finished product quality evaluation, batch stability analysis and authenticity identification.

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