Traditional Chinese medicine fingerprint detection method for polygonum viviparum preparation

Abstract

The invention relates to the technical field of medicine component content determination, in particular to a traditional Chinese medicine fingerprint spectrum detection method of a serpentgrass preparation, which finally determines a chromatographic column: Agilent color type: ZORBAX Eclipse Plus C18 4.6 * 250mm * 5mu m, a mobile phase A being 0.1% phosphoric acid water, a mobile phase B being methanol and a column temperature being 30 DEG C by combining repeated exploration and saving time and reagent cost; the sample size is 10 mu L, the chromatographic peak shape of each component is relatively good, the peak appearance time is stable, the linear relation, the precision, the repeatability and the stability are good, and the average recovery rate of luteolin in a sample of a sample adding recovery experiment is 95.2%; the method can be used for guiding feeding in the production and preparation process of the polygonum viviparum preparation and standardizing production operation, so that the integrity and controllability of quality control in the whole process are ensured, and the safety, effectiveness and stability of clinical medication are ensured.

Description

Technical Field

[0001] The invention relates to the technical field of drug component content determination, and in particular to a traditional Chinese medicine fingerprint detection method for a Polygonum villosa preparation. Background Art

[0002] Polygonum viviparum L. is a commonly used Chinese herbal medicine. It belongs to the Polygonum genus of the Polygonaceae family and is a perennial herb. Studies have shown that Polygonum viviparum contains a variety of active ingredients, including flavonoids, phenolics, polysaccharides, and volatile oils. Numerous studies have demonstrated that Polygonum viviparum exhibits anti-inflammatory, antioxidant, antibacterial, and antitumor effects. Currently, research is underway to determine the quality standards for Polygonum viviparum. Hu Ruilong et al. established methods for the RP-HPLC determination of quercetin content and the TLC identification of β-sitosterol in Polygonum viviparum, improving upon the 1995 version of the Polygonum viviparum identification standard. These methods are effective in controlling the quality of Polygonum viviparum. However, with the advancement of chromatographic detection technology and the demands of production, these methods have significant limitations in terms of accuracy, practicality, and comprehensive quality control of Polygonum viviparum components. In particular, the development of comprehensive qualitative and quantitative control methods for the quality standards of Polygonum viviparum preparations is urgently needed.

[0003] Traditional Chinese medicine (TCM) fingerprints are a comprehensive, quantifiable identification method based on systematic research on the chemical components of TCM. They are primarily used to evaluate the authenticity, quality, and stability of TCM materials and semi-finished TCM preparations. Currently, the use of fingerprints as a quality control method for TCM extracts and preparations has become an international consensus, and various fingerprint control technology systems tailored to the characteristics of TCM are being researched and established. TCMs and their preparations are complex, multi-component systems, and their quality assessment requires appropriate detection methods that provide rich identification information. However, current methods such as microscopic identification, physicochemical identification, and content determination are insufficient to address this issue. Establishing TCM fingerprints will provide a more comprehensive picture of the types and quantities of chemical components present in TCMs and their preparations, thereby enabling a holistic description and evaluation of drug quality. In summary, the research and development of TCM fingerprints is of great significance for improving the quality of TCMs and promoting their modernization. Summary of the Invention

[0004] The primary purpose of the present invention is to provide an HPLC detection method for a Polygonum villosa preparation, wherein the Polygonum villosa preparation is extracted to prepare a test solution, and the test solution is determined by HPLC; the HPLC determination conditions are:

[0005] Chromatographic column: ZORBAX Eclipse Plus C18 4.6 × 250 mm × 5 μm;

[0006] Mobile phase A is 0.1% phosphoric acid or 0.1%-0.5% formic acid, and B is methanol or acetonitrile;

[0007] The mobile phase conditions are as follows:

[0008]

[0009] Flow rate: 0.5mL / min-1.0mL / min;

[0010] Column temperature 30℃-50℃;

[0011] Injection volume 1μL-120μL.

[0012] Preferably, the mobile phase A is 0.1% phosphoric acid water, and B is methanol.

[0013] Preferably, the flow rate is 0.5 mL / min and the detection wavelength is 210-240 nm.

[0014] Preferably, the column temperature is 30°C.

[0015] Preferably, the injection volume is 10 μL.

[0016] The second object of the present invention is to provide a method for establishing an HPLC fingerprint of a Polygonum vicifolium preparation, the method comprising: (1) preparing a reference solution: taking an appropriate amount of a reference substance, accurately weighing it, and preparing a 1 mg / mL solution;

[0017] (2) Preparation of test solution: Take an appropriate amount of Polygonum vicifolium preparation sample and add methanol to prepare a 1 mg / mL solution;

[0018] (3) HPLC detection: The test sample solution and the reference sample solution of (1) and (2) above were detected using the HPLC detection method for detecting the Polygonum vitripennis preparation to obtain the test sample fingerprint chromatogram and the reference sample fingerprint chromatogram.

[0019] Preferably, the reference substances in step (1) include chlorogenic acid, quercetin, quercetin, isoquercetin, rutin, luteolin, kaempferol, catechin, and β-sitosterol.

[0020] The third object of the present invention is to provide the application of the method for establishing the HPLC fingerprint of the Polygonum vitripennis preparation in the detection and identification of the Polygonum vitripennis preparation.

[0021] The fourth object of the present invention is to provide a fingerprint obtained by the method for establishing the HPLC fingerprint of the Polygonum vicifolium preparation.

[0022] The beneficial effects of the present invention are:

[0023] (1) The present invention provides an HPLC detection method for a Polygonum vitripennis preparation. Through repeated exploration combined with saving time and reagent costs, the chromatographic column is finally determined: Agilent column type: ZORBAX Eclipse Plus C18 4.6×250mm×5μm, the mobile phase A is 0.1% phosphoric acid water, B is methanol, the column temperature is 30°C; the injection volume is 10μL. When used as the extraction conditions, the chromatographic peaks of each component have good peak shapes and stable peak elution times, which can more comprehensively and accurately reflect the chemical information of the Polygonum vitripennis preparation.

[0024] (2) Linear relationship: The concentration of luteolin in the range of 6.25-100 μg / mL showed a good linear relationship with the peak area;

[0025] (3) Precision: RSD (n=6) was 0.274%, indicating that this method had good precision, met the requirements of methodology validation, and could meet the requirements of content determination.

[0026] (4) Repeatability: The average peak area of ​​6 samples of the same batch of Polygonum villosa preparation was 332683, and the luteolin content was calculated to be 15.2 μg / mL with an RSD of 0.613%, indicating that the detection method has good repeatability and meets the content determination requirements.

[0027] (5) Sample recovery experiment: The average recovery rate of luteolin in the samples was 95.2%, and the RSD was 3.57%.

[0028] (6) Stability test: The luteolin content was calculated to be 15.65 μg / mL, which is 0.739, indicating that the Polygonum vicifolium preparation was stable within 24 h. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 HPLC chromatogram of optimized mobile phase composition

[0030] Figure 2 Optimization of gradient elution program

[0031] Figure 3 HPLC fingerprints of 9 batches of Polygonum villosa preparations

[0032] Figure 4 Spectra of Polygonum villosa preparation and reference substance

[0033] Figure 5 Luteolin standard curve DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0035] In the following examples, unless otherwise specified, all reagents are commercially available.

[0036] In the following examples, unless otherwise specified, the methods used are all conventional methods.

[0037] Example 1: Optimization of HPLC Detection Method and Establishment of Fingerprint of Polygonum villosa Preparation

[0038] 1. Materials

[0039] 1.1 Polygonum villosa preparations

[0040] Polygonum villosa slices were purchased from Gansu Fuxinghou Biopharmaceutical Technology Co., Ltd., batch number Y2206020. Polygonum villosa preparations were prepared by the Natural Veterinary Medicine and Antibiotic Alternatives Team at the Lanzhou Institute of Animal Husbandry and Veterinary Pharmacy, Chinese Academy of Agricultural Sciences, and stored at -4°C until ready for use. Polygonum villosa slices were extracted with ethanol at reflux for 120 min, using a solid-liquid ratio of 1:10, three times to a flavonoid content of ≥67%. The extracts were then concentrated under reduced pressure and purified using AB-8 macroporous resin, eluted with 50-70% ethanol, and supplemented with 5% propylene glycol and 0.2% potassium sorbate.

[0041] 1.2 Standards

[0042] Anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd., production batch number: 10009218), acetonitrile (Sinopharm Chemical Reagent Co., Ltd., production batch number: 400641646), methanol (Sinopharm Chemical Reagent Co., Ltd., production batch number: 10014108), formic acid (Sinopharm Chemical Reagent Co., Ltd., production batch number: 40023774), phosphoric acid (Sinopharm Chemical Reagent Co., Ltd., production batch number: 10015408), chlorogenic acid (China Food and Drug Administration, production batch number: TC01811G), quercetin (China Food and Drug Administration, production batch number: TQ011225G), quercetin (China Food and Drug Administration, production batch number: 1811020), isoquercetin (China Food and Drug Inspection Institute, production batch number: SDDRS11441020); rutin (China Food and Drug Inspection Institute, production batch number: XW01531842), luteolin (China Food and Drug Inspection Institute, XW0149170304), kaempferol (China Food and Drug Inspection Institute, production batch number: XW0152018301), catechin (China Food and Drug Inspection Institute, production batch number: XW01125708501), β-sitosterol (China Food and Drug Inspection Institute, production batch number: XW018346505).

[0043] 2. Methods

[0044] 2.1 Preparation of reference solution

[0045] Take appropriate amounts of the reference substances chlorogenic acid, quercetin, quercetin, isoquercetin, rutin, luteolin, kaempferol, catechin, and β-sitosterol, accurately weigh them, and prepare a 1 mg / mL solution.

[0046] 2.2 Preparation of test solution

[0047] Take an appropriate amount of the Polygonum villosa preparation sample, add methanol, and prepare a 1 mg / mL solution.

[0048] 2.3 Optimization of chromatographic conditions

[0049] 2.3.1 Column selection

[0050] When other conditions remain unchanged, a screening experiment is conducted on the chromatographic columns. The chromatographic columns used in the experiment include reverse phase chromatographic columns, normal phase chromatographic columns, ion exchange columns, and HILIC columns.

[0051] Based on research progress and preliminary experiments, a C18 reversed-phase column was selected. C18 has a wide pH tolerance range (pH 2-8), is compatible with common organic solvents such as acetonitrile and methanol, and provides strong hydrophobicity, making it suitable for over 80% of small molecules. It also offers good column efficiency, and high carbon loading (such as the Agilent ZORBAX SB-C18) enhances retention.

[0052] 2.3.2 Mobile phase optimization

[0053] With other conditions unchanged, the mobile phases were screened. Mobile phase A included 0.1% formic acid, 0.5% formic acid and 0.1% phosphoric acid, and mobile phase B included methanol and acetonitrile.

[0054] The screening results of mobile phase A are as follows Figure 1 As shown, after comparing 0.1% formic acid, 0.5% formic acid and 0.1% phosphoric acid, it was found that the peak shape symmetry and separation degree of 0.1% phosphoric acid aqueous solution were better, and finally the mobile phase A was determined to be 0.1% phosphoric acid aqueous solution;

[0055] Methanol and acetonitrile were screened for mobile phase B, and it was found that methanol had better baseline stability and target response value than acetonitrile. Therefore, methanol was selected as mobile phase B. The chromatographic behavior of both was stable under gradient elution conditions, meeting the analysis requirements.

[0056] 2.3.3 Optimization of gradient elution program

[0057] Gradient elution is key to balancing resolution and analysis time, and optimal gradient conditions must be determined experimentally. Initial gradient testing begins with a wide gradient (0–100% mobile phase B, 20 min). The retention time and separation of the target compound are observed to determine the approximate retention window. Compound peaks are concentrated around 10–15 min. The gradient range is narrowed for optimization. Due to overlapping peaks of multiple compounds, the gradient time is extended and the gradient slope is slowed to improve resolution. The organic phase (phase B) ratio is adjusted to control compound retention. Initial gradient screening indicates that the target compound elutes concentratedly between 10 and 25 min, so a fine gradient is set in this range. 0-5min (mobile phase B 5% → 20%) is a slow gradient (3% B / min) for separating highly polar impurities and avoiding early co-elution; 5-15min (mobile phase B 20% → 40%) is to extend the main component separation window; 15-25min (mobile phase B 40% → 50%) is an ultra-low slope of 0.5% B / min to improve the separation of structural analogs; 28-30min (mobile phase B 100% → 5%) is to quickly regenerate the chromatographic column ( Figure 2 ).

[0058] 2.3.4 Column temperature optimization

[0059] The separation effects at 30°C, 40°C, and 50°C were tested to evaluate the effects of column temperature on the retention time, resolution, and column pressure of the target compounds.

[0060] The final temperature selected was 30°C, which achieved the required separation, optimal peak shape, and stability, avoiding degradation of heat-sensitive compounds caused by high temperature.

[0061] 2.3.5 Optimization of flow rate and injection volume

[0062] Flow rates of 0.5, 0.8, and 1.0 mL / min were screened, and 0.5 mL / min was ultimately selected based on the fact that it provided the best separation efficiency and the lowest column pressure to avoid high-pressure damage to the chromatographic column.

[0063] Injection volumes of 1, 5, 10, 15, and 20 μL were screened, and 10 μL was finally selected based on the fact that it was controllable within the linear range, the peak area was proportional to the injection volume, and there was no column overloading; and the reproducibility met the standards, with peak area RSD < 2%.

[0064] 2.3.6 Final optimization conditions

[0065] Chromatographic column: Agilent column type: ZORBAX Eclipse Plus C18 4.6×250mm×5μm.

[0066] Mobile phase A was 0.1% phosphoric acid in water, and mobile phase B was methanol.

[0067] The flow rate was 0.5 mL / min;

[0068] Column temperature 30°C;

[0069] Injection volume: 10 μL.

[0070] Table 1 Liquid chromatography mobile phase conditions

[0071]

[0072] 2.4 Linear range investigation

[0073] Accurately weigh the luteolin standard to prepare a stock solution at a concentration of 200 μg / mL. Serially dilute the stock solution to obtain luteolin standard solutions at concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL. Inject 10 μL of each solution according to the chromatographic conditions described in 2.3.6. Regress the measured peak area against the luteolin concentration. Plot a standard curve using luteolin concentration as the abscissa (X) and peak area as the ordinate (Y) for linear regression, and determine the regression equation.

[0074] 2.5 Precision test

[0075] A 40 μg / mL luteolin standard solution was injected six times continuously, and the relative standard deviation (RSD%) of the peak area was determined to verify the repeatability of the instrument.

[0076] 2.6 Stability investigation

[0077] Prepare solutions from the same batch of samples according to the method in 2.2. Inject 10 μL of the sample at 0, 4, 8, 12, and 24 hours under the chromatographic conditions in 2.3 to determine the peak area. Calculate the RSD (%) of the peak area at different time points to determine the stability of the sample solution over 24 hours.

[0078] 2.7 Repeatability test

[0079] Six samples from the same batch were processed in parallel. Solutions were prepared according to the method in 2.2. Test solutions were prepared and measured under the chromatographic conditions in 2.3. The repeatability of the method was confirmed to meet the requirements for quantitative analysis by calculating the RSD (%) of the peak area and content.

[0080] 2.8 Sample recovery test

[0081] Accurately weigh six samples (10 g each) of known concentration and quantitatively add 1 mL of a luteolin standard solution of known concentration. Prepare the test solution according to the method in 2.1 and analyze the sample. Calculate the recovery based on the measured peak area, and evaluate the accuracy of the method using the RSD (%).

[0082] 3. Test results

[0083] 3.1 Fingerprint

[0084] Nine batches of Polygonum vicifolium preparations (S1-S9) were scanned at full wavelength, and automatic peak matching and baseline correction were performed using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012A Edition) to establish a common pattern digital fingerprint. The angle cosine method was used to calculate the similarity between batches, and the threshold value >0.90 was set as the qualification standard. The data were analyzed using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) software. With Y1 as the reference spectrum, the median method was used, the time width was set to 0.1min, and multi-point correction was performed before automatic matching to generate superimposed fingerprints and control fingerprints. The similarity of the nine batches of samples relative to the control fingerprint R was greater than 0.95, and the results are as follows: Figure 3 , Table 2. By comparing with the reference substances, 8 common peaks were identified, namely luteolin, luteolin, chlorogenic acid, quercetin, rutin, vitexin, kaempferol, and β-sitosterol. Figure 4 .

[0085] Table 2 Similarity evaluation results

[0086]

[0087] 3.2 Content determination

[0088] 3.2.1 Linear range investigation

[0089] With the concentration of luteolin reference substance as the horizontal axis (x) and the chromatographic peak area as the vertical axis (y), linear regression was performed to obtain the regression equation y=33324x-17021, R 2 =0.9997. The results showed that the luteolin concentration in the range of 6.25-100 μg / mL had a good linear relationship with the peak area. Figure 5 .

[0090] 3.2.2 Precision test

[0091] The test results are shown in Table 3. The concentration of the prepared standard was 100 μg / mL, and the RSD (n=6) was calculated to be 0.274%, indicating that this method has good precision, meets the methodological validation requirements, and can meet the content determination requirements.

[0092] Table 3 Precision test

[0093]

[0094] 3.2.3 Repeatability test

[0095] The test results are shown in Table 4. The average peak area of ​​6 samples of the same batch of Polygonum villosa preparation was 332683. The calculated luteolin content was 15.2 μg / mL, and the RSD was 0.613%, indicating that the detection method had good repeatability and met the content determination requirements.

[0096] Table 4 Repeatability test

[0097]

[0098] 3.2.4 Stability test

[0099] The test results are shown in Table 5. The average peak area was measured to be 348633, and the luteolin content was calculated to be 15.65 μg / mL, which was 0.739, indicating that the Polygonum vicifolium preparation was stable within 24 h.

[0100] Table 5 Repeatability test

[0101]

[0102] 3.2.5 Sample recovery test

[0103] The test results are shown in Table 6. According to the previous test, the samples with determined concentrations were prepared and the recovery rate and RSD were calculated. The average recovery rate of luteolin in the samples was 95.2% and the RSD was 3.57%.

[0104] The recovery rate calculation formula is: Recovery rate % = (CA) / B×100%

[0105] A is the amount of the measured component contained in the sample; B is the amount of the reference substance added; C is the actual measurement;

[0106] Table 6 Sample recovery test

[0107]

[0108] The thin-layer chromatography method established by the present invention has strong specificity and high fingerprint similarity (>0.95), and the linearity, precision, stability, repeatability and accuracy of the HPLC method for determining the content of luteolin are verified and evaluated. Studies have shown that the use of HPLC fingerprints can effectively control the quality of Polygonum vitripennis preparations. The eight common characteristic peaks in the fingerprint are luteolin, luteolin, chlorogenic acid, quercetin, rutin, vitexin, kaempferol, and β-sitosterol. Through the establishment of this fingerprint, the types and quantities of chemical components contained in Polygonum vitripennis and its preparations can be more comprehensively reflected, and the quality of Polygonum vitripennis preparations can be described and evaluated as a whole. It is a comprehensive, reliable, and quantifiable quality identification method. The establishment of the HPLC content determination method for luteolin provides key technical parameters for subsequent standardized production. In the future, it can be combined with pharmacological experiments to associate key components with efficacy and optimize quality control indicators. This method can comprehensively evaluate the content of key components of Polygonum vitripennis preparations and provide a scientific basis for their quality control and clinical application.

Claims

1. A HPLC detection method for a Polygonum villosa preparation, characterized in that: The test solution was prepared after extracting the Polygonum villosa preparation, and the solution was determined by HPLC; the HPLC determination conditions were: Chromatographic column: ZORBAX Eclipse Plus C18 4.6 × 250 mm × 5 μm; Mobile phase A is 0.1% phosphoric acid or 0.1%-0.5% formic acid, and B is methanol or acetonitrile; The mobile phase conditions are as follows: Flow rate: 0.5mL / min-1.0mL / min; Column temperature 30℃-50℃; Injection volume 1μL-20μL.

2. The method according to claim 1, wherein The mobile phase A is 0.1% phosphoric acid, and the mobile phase B is methanol.

3. The method according to claim 1, wherein The flow rate is 0.5 mL / min.

4. The method according to claim 1, wherein The column temperature is 30°C.

5. The method according to claim 1, wherein The injection volume is 10 μL.

6. A method for establishing an HPLC fingerprint of a Polygonum vicifolium preparation, characterized in that: The method includes: (1) Preparation of reference solution: Take an appropriate amount of each reference substance, accurately weigh it, and prepare a 1 mg / mL solution; (2) Preparation of test solution: Take an appropriate amount of Polygonum vicifolium preparation sample and add methanol to prepare a 1 mg / mL solution; (3) HPLC detection: The test sample solution and the reference sample solution of (1) and (2) above were detected using the HPLC detection method for detecting the Polygonum vitripennis preparation according to claim 1 to obtain the test sample fingerprint chromatogram and the reference sample fingerprint chromatogram.

7. The method according to claim 6, wherein The reference substances in step (1) include chlorogenic acid, quercetin, quercetin, isoquercetin, rutin, luteolin, kaempferol, catechin, and β-sitosterol.

8. Use of the method for establishing the HPLC fingerprint of the Polygonum vitripennis preparation according to claim 6 or 7 in detecting and identifying the Polygonum vitripennis preparation.

9. The fingerprint obtained by the method for establishing the HPLC fingerprint of the Polygonum vicifolium preparation according to claim 6 or 7.

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