Peucedanum praeruptorum dunn quality control method based on HPLC fingerprint spectrum

Abstract

The invention relates to the technical field of quality control of medicinal materials, in particular to a peucedanum praeruptorum dunn quality control method based on an HPLC (High Performance Liquid Chromatography) fingerprint spectrum. The method comprises the following steps: establishing fingerprints of peucedanum praeruptorum from different producing areas, and marking common peaks and known component peaks; the obtained data is systematically analyzed by applying chemical identification technologies such as a similarity evaluation method, a system clustering method and a principal component analysis method, and a quality control system of the peucedanum praeruptorum dunn medicinal material is perfected, so that the safety and effectiveness of clinical medication of the peucedanum praeruptorum dunn medicinal material are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of quality control of medicinal materials, and particularly to a method for quality control of Peucedanum praeruptorum Dunn based on HPLC fingerprint. Background Art

[0002] Peucedanum praeruptorum Dunn is a perennial herb of the genus Peucedanum in the family Umbelliferae. The application history of Peucedanum praeruptorum Dunn in Chinese medicine is long, and its record can be traced back to "Materia Medica of Famous Physicians" at the earliest. Peucedanum praeruptorum Dunn mainly uses its roots as medicine. It is bitter, pungent, slightly cold, and belongs to the lung meridian, with the effects of dispersing wind and clearing heat, descending qi and resolving phlegm. It is mainly used to treat symptoms such as thick yellow phlegm, phlegm-heat asthma, and excessive phlegm in wind-heat cough.

[0003] In recent years, there have been many research reports on the quality evaluation of Peucedanum praeruptorum Dunn using high performance liquid chromatography fingerprint, and the related conditions and methods have also tended to be mature. However, there is no report on the quality research of Peucedanum praeruptorum Dunn from different producing areas. Summary of the Invention

[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a method for quality control of Peucedanum praeruptorum Dunn based on HPLC fingerprint to solve the problems in the prior art. The present invention establishes the fingerprint of Peucedanum praeruptorum Dunn from different producing areas in Guizhou Province, marks the common peaks and known component peaks; uses chemical recognition technologies such as similarity evaluation method, hierarchical clustering method, and principal component analysis method to systematically analyze the obtained data, improve the quality control system of Peucedanum praeruptorum Dunn in Guizhou Province, ensure the safety and effectiveness of clinical medication of Peucedanum praeruptorum Dunn, and lay a solid foundation for the further development and utilization of Peucedanum praeruptorum Dunn in Guizhou Province.

[0005] To achieve the above purpose and other related purposes,

[0006] In the first aspect of the present invention, there is provided a method for quality control of Peucedanum praeruptorum Dunn based on HPLC fingerprint, including the following steps:

[0007] Step 1: Mix Peucedanum praeruptorum Dunn with a solvent to prepare a mixed solution of (0.1-10) g / ml, perform ultrasonic treatment, and collect the filtrate of Peucedanum praeruptorum Dunn after filtration;

[0008] Step 2: Determine the filtrate of Peucedanum praeruptorum Dunn by high performance liquid chromatography. The chromatographic conditions are as follows: chromatographic column 250 m×4.6 mm, 5 μm; flow rate 0.5-1.5 mL / min -1 ; detection wavelength: 321 nm; column temperature: 25-45 °C; injection volume: 10 μL; the gradient elution program of the mobile phase is:

[0009]

[0010]

[0011] Step 3: There are at least characteristic peaks of qianhucoumarin I, praeruptorin A, praeruptorin B, and praeruptorin E in the obtained chromatogram.

[0012] In the embodiment of the present invention, the solvent in Step 1 is methanol.

[0013] In the embodiment of the present invention, the preparation process of the praeruptorum decoction filtrate is as follows: Praeruptorum is ground into powder, and the praeruptorum powder is mixed with methanol to form a mixed solution with a concentration of (0.1 - 5) g / ml. The mixed solution is ultrasonically treated for 5 - 30 min under the conditions of a power of 200 - 1000 W and a frequency of 20 - 60 kHz, and the praeruptorum decoction filtrate is collected after filtration.

[0014] In the embodiment of the present invention, the preparation process of the praeruptorum decoction filtrate is as follows: Praeruptorum is ground into powder and passed through a 50 - mesh sieve. The praeruptorum powder is mixed with methanol to form a mixed solution with a concentration of (0.5 - 2) g / ml. The mixed solution is ultrasonically treated for 8 - 15 min under the conditions of a power of 400 - 600 W and a frequency of 35 - 45 kHz, and the praeruptorum decoction filtrate is collected after filtration.

[0015] In the embodiment of the present invention, the chromatographic conditions in Step 2 are as follows: The flow rate is 0.5 - 1.0 mL / min -1 ; Column temperature: 30 - 40 °C.

[0016] In the embodiment of the present invention, the gradient elution program of the mobile phase in Step 2 is as follows:

[0017]

[0018] In the embodiment of the present invention, in Step 3: There are at least characteristic peaks of Pd - Ib, qianhucoumarin I, praeruptorin A, 3 - Hydroxy - dihydroseselin - isovalera, praeruptorin B, and praeruptorin A E in the chromatogram.

[0019] In the embodiments of the present invention, the retention time of Pd-Ib is 34.5 min to 35.0 min; the retention time of qianhucoumarin I is 42.0 min to 42.5 min; the retention time of praeruptorin A is 44.8 to 45.3 min; the retention time of 3-Hydroxy-dihydroseselin-isovalera is 48.5 to 49.0 min; the retention time of praeruptorin B is 60.5 to 61.0 min; the retention time of praeruptorin E is 64.0 to 65 min.

[0020] As described above, a method for controlling the quality of Peucedanum praeruptorum Dunn based on HPLC fingerprint has the following beneficial effects: By comparing the retention times of 49 batches of Peucedanum praeruptorum Dunn samples with those of the reference substance chromatogram, a total of 6 common peaks were co-identified and 25 characteristic peaks were marked. The establishment of this method provides an analytical method and data support for the quality evaluation of Peucedanum praeruptorum Dunn medicinal materials. Description of the Drawings

[0021] Figure 1 It is the HPLC fingerprint of 49 batches of Peucedanum praeruptorum Dunn medicinal materials.

[0022] Figure 2 It is the HPLC reference fingerprint of Peucedanum praeruptorum Dunn medicinal materials.

[0023] Figure 3 It is the common pattern chromatogram of Peucedanum praeruptorum Dunn medicinal materials and the HPLC of reference substances, where Figure 3 A is the HPLC chromatogram of the methanol extract of Peucedanum praeruptorum Dunn, Figure 3 B is the HPLC chromatogram of the mixed reference substances.

[0024] Figure 4 It is the clustering analysis result of the peak areas of the common peaks of 49 batches of medicinal materials.

[0025] Figure 5 It is the scree plot of the principal component analysis. Detailed Embodiments

[0026] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification.

[0027] 1 Materials

[0028] Peucedanum praeruptoside A reference substance (batch number PCS-2105278, purity ≥ 98%, Chengdu Zhibiao Huachun Biological Company), Peucedanum praeruptoside B reference substance (batch number PCS-210508, purity ≥ 98%, Chengdu Zhibiao Huachun Biological Company), Peucedanum praeruptoside E reference substance (batch number RFS-B04401902026, purity ≥ 99.98%, Chengdu Ruifensi Biotechnology Co., Ltd.); the methanol and acetonitrile used in the analysis of the samples were chromatographically pure; the methanol used in the preparation of the samples was analytically pure; the water was Wahaha purified water.

[0029] 2. Sample information

[0030] In this experiment, a total of 49 batches of Peucedanum peucedanum samples were collected from different regions of Guizhou Province, including 19 batches of samples collected in 2022 and 30 batches of samples collected in 2023. See Table 1 for details.

[0031] Table 1 Sample collection information table

[0032]

[0033]

[0034] Example 1

[0035] Quality control method of Peucedanum peucedanum based on HPLC fingerprint

[0036] 1HPLC detection method of Peucedanum peucedanum

[0037] Step 1: Preparation of reference solution

[0038] Accurately weigh an appropriate amount of each reference substance, add methanol to prepare a reference substance stock solution with a concentration of 50 μg / ml.

[0039] Step 2: Preparation of test solution

[0040] Grind each batch of dried Peucedanum peucedanum medicinal materials to make them pass through a No. 3 sieve, take about 1.0 g of each medicinal material powder, accurately weigh it, put it in a stoppered conical flask, accurately add 10 mL of methanol solution, stopper it, weigh it, treat it with ultrasound (power 500 W, frequency 40 kHz) for 10 minutes, let it cool, weigh it again, add methanol to make up for the lost weight, shake it well, let it stand, aspirate the supernatant and filter it with a 0.45 μm microporous membrane to obtain the product.

[0041] Step 3: Chromatographic conditions of Peucedanum peucedanum

[0042] Chromatographic column: SuperLu C18 (2) chromatographic column (250mm×4.6mm, 5μm); flow rate: 0.8mLmin-1; detection wavelength: 321nm; column temperature: 35℃; injection volume: 10μL; mobile phase: water (A)-methanol (B); gradient elution: 0-15min, 50%-55%B; 15-16min, 55%-65%B; 16-30min, 65%-70%B; 30-55min, 70%-80%B; 55-70min, 80%-100%B; 70-75min, 100%-50%B. A total of 75min was collected.

[0043] 2 Methodological investigation

[0044] 2.1 Precision test

[0045] Take 1 portion of sample powder (No.: S40), accurately weigh it, prepare the test solution according to the above method, inject the test solution 6 times continuously according to the above chromatographic conditions, and record the relative peak area and relative retention time of each main chromatographic peak. The HPLC spectrum was imported into the similarity software for analysis. The results showed that the similarity of the 6 injection peaks was greater than 0.90, and the RSD of the peak area of ​​each main chromatographic peak was less than 2%, indicating that the precision of the instrument was good. The results are shown in Table 2.

[0046] Table 2 Precision test results (n = 6)

[0047]

[0048] 2.2 Repeatability test

[0049] Take 6 samples of the same batch of white flower peucedanum medicinal materials (No.: S40), prepare each test solution according to the above preparation method, and measure according to the above chromatographic conditions, and record the common peak area of ​​each sample. The obtained HPLC spectrum is imported into the similarity evaluation software for analysis. The results show that the RSD value of the relative peak area of ​​each chromatographic peak is less than 2%, indicating that the repeatability of this method is good. The results are shown in Table 3.

[0050] Table 3 Repeatability test results (n=6)

[0051]

[0052]

[0053] 2.3 Stability test

[0054] Take the sample (No.: S40) as the object of investigation, weigh it accurately, prepare each test solution according to the above method, and determine the samples at 0, 3, 6, 9, 12, and 24 hours respectively according to the above chromatographic conditions. Record the peak areas to investigate its stability. Import the HPLC chromatograms into the similarity software for analysis. The results show that the RSD values of the relative peak areas of each common peak in the sample are all less than 2%, indicating that the test solution has good stability within 24 hours. The results are shown in Table 4.

[0055] Table 4 Results of the stability test (n = 6)

[0056]

[0057] 3 Establishment of fingerprint and similarity evaluation

[0058] 3.1 Establishment of fingerprint

[0059] According to the above method for preparing the test solution, prepare 49 batches of Peucedani Radix samples into test solutions, and determine them according to the above chromatographic conditions. The recording time of the chromatogram is 75 min. Import the obtained HPLC chromatograms in AIA format into the software of "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (Version 2.0)". Since gradient elution is used in the experiment, the retention times of the chromatographic peaks in the HPLC chromatograms of 49 batches of Peucedani Radix may vary slightly. Therefore, the multi-point correction method is used for peak similarity calculation. Select S1 as the reference chromatogram, the median method, the time window width is 0.1 min, and the full-spectrum peak matching mode is used to establish the superimposed chromatogram and the reference chromatogram of HPLC of Peucedani Radix from different producing areas in Guizhou Province.

[0060] Figure 1 The HPLC fingerprint of 49 batches of Peucedani Radix samples. The results show that a total of 11 common peaks are determined in the HPLC fingerprints of 49 batches of Peucedani Radix samples.

[0061] 3.2 Identification of common peaks and calibration of characteristic peaks

[0062] Detect each prepared reference solution according to the above chromatographic conditions and record the chromatogram.

[0063] Figure 2 The HPLC reference fingerprint of Peucedani Radix samples. By comparing the retention times with the sample chromatograms, a total of 6 common peaks are identified. That is, peak 1 is Pd-Ib (compound 6); peak 3 is qianhucoumarin I (compound 9)); peak 4 is praeruptorin A (compound 4); peak 5 is 3-Hydroxy-dihydroseselin-isovalera (compound 13); peak 8 is praeruptorin B (compound 2); peak 10 is peucedanin E (compound 10).

[0064] The CAS number of compound 6 is 477-33-8, and its structural formula is shown as follows:

[0065] The CAS number of compound 9 is 53023-17-9, and its structural formula is shown as follows:

[0066] The CAS number of compound 13 is 19380-07-05, and its structural formula is shown as follows:

[0067] Figure 3 It is the common pattern chromatogram of Peucedani Radix and the reference substance HPLC, among which Figure 3 A is the HPLC chromatogram of the methanol extract of Peucedani Radix, Figure 3 B is the HPLC chromatogram of the mixed reference substances. By comparing the retention times of the HPLC chromatograms of the mixed reference substances and the methanol extract of Peucedani Radix, 25 characteristic peaks were marked out. That is, the retention time of compound 1 is 34.748 min; the retention time of compound 2 is 60.767 min; the retention time of compound 3 is 64.424 min; the retention time of compound 4 is 45.047 min; the retention time of compound 5 is 27.093 min; the retention time of compound 6 is 34.794 min; the retention time of compound 7 is 45.373 min; the retention time of compound 8 is 43.719 min; the retention time of compound 9 is 42.323 min; the retention time of compound 10 is 47.798 min; the retention time of compound 11 is 37.715 min; the retention time of compound 12 is 44.905 min; the retention time of compound 13 is 48.646 min; the retention time of compound 14 is 64.335 min; the retention time of compound 15 is 47.319 min; the retention time of compound 19 is 19.009 min; the retention time of compound 20 is 22.719 min; the retention time of compound 21 is 20.297 min; the retention time of compound 22 is 23.716 min; the retention time of compound 23 is 35.773 min; the retention time of compound 24 is 18.977 min; the retention time of compound 27 is 7.931 min; the retention time of compound 28 is 7.412 min; the retention time of compound 29 is 20.239 min.

[0068] 3.3 Fingerprint chromatogram similarity analysis

[0069] Taking the control spectrum R as the reference spectrum, the fingerprint spectra of 49 batches of Peucedanum praeruptorum Dunn samples were compared with the control fingerprint spectrum, and the similarity values of the fingerprint spectra of Peucedanum praeruptorum Dunn medicinal materials in each batch were calculated. The results showed that the similarities of 11 common peaks in the HPLC fingerprint spectra of 49 batches of Peucedanum praeruptorum Dunn samples were all greater than 0.9. The similarity results are shown in Table 5.

[0070] Table 5 Similarity results of Peucedanum praeruptorum Dunn from different origins

[0071]

[0072]

[0073] 3.4 Cluster analysis of fingerprint spectra of Peucedanum praeruptorum Dunn medicinal materials from different origins

[0074] Excel was used to quantify the peak areas of the obtained common peaks, and the data were imported into SPSS 26.0 software. The Ward method was selected, and cluster analysis was carried out by the measurement method of squared Euclidean distance. The results are shown in Figure 4 . Figure 4 It is the cluster analysis result of the peak areas of common peaks of 49 batches of medicinal materials.

[0075] The cluster analysis results showed that when the distance was 20, the Peucedanum praeruptorum Dunn medicinal materials from 49 different origins in Guizhou Province were divided into two categories: S4, S17, S21, S22, S7, S39, S25 were grouped into one category, and S29, S42, S15, S35, S49, S9, S28, S27, S41, S2, S26, S36, S18, S23, S5, S19, S40, S11, S24, S33, S8, S43, S30, S10, S45, S46, S44, S47, S16, S20, S34, S31, S37, S6, S13, S38, S48, S12, S32, S1, S14, S3 samples were grouped into one category. When the distance was 10, the Peucedanum praeruptorum Dunn medicinal materials from 49 different origins in Guizhou Province were divided into five categories: S29, S42, S15, S35, S49, S9, S28, S27, S41, S2, S26, S36, S18, S23, S5, S19, S40, S11, S24, S33 were grouped into one category; S8, S43, S30, S10 were grouped into one category; S45, S46, S44, S47, S16, S20, S34, S31, S37, S6, S13, S38, S48, S12, S32, S1, S14, S3 were grouped into one category; S4, S17, S21, S22, S7, S39 were grouped into one category; S25 formed a separate category. According to the results of the cluster analysis, it can be seen that the 49 batches of Peucedanum praeruptorum Dunn samples are not completely the same, and there are certain differences in the quality and component contents of Peucedanum praeruptorum Dunn medicinal materials from different origins in Guizhou Province.

[0076] 3.5 PCA of Peucedani Radix Medicinal Materials from Different Origins

[0077] Based on the liquid chromatography maps of 49 batches of Peucedani Radix from different origins, common peaks were screened out, and principal component analysis was performed on the peak areas of all common peaks using SPSS 26.0 software.

[0078] The results are shown in Table 6 Figure 5 which is the scree plot of principal component analysis. When the eigenvalue is not less than 1, the cumulative variance contribution rate of the first 4 principal components is 74.927%. From Figure 5 the scree plot of principal component analysis, it can be seen that the slopes of the first 4 principal components are relatively steep, and the subsequent slopes tend to be gentle, indicating that the first 4 principal components can represent the quality of Peucedani Radix medicinal materials. Therefore, these 4 components can be extracted as the main components to evaluate the quality of 49 batches of Peucedani Radix samples.

[0079] Table 6 Eigenvalues and Cumulative Variance Contribution Rates

[0080]

[0081]

[0082] Analysis was carried out on the first 4 principal components to obtain the common peak factor loading matrix, as shown in Table 7. The main influencing factor of principal component 1 mainly comes from peak 10, i.e., peucedanin E; the main influencing factor of principal component 2 mainly comes from peak 8, i.e., peucedanidin B; the main influencing factor of principal component 3 mainly comes from the chemical components of peak 6; the main influencing factor of principal component 4 mainly comes from the chemical components of peak 3; among them, the correlation coefficient of praeruptorin A is the largest in principal component 2. Thus, it can be seen that the quality differences of Peucedani Radix from different origins in Guizhou Province may be related to the contents of different chemical components.

[0083] Table 7 Principal Component Loading Matrix

[0084]

[0085] In summary, the present invention has established a high-performance liquid chromatography fingerprint of 49 batches of Peucedanum praeruptorum Dunn from different producing areas in Guizhou Province. The similarity results show that the similarity of the fingerprint peaks of Peucedanum praeruptorum Dunn from 49 different producing areas is above 0.9, indicating that the established fingerprint method is feasible. By comparing the retention times of 49 batches of Peucedanum praeruptorum Dunn samples with the reference substance chromatogram, 6 common peaks and 25 characteristic peaks were identified. According to the results of cluster analysis and principal component analysis, there are certain differences in the quality and component content of Peucedanum praeruptorum Dunn from different producing areas in Guizhou Province. By comprehensively considering the peak area information of the fingerprint chromatogram, it is found that the quality of Peucedanum praeruptorum Dunn from Liupanshui, Guizhou is relatively good. The quality differences of the medicinal materials from different producing areas may be related to the content of different chemical components. The establishment of this method provides an analytical method and data support for the quality evaluation of Peucedanum praeruptorum Dunn medicinal materials.

[0086] Therefore, the present invention effectively overcomes various disadvantages in the prior art and has high industrial utilization value.

[0087] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A quality control method for Peucedanum peucedanum based on HPLC fingerprint, characterized in that: The steps include: Step 1, mixing Peucedanum peucedanum with a solvent to prepare a mixed solution of (0.1-10) g / ml, ultrasonically treating, filtering and collecting the Peucedanum peucedanum filtrate; Step 2: The filtrate of Peucedanum peucedanum was determined by high performance liquid chromatography. The chromatographic conditions were: chromatographic column 250m×4.6mm, 5μm; flow rate 0.5~1.5mL.min -1 ; Detection wavelength: 321nm; Column temperature: 25-45°C; injection volume: 10 μL; gradient elution program of mobile phase: Step 3: The obtained chromatogram contains at least characteristic peaks of qianhucoumarin I, qianhucoumarin A, qianhucoumarin B and qianhucoumarin E.

2. A method for quality control of Peucedanum peucedanum based on HPLC fingerprint according to claim 1, characterized in that: The solvent in step 1 is methanol.

3. A method for controlling the quality of Peucedanum peucedanum based on HPLC fingerprint according to claim 2, characterized in that: The preparation process of the peucedanum peucedanum filtrate is as follows: the peucedanum peucedanum powder is ground into powder, the peucedanum peucedanum powder is mixed with methanol to prepare a mixed solution of (0.1-5) g / ml, the mixed solution is ultrasonically treated for 5-30 minutes under the conditions of power of 200-1000 W and frequency of 20-60 kHz, and the peucedanum peucedanum filtrate is collected after filtration.

4. A method for controlling the quality of Peucedanum peucedanum based on HPLC fingerprint according to claim 3, characterized in that: The preparation process of the peucedanum peucedanum filtrate is as follows: the peucedanum peucedanum powder is ground into powder and passed through a 50-mesh sieve, the peucedanum peucedanum powder is mixed with methanol to prepare a mixed solution of (0.5-2) g / ml, the mixed solution is ultrasonically treated for 8-15 minutes under the conditions of a power of 400-600 W and a frequency of 35-45 kHz, and the peucedanum peucedanum filtrate is collected after filtration.

5. A method for controlling the quality of Peucedanum peucedanum based on HPLC fingerprint according to claim 1, characterized in that: The chromatographic conditions of step 2 are: flow rate 0.5-1.0 mL.min -1 ; Column temperature: 30~40℃.

6. A method for controlling the quality of Peucedanum peucedanum based on HPLC fingerprint according to claim 1, characterized in that: The gradient elution procedure of the mobile phase in step 2 is:

7. A method for controlling the quality of Peucedanum peucedanum based on HPLC fingerprint according to claim 1, characterized in that: In the step 3, the chromatogram contains at least characteristic peaks of Pd-Ib, qianhucoumarin I, peucedanum purpurogenum A, 3-Hydroxy-dihydroseselin-isovalera, peucedanum purpurogenum B, and peucedanum purpurogenum A E.

8. A method for controlling the quality of Peucedanum peucedanum based on HPLC fingerprint according to claim 7, characterized in that: The retention time of Pd-Ib is 34.5min to 35.0min; The retention time of qianhucoumarin I is 42.0 min to 42.5 min; The retention time of praeruptorin A is 44.8-45.3 min; The retention time of the 3-Hydroxy-dihydroseselin-isovalera is 48.5-49.0 min; The retention time of peucedanum acetogenin is 60.5-61.0 min; The retention time of peucedanum praeruptorum E is 64.0-65 ​​min.

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