Method for establishing fingerprint spectrum of radix pseudostellariae medicinal material

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for establishing a fingerprint spectrum of a radix pseudostellariae medicinal material. The establishment method comprises the following steps: (1) preparation of a test solution: taking a radix pseudostellariae medicinal material, extracting with 95% ethanol, filtering, drying filtrate, and adding an acetonitrile solution for redissolving to obtain the test solution; and (2) sample introduction analysis: injecting the test solution into a high performance liquid chromatograph for analysis, and constructing the fingerprint spectrum of the radix pseudostellariae medicinal material by taking a common chromatographic peak as a common characteristic peak. The method has the advantages of being good in peak separation effect, large in number of common peaks and high in accuracy and similarity.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for establishing a fingerprint spectrum of Radix Pseudostellariae Radix. Background Art

[0002] The traditional Chinese medicine Pseudostellaria heterophylla is the dried root of Pseudostellaria heterophylla (Miq.) Pax exPax et Hoffm. of the Caryophyllaceae family. It has the effects of invigorating qi and strengthening the spleen, promoting body fluid and moistening the lungs. It also has pharmacological activities such as immunomodulation, anti-tumor, and hypoglycemic effects.

[0003] Traditional Chinese medicine fingerprint is based on the identification of multiple chemical components and can be used to comprehensively evaluate the quality of traditional Chinese medicine. Currently, there are many literatures and patent applications reporting and publicizing the fingerprint technology of Radix Pseudostellariae Radix, but there are problems such as poor peak separation, small number of common peaks that can be used for comparison, and low accuracy of fingerprint.

[0004] For example, prior art 1 (Liu Weixiang, Hu Haiyun, Liu Xichun, et al. Study on HPLC Chromatographic Fingerprint of Radix Pseudostellariae [J]. Chinese Herbal Medicine, 2007, 38 (5): 4) discloses the study on HPLC chromatographic fingerprint of Radix Pseudostellariae, and its chromatographic conditions are as follows: chromatographic column: DikmaKromasil C18 (250mm×4.6mm, 5pm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution program (A): 0-25min: 0%-15%; 25-40min: 15%-16%; 40-45min: 16%-25%; 45-75min: 25%-50%. Volume flow rate: 0.8mL / min; column temperature: 25℃; injection volume: 10μL; detection wavelength: 203nm. The preparation of the test solution includes: accurately weighing 1.0g of Pseudostellariae Radix (coarse powder), adding 50mL of 50% ethanol, ultrasonicating for 30min (300W), filtering, evaporating the filtrate to dryness, dissolving the residue in 10mL of water, extracting with 30mL of n-butanol saturated with water, washing the extract with 20mL of water saturated with n-butanol, evaporating the n-butanol solution, dissolving the residue in 50% methanol, and making up to 5mL, shaking well. Pass through a 0.45um microporous filter membrane to obtain. Its fingerprint is as follows Figure 1 As shown. Figure 1 It can be seen that the separation degree of peak 4 and peak 5 of its fingerprint spectrum is poor, and the number of common peaks is small, only 7 peaks, and the accuracy of the fingerprint spectrum needs to be further improved.

[0005] Chinese patent application CN114720568A discloses a fingerprint establishment method and application of Radix Pseudostellariae Radix, which adopts high performance liquid chromatography, and the conditions of the high performance liquid chromatography include: using octadecylsilane bonded silica gel as filler; using acetonitrile as mobile phase A and water as mobile phase B to form a mixed mobile phase, eluting according to the gradient of the volume ratio of mobile phase A to mobile phase B in the mixed mobile phase from 5:95 to 90:10 within 0 to 55 minutes; the detection wavelength is 203nm. However, it also has only 12 common peaks, and the accuracy of the fingerprint spectrum needs to be further improved.

[0006] Therefore, it is very necessary to develop a method for establishing the fingerprint of Radix Pseudostellariae Radix that can solve the above technical problems. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for establishing a fingerprint spectrum of Radix Pseudostellariae with better peak separation effect and a larger number of common peaks.

[0008] The present invention is achieved through the following technical solutions:

[0009] A method for establishing a fingerprint spectrum of Radix Pseudostellariae Radix, comprising the following steps:

[0010] (1) Preparation of test solution: Extract Radix Pseudostellariae Radix with 95% ethanol, filter, dry the filtrate and re-dissolve it in acetonitrile solution to obtain the test solution;

[0011] (2) Sampling analysis: The sample solution was injected into a high performance liquid chromatograph for analysis, and the common chromatographic peaks were used as common characteristic peaks to construct the fingerprint of Pseudostigma pseudoginseng.

[0012] In some embodiments of the present invention, the volume concentration of the acetonitrile solution in step (1) is 20%.

[0013] In some embodiments of the present invention, the extraction method in step (1) is heating reflux extraction.

[0014] In some embodiments of the present invention, the dosage ratio of the Radix Pseudostellariae Radix and 95% ethanol in step (1) is 1 g:17.5 mL.

[0015] In some embodiments of the present invention, the extraction time in step (1) is 1 hour.

[0016] In some embodiments of the present invention, the dosage ratio of Radix Pseudostellariae Radix to acetonitrile solution in step (1) is 4 g:5 mL.

[0017] In some embodiments of the present invention, in step (1), the Radix Pseudostellariae Radix is ​​first ground into powder and then extracted with 95% ethanol.

[0018] In some preferred embodiments of the present invention, the powder in step (1) is passed through a No. 5 sieve.

[0019] In some embodiments of the present invention, the establishment method also includes the preparation of a reference solution: selecting Radix Pseudostellariae Cyclic Peptide A, Radix Pseudostellariae Cyclic Peptide B, Radix Pseudostellariae Cyclic Peptide D, Radix Pseudostellariae Cyclic Peptide A, Radix Pseudostellariae Cyclic Peptide B, Radix Pseudostellariae Cyclic Peptide D, Radix Pseudostellariae Cyclic Peptide E and Radix Pseudostellariae Cyclic Peptide H as reference substances, adding methanol to dissolve them, and preparing a reference solution; in step (2), the test solution and the reference solution are injected into a high performance liquid chromatograph for analysis, and the common chromatographic peaks are used as common characteristic peaks to construct a Radix Pseudostellariae medicinal material fingerprint.

[0020] In some embodiments of the present invention, the Pseudostellariae Radix fingerprint spectrum includes at least 20 common characteristic peaks, and the relative retention time ranges of the peak Pseudostellariae Radix Cyclopeptide B are as follows: 0.0642-0.0643, 0.0806-0.0807, 0.0945-0.0947, 0.1191-0.1193, 0.1637-0.1643, 0.1917-0.1923, 0.2474-0.2480, 0.3300-0. 0.3314, 0.3791-0.3804, 0.3967-0.3983, 0.4433-0.4452, 0.4642-0.4662, 0.7646-0.7664, 0.7946-0.7965, 1.0000, 1.0854-1.0890, 1.1044-1.1068, 1.1550-1.1591, 1.2282-1.2320, 1.2520-1.2564.

[0021] In some embodiments of the present invention, the test conditions of the high performance liquid chromatograph in step (2) include: using variable wavelength detection, specifically 0-10.5 min, 220 nm; 10.5-35 min, 270 nm; 35-135 min, 203 nm.

[0022] In some embodiments of the present invention, the test conditions of the high performance liquid chromatograph in step (2) include

[0023] In summary, acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B.

[0024] In some embodiments of the present invention, the test conditions of the high performance liquid chromatograph in step (2) also include: the elution method is gradient elution, specifically: 0-30 min, 10%-16% A; 30-35 min, 16%-23% A; 35-85 min, 23%-43% A; 85-91 min, 43%-67% A; 91-115 min, 67%-95% A; 115-135 min, 95% A.

[0025] In some embodiments of the present invention, the test solution and the reference solution are filtered through a 0.22 μm microporous filter membrane before injection in step (2).

[0026] In some embodiments of the present invention, the test conditions of the high performance liquid chromatograph include:

[0027] Chromatographic column: Agilent ZORBAX Eclipse Plus C18, 5 μm, 4.6 × 250 mm;

[0028] Flow rate: 1ml / min;

[0029] Column temperature: 25°C;

[0030] Injection volume: 20 μL.

[0031] The beneficial effects of the present invention are:

[0032] The present invention optimizes the extraction solvent, and the chromatogram finally obtained has good and clear peak shape and high separation degree, which is conducive to constructing the fingerprint spectrum and improving the evaluation accuracy.

[0033] The present invention screens the re-dissolving solvent and finds that only the acetonitrile solution can be used to re-dissolve the extracted substance.

[0034] The present invention further optimizes the elution procedure. The chromatographic peaks obtained by adopting the elution procedure of the present invention are more evenly distributed, have more peaks, and have better separation.

[0035] The present invention adopts an ultraviolet detector to perform detection under the three wavelength switching conditions of 203nm, 220nm and 270nm, and the obtained chromatographic peak separation is good and the number of peaks is large.

[0036] The number of common peaks obtained by the fingerprint spectrum constructed by the present invention is as high as 20, and the precision, stability and repeatability are good. The similarity evaluation results of 49 batches of Radix Pseudostellariae Radix are relatively high. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the fingerprint of prior art 1.

[0038] Figure 2This is the screening result of the elution program.

[0039] Figure 3 This is the screening result of the detection wavelength.

[0040] Figure 4 It shows the screening results of extraction solvents and the local magnified images of peaks 1, 2, and 3.

[0041] Figure 5 This is the chromatogram of the S1 sample and the local enlarged image of peaks 1, 2, and 3.

[0042] Figure 6 The following are chromatograms of 8 reference substances. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0044] Example 1

[0045] 1. Purpose of the experiment

[0046] A new HPLC fingerprint method for Pseudostellariae Radix was established, and the Pseudostellariae Radix produced in Hebei was included in the quality assessment scope. On the basis of sufficient batches of medicinal materials, as many common chromatographic peaks as possible were obtained to facilitate quality evaluation, and the chromatographic peaks were identified by qualitative method based on retention time using reference substances.

[0047] 2. Implementation steps

[0048] 2.1 Step 1: Preparation of Pseudostellariae Radix Test Solution. Take about 4.00 g of Pseudostellariae Radix powder (passed through No. 5 sieve), weigh accurately, place in a stoppered conical flask, add 70 mL of 95% (v / v) ethanol, heat and reflux for 1 h, cool, filter, wash with 95% (v / v) ethanol several times, combine the filtrate and washings, evaporate to dryness, add 20% (v / v) acetonitrile solution to dissolve, transfer to a 5 mL volumetric flask, and dilute to volume.

[0049] 2.2 Step 2: Preparation of reference solution. Take Heterophyllin A (Pseudostellarin A, HA), Heterophyllin B (Pseudostellarin B, HB), Heterophyllin D (Pseudostellarin D, HD), Pseudostellarin A (Pseudostellarin A, PA), Pseudostellarin B (Pseudostellarin B, PB), Pseudostellarin D (Pseudostellarin D, PD), Pseudostellarin E (Pseudostellarin E, PE), Pseudostellarin G (Pseudostellariae cyclotide heptad, PG) 8 kinds of Pseudostellariae cyclotide reference substances were accurately weighed in appropriate amounts, and methanol was added to make up to volume to obtain mixed solutions of Pseudostellariae cyclotide reference substances with concentrations of 116.3666μg / mL, 105.2800μg / mL, 101.2121μg / mL, 120.1714μg / mL, 101.6322μg / mL, 102.7910μg / mL, 110.0299μg / mL, and 102.6577μg / mL, respectively.

[0050] 2.3 Step 3: Sample injection and analysis. The test solution and reference solution were filtered through a 0.22 μm microporous membrane and injected into a high performance liquid chromatograph to record the chromatogram.

[0051] The test conditions of the high performance liquid chromatograph include: chromatographic column: Agilent ZORBAX Eclipse Plus C18, 5μm, 4.6×250mm. Mobile phase: acetonitrile (A), 0.1% phosphoric acid aqueous solution (B). Elution program: 0-30min, 10%-16%A; 30-35min, 16%-23%A; 35-85min, 23%-43%A; 85-91min, 43%-67%A; 91-115min, 67%-95%A; 115-135min, 95%A. Wavelength: 0-10.5min, 220nm; 10.5-35min, 270nm; 35-135min, 203nm. Flow rate: 1ml / min. Column temperature: 25℃. Injection volume: 20μL.

[0052] 2.4 Step 4: Evaluate similarity. Import the obtained chromatogram into the Chinese medicine chromatographic fingerprint similarity system to evaluate the similarity.

[0053] 3. Solution Optimization

[0054] 3.1 Screening of elution procedures

[0055] Take Radix Pseudostellariae Radix powder, extract, inject, and perform gradient elution according to the three programs in Table 1. The detection wavelength is 203nm. The rest is the same as "2. Implementation steps". Export the chromatogram. The results are shown in Table 1 and Figure 2 As shown, it was found that elution procedure 3 obtained more chromatographic peaks and better separation.

[0056] Table 1 Screening results of elution program

[0057]

[0058]

[0059] 3.2 Screening of detection wavelength

[0060] Take Radix Pseudostellariae Radix powder, extract, and analyze 4 times. Use UV detector to detect under 203nm, 220nm, 270nm, and 3 wavelength switching conditions. The rest is the same as "2. Implementation steps". The results are shown in Table 2 and Figure 3 As shown, it was found that a large number of clear chromatographic peaks could be retained by wavelength switching technology.

[0061] Table 2 Screening results of detection wavelength

[0062]

[0063] 3.3 Screening of extraction solvent

[0064] Take 5 portions of the same batch of Radix Pseudostellariae Radix powder, extract them with methanol, 95% ethanol, 80% methanol, 75% ethanol, and 50% ethanol, respectively, inject and analyze, and the rest is the same as "2. Implementation steps". Export the chromatogram, the results are shown in Table 3 and Figure 4 As shown, it was found that the chromatographic peaks obtained by extraction with 95% ethanol were more numerous and clear, so the extraction solvent was determined to be 95% ethanol.

[0065] Table 3 Screening results of extraction solvents

[0066] Extraction solvent Chromatogram information Methanol Peaks 1, 2, and 3 have poor peak shapes or are invisible, and the separation is low. 95% ethanol Peaks 1, 2, and 3 have good and clear shapes and high separation 80% Methanol Peaks 1, 2, and 3 have poor peak shapes or are invisible, and the separation is low. 75% ethanol Peaks 1, 2, and 3 have poor peak shapes or are invisible, and the separation is low. 50% ethanol Peaks 1, 2, and 3 have poor peak shapes or are invisible, and the separation is low.

[0067] 3.4 Screening of reconstitution solvents

[0068] Take Radix Pseudostellariae powder, extract it, and reconstitute it with 80% methanol, 50% methanol, and 20% acetonitrile respectively. The rest is the same as "2. Implementation Steps". It turns out that it is extremely difficult to reconstitute the evaporated sample with 80% methanol and 50% methanol.

[0069] 4. Methodological investigation

[0070] 4.1 Collecting samples

[0071] A total of 49 batches of Pseudostellariae Radix commercial products of different specifications and grades from three production areas, Fujian, Guizhou and Hebei, were collected from markets, medicinal material companies and production areas. The specific information is shown in Table 4.

[0072] Table 449 Batch information of medicinal materials

[0073]

[0074]

[0075]

[0076] 4.2 Determination of common peaks and identification of chromatographic peaks

[0077] 49 batches of Pseudostellariae Radix commercial products were used to prepare the test solution, and the sample was injected for analysis. According to the results, 20 common chromatographic peaks were screened out ( Figure 5 ). Using S1 as reference, match the chromatogram of the mixed reference substance ( Figure 6 ), the common chromatographic peaks were identified, and peak 13, peak 15, peak 17, peak 18, peak 19, and peak 20 were identified as PA, HB, HD, HA, PE, and PG, respectively.

[0078] 4.3 Precision investigation

[0079] The samples of Radix Pseudostellariae Radix were randomly sampled to prepare the test solution, and the analysis was repeated 6 times. The relative peak area and relative retention time of each common peak were counted with peak 15 as the reference, and the RSD value was calculated. See Table 5-Table 6 for details.

[0080] Table 5 Relative retention time and RSD value of each common peak

[0081]

[0082]

[0083] Table 6 Relative peak areas and RSD values ​​of common peaks

[0084]

[0085]

[0086] 4.4 Stability investigation

[0087] The samples of Radix Pseudostellariae Radix were randomly sampled to prepare the test solution, which was analyzed 6 times at 0h, 3h, 6h, 12h, 18h and 24h, and the relative peak area and relative retention time of each common peak were counted with peak 15 as reference, and the RSD value was calculated. See Table 7-Table 8 for details.

[0088] Table 7 Relative retention time and RSD value of each common peak

[0089]

[0090] Table 8 Relative peak areas and RSD values ​​of common peaks

[0091]

[0092]

[0093] 4.5 Repeatability study

[0094] Six portions of the same batch of Radix Pseudostellariae Radix were randomly sampled to prepare six test solutions, which were analyzed six times. Peak 15 was used as a reference to calculate the relative peak area and relative retention time of each common peak and the RSD value. See Table 9-Table 10 for details.

[0095] Table 9 Relative retention time and RSD value of each common peak

[0096]

[0097]

[0098] Table 10 Relative peak areas and RSD values ​​of common peaks

[0099]

[0100]

[0101] 4.6 Similarity Evaluation

[0102] The 49 batches of Pseudostellariae Radix analyzed spectra were imported into the similarity evaluation software of traditional Chinese medicine fingerprint spectra, the interference peaks in the first 3 minutes were cut off, S1 was used as the reference spectrum, and the 20 common peaks were matched as Mark peaks to obtain the reference spectrum, and the similarity was evaluated. The results are shown in Table 11. The results show that compared with the reference spectrum, the similarity is above 0.888, indicating that the quality consistency of each batch of Pseudostellariae Radix is ​​good.

[0103] Table 11 Similarity evaluation results

[0104]

[0105]

[0106] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for establishing a fingerprint of Radix Pseudostellariae Radix, characterized in that: The steps include: (1) Preparation of test solution: Extract Radix Pseudostellariae Radix with 95% ethanol, filter, dry the filtrate and re-dissolve it in acetonitrile solution to obtain the test solution; (2) Sampling analysis: The sample solution was injected into a high performance liquid chromatograph for analysis, and the common chromatographic peaks were used as common characteristic peaks to construct the fingerprint of Pseudoradix pseudoginseng.

2. The establishment method according to claim 1, characterized in that: The test conditions of the high performance liquid chromatograph in step (2) include: using variable wavelength detection, specifically 0-10.5 min, 220 nm; 10.5-35 min, 270 nm; 35-135 min, 203 nm.

3. The establishment method according to claim 1, characterized in that: The test conditions of the high performance liquid chromatograph in step (2) include: using acetonitrile as mobile phase A and using 0.1% phosphoric acid aqueous solution as mobile phase B.

4. The establishment method according to claim 3, characterized in that: The elution method is gradient elution, specifically: 0-30 min, 10%-16% A; 30-35 min, 16%-23% A; 35-85 min, 23%-43% A; 85-91 min, 43%-67% A; 91-115 min, 67%-95% A; 115-135 min, 95% A.

5. The establishment method according to claim 1, characterized in that: The extraction method in step (1) is heating reflux extraction.

6. The establishment method according to claim 1, characterized in that: The volume concentration of the acetonitrile solution in step (1) is 20%.

7. The establishment method according to claim 1, characterized in that: The method also includes the preparation of a reference solution: selecting Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix D, Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix D, Pseudostellariae Radix E and Pseudostellariae Radix H as reference substances, adding methanol to dissolve, and preparing a reference solution; In step (2), the test solution and the reference solution are injected into a high performance liquid chromatograph for analysis, and the common chromatographic peaks are used as common characteristic peaks to construct a fingerprint of Radix Pseudostellariae Radix.

8. The establishment method according to claim 7, characterized in that: The fingerprint spectrum of Radix Pseudostellariae Radix includes at least 20 common characteristic peaks, and the relative retention time ranges of the fingerprint spectrum of the Radix Pseudostellariae Radix Cyclopeptide B relative to the reference peak are: 0.0642-0.0643, 0.0806-0.0807, 0.0945-0.0947, 0.1191-0.1193, 0.1637-0.1643, 0.1917-0.1923, 0.2474-0.2480, 0.3300-0.3314 、0.3791-0.3804、0.3967-0.3983、0.4433-0.4452、0.4642-0.4662、0.7646-0.7664、0.7946-0.7965、1.0000、1.0854-1.0890、1.1044-1.1068、1.1550-1.1591、1.2282-1.2320、1.2520-1.2564.

9. The establishment method according to claim 1, characterized in that: In step (1), the ratio of Radix Pseudostellariae medicinal material to 95% ethanol is 1 g:17.5 mL; the extraction time is 1 h; the ratio of Radix Pseudostellariae medicinal material to acetonitrile solution is 4 g:5 mL.

10. The establishment method according to claim 7, characterized in that: Before injection in step (2), the test solution and the reference solution were filtered through a 0.22 μm microporous filter membrane; The test conditions of the high performance liquid chromatograph include: Chromatographic column: Agilent ZORBAX Eclipse Plus C18, 5 μm, 4.6 × 250 mm; Flow rate: 1ml / min; Column temperature: 25°C; Injection volume: 20 μL.

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