Fingerprint detection method of traditional Chinese medicine composition with hypoglycemic effect

Abstract

The present application relates to the technical field of detection method, and proposes a fingerprint detection method for traditional Chinese medicine composition with blood sugar lowering effect, comprising the following steps: taking control solution and test solution to perform high performance liquid chromatography detection to obtain the fingerprint of the traditional Chinese medicine composition; during high performance liquid chromatography detection, using acetonitrile as mobile phase A, using phosphoric acid aqueous solution as mobile phase B, and adopting gradient elution. Through the above technical scheme, the problem of poor reproducibility and unstable precision of the detection method for traditional Chinese medicine composition in related technologies is solved.

Description

Technical Field

[0001] This invention relates to the field of detection methods, specifically to a fingerprint spectrum detection method for a traditional Chinese medicine composition with hypoglycemic effect. Background Technology

[0002] Diabetes mellitus is a chronic disease characterized by hyperglycemia, caused by absolute or relative insulin deficiency and impaired insulin use. It is one of the three major chronic diseases, and its core harm often lies not in the abnormal blood sugar state itself, but in the complications resulting from long-term glucose metabolism disorders. These complications have a greater impact on patients' quality of life. Based on the etiology and mechanisms of diabetes and current medical research directions, and grounded in the theory of mutual generation and restraint of the Five Elements in Traditional Chinese Medicine, a refined herbal formula, Jingyan Shenyang Pian, is proposed. This formula comprises multiple Chinese herbs including kudzu root, yam, bitter melon peptide, astragalus, raspberry, bee flower, ginseng, oyster shell, polygonatum, and selenium-enriched black wheat peptide. It is a Chinese herbal combination for treating diabetes, showing significant efficacy against trend indicators and complications of diabetes.

[0003] However, current research on the quality of this traditional Chinese medicine composition is limited to the determination of the content of individual components. Physicochemical tests only characterize the content of total saponins, total flavonoids, and total polysaccharides, which cannot comprehensively characterize the chemical characteristics and overall quality of the traditional Chinese medicine composition. Therefore, it is necessary to study how to establish a quality testing method for this traditional Chinese medicine composition in order to better ensure its quality and efficacy.

[0004] However, the components of traditional Chinese medicine (TCM) compositions are numerous and complex, and reactions may occur between different components during extraction, further complicating the composition. Compared to conventional quality testing methods for these compositions, the information provided is limited, and it is difficult to comprehensively reflect their overall chemical characteristics. With the development of analytical chemistry techniques, fingerprinting has become an effective means of evaluating the overall quality of multi-component TCM compositions. Fingerprinting research is of great practical significance for ensuring the efficacy of TCM compositions, improving the overall level of the TCM industry, promoting the modernization of TCM agriculture, and driving TCM's global reach. Furthermore, the establishment of fingerprint profiles for TCM compositions should be based on analytical science, separation science, bioinformatics, and TCM principles, reflecting the comprehensiveness, integrity, hierarchy, correlation, and dynamism of fingerprint profiles. This will ensure the widespread adoption and application of fingerprinting.

[0005] While high-performance liquid chromatography (HPLC) fingerprinting is widely used, existing methods still suffer from poor reproducibility and unstable precision. Summary of the Invention

[0006] This invention proposes a fingerprint spectrum detection method for traditional Chinese medicine compositions with hypoglycemic effects, which solves the problems of poor reproducibility and unstable precision in the detection methods of traditional Chinese medicine compositions in related technologies.

[0007] The technical solution of the present invention is as follows:

[0008] This invention proposes a fingerprint spectrum detection method for a traditional Chinese medicine composition with hypoglycemic effect, comprising the following steps: taking a reference solution and a test solution for high performance liquid chromatography detection to obtain the fingerprint spectrum of the traditional Chinese medicine composition;

[0009] During the high-performance liquid chromatography detection, acetonitrile was used as mobile phase A and phosphoric acid aqueous solution was used as mobile phase B, with gradient elution employed.

[0010] The specific procedure for gradient elution is as follows:

[0011] From 0 min to 18 min, the volume fraction of mobile phase A changed from 2% to 5%, and the volume fraction of mobile phase B changed from 98% to 95%.

[0012] From 18 min to 45 min, the volume fraction of mobile phase A changed from 5% to 16%, and the volume fraction of mobile phase B changed from 95% to 84%.

[0013] From 45 min to 80 min, the volume fraction of mobile phase A changed from 16% to 45%, and the volume fraction of mobile phase B changed from 84% to 55%.

[0014] From 80 to 100 minutes, the volume fraction of mobile phase A changed from 45% to 80%, and the volume fraction of mobile phase B changed from 55% to 20%.

[0015] From 100 to 115 minutes, the volume fraction of mobile phase A changed from 80% to 2%, and the volume fraction of mobile phase B changed from 20% to 98%.

[0016] From 115 min to 135 min, the volume fraction of mobile phase A was 2%, and the volume fraction of mobile phase B was 98%.

[0017] Constant gradient elution is commonly used for determining the content of sample components because the determination of a single component requires a clear peak response, moderate retention time, and symmetrical peak shape, which constant gradient elution can meet. Gradient elution is often used to characterize the components in a sample in a chromatogram. Fingerprint chromatogram establishment refers to the process of characterizing all components of a sample solution, after certain treatment methods, under specific chromatographic conditions. Therefore, this invention chooses a mobile gradient elution method to establish a fingerprint chromatogram.

[0018] As a further technical solution, the volume fraction of the phosphoric acid aqueous solution is 0.05%~0.15%.

[0019] As a further technical solution, a C18 reverse-phase column is used for the high-performance liquid chromatography detection.

[0020] As a further technical solution, the flow rate during high-performance liquid chromatography detection is 0.8~1.2 mL·min. -1 The column temperature is 25~35℃, and the injection volume is 10~30μL.

[0021] As a further technical solution, the detection wavelength during high-performance liquid chromatography is 240~270nm.

[0022] As a further technical solution, the method for preparing the reference solution includes the following steps: dissolving the reference standard in methanol, filtering, and obtaining the reference solution.

[0023] As a further technical solution, the reference solution includes puerarin reference solution, puerarin apigenin reference solution, daidzein reference solution, daidzein reference solution, and genistein reference solution.

[0024] As a further technical solution, the puerarin reference solution contains 0.43 mg / mL of puerarin. -1 ;

[0025] The puerarin-apigenin reference solution contained 0.41 mg / mL of puerarin-apigenin. -1 ;

[0026] The daidzein reference solution contained 0.481 mg / mL of daidzein. -1 ;

[0027] The daidzein reference solution contained 0.66 mg / mL of daidzein. -1 ;

[0028] The genistein reference solution contained 0.64 mg / mL of genistein. -1 .

[0029] As a further technical solution, the preparation method of the test solution includes the following steps: solvent extraction of the traditional Chinese medicine composition, filtration, and obtaining the test solution.

[0030] As a further technical solution, the mass-to-volume ratio of the traditional Chinese medicine composition to the solvent is 2-3g:25mL.

[0031] As a further technical solution, in the preparation methods of the reference solution and the test solution, filtration is carried out independently using microporous membranes with a pore size of 0.22 μm.

[0032] As a further technical solution, the components of the traditional Chinese medicine composition include kudzu root, yam, bitter melon peptide, astragalus, raspberry, bee flower, ginseng, oyster, polygonatum, and selenium-enriched black wheat peptide.

[0033] As a further technical solution, the solvent includes an aqueous solution of ethanol with a volume fraction of 50% to 75%;

[0034] The extraction methods include ultrasonic extraction or reflux extraction.

[0035] As a further technical solution, the extraction method is ultrasonic extraction.

[0036] As a further technical solution, the ultrasonic extraction frequency is 100Hz, the power is 40W, and the time is 20~40min.

[0037] As a further technical solution, the fingerprint spectrum of the traditional Chinese medicine composition contains 20 common peaks, with peak 13 as the reference peak; the relative retention time of the common peaks is (AVG±SD):

[0038] Peak 1: 0.2538 ± 0.0003; Peak 2: 0.4349 ± 0.00059; Peak 3: 0.46039 ± 0.00049;

[0039] Peak 4: 0.4993 ± 0.0004; Peak 5: 0.5497 ± 0.0004; Peak 6: 0.6137 ± 0.0004;

[0040] Peak 7: 0.6386±0.0004; Peak 8: 0.6647±0.0004; Peak 9: 0.6872±0.0004;

[0041] Peak 10: 0.7305±0.0004; Peak 11: 0.9097±0.0004; Peak 12: 0.9783±0.0002;

[0042] Peak 14: 1.1492 ± 0.0002; Peak 15: 1.2132 ± 0.0004; Peak 16: 1.2636 ± 0.0005;

[0043] Peak 17: 1.3079 ± 0.0004; Peak 18: 1.3572 ± 0.0005; Peak 19: 1.4482 ± 0.0004;

[0044] Peak 20: 1.4624 ± 0.001.

[0045] The relative peak area of ​​the common peak is (AVG±SD):

[0046] Peak 1: 0.1816 ± 0.0051; Peak 2: 6.0200 ± 0.1291; Peak 3: 0.0820 ± 0.0023;

[0047] Peak 4: 0.9319±0.0114; Peak 5: 1.7246±0.0291; Peak 6: 0.1618±0.0044;

[0048] Peak 7: 0.2179 ± 0.0062; Peak 8: 0.1244 ± 0.0036; Peak 9: 0.2341 ± 0.0061;

[0049] Peak 10: 0.2241 ± 0.0064; Peak 11: 0.1594 ± 0.0047; Peak 12: 0.0791 ± 0.0022;

[0050] Peak 14: 0.4504 ± 0.0135; Peak 15: 0.0439 ± 0.0012; Peak 16: 0.1002 ± 0.0025;

[0051] Peak 17: 0.5077±0.0151; Peak 18: 0.0926±0.0027; Peak 19: 0.0831±0.0024;

[0052] Peak 20: 0.2263 ± 0.0064.

[0053] Ultimately, five common peaks were identified: peak 2 was puerarin, peak 4 was puerarin apigenin, peak 5 was daidzin, peak 13 was daidzinogen, and peak 14 was genistein.

[0054] The working principle and beneficial effects of this invention are as follows:

[0055] 1. The gradient elution program of the present invention avoids the problems of too small intervals between the peaks of each substance during detection, incomplete separation, and too low response values. The peaks of each substance in the chromatogram have the advantages of good separation and symmetrical peak shape.

[0056] 2. The detection method for fingerprint spectrum of traditional Chinese medicine composition established in this invention has the characteristics of being simple and reliable, having stable samples, good reproducibility, and stable precision of equipment.

[0057] 3. The fingerprint spectrum of the traditional Chinese medicine composition of the present invention has a common pattern, which identifies 20 common peaks and identifies 5 common characteristic peaks. This method selects daidzein No. 13 as the reference peak of the fingerprint spectrum and determines the relative retention time and relative peak area of ​​each common peak.

[0058] 4. The fingerprinting method established in this invention is used for quality monitoring of the traditional Chinese medicine composition. It can comprehensively and effectively control the quality of the traditional Chinese medicine composition as a whole, thereby reflecting the stability, reliability, and safety of the product. Attached Figure Description

[0059] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0060] Figure 1 These are the detection chromatograms of the blank solvent, reference solution, and traditional Chinese medicine composition sample in Example 1 of this invention;

[0061] Figure 2 These are the reference chromatograms (R) and HPLC fingerprints of 11 batches of traditional Chinese medicine composition samples in Example 1 of this invention;

[0062] Figure 3 The fingerprint spectra of the test samples with different extraction solvents of this invention;

[0063] Figure 4 These are fingerprint spectra of the test samples under different extraction methods according to the present invention;

[0064] Figure 5 The fingerprint spectra of the test sample solutions in Examples 1, 9-10 of this invention are shown.

[0065] Figure 6 The fingerprint spectra of the test sample solutions in Examples 1 and 1-3 of this invention are shown below.

[0066] Figure 7 The fingerprint spectra of the test sample solutions in Examples 1 and 4-5 of this invention are shown.

[0067] Figure 8 This is the fingerprint spectrum of the test solution of Comparative Example 6 of the present invention;

[0068] Figure 9 This is the fingerprint spectrum of the test solution of Comparative Example 7 of the present invention;

[0069] Figure 10 This is the fingerprint spectrum of the test solution of Comparative Example 8 of the present invention;

[0070] Figure 11 This is the fingerprint spectrum of the test solution of Comparative Example 9 of the present invention;

[0071] Figure 12 This is the fingerprint spectrum of the test solution of Comparative Example 10 of the present invention;

[0072] Figure 13 This is the fingerprint spectrum of the test solution in Example 1 of the present invention;

[0073] Figure 14 3D view of the test solution prepared in Example 1 of the present invention at a full wavelength of 190~400nm;

[0074] Figure 15This is a full-wavelength scan of the test solution in Example 1 of the present invention;

[0075] Figure 16 This is a scan of the optimal wavelength range of the test solution prepared in Example 1 of the present invention;

[0076] Figure 17 The fingerprint spectra are of the test solutions prepared in Example 1 at different wavelengths according to the present invention. Detailed Implementation

[0077] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0078] In the following embodiments and comparative examples:

[0079] 1. Instruments

[0080] High-performance liquid chromatograph (Waters ARC, PDA detector), electronic analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd., BCE224-1CCN), portable vacuum pump (Tianjin Aotesaiens Instruments Co., Ltd. AP-01P), ultrasonic cleaner (Kunshan Jelime Ultrasonic Instruments Co., Ltd., KS-600DE), multi-functional grinder (Yongkang Minye Industry and Trade Co., Ltd., 2500T); ultrapure water system (Sichuan Youpu Ultrapure Technology Co., Ltd., UPR-II-10T); constant temperature water bath (Changzhou Tianrui Instruments Co., Ltd., XMTE-206); low-speed benchtop centrifuge (Beijing Shidai Beili Centrifuge Co., Ltd., DT5-2B); vortex mixer (Germany, Vortex 2).

[0081] 2. The traditional Chinese medicine composition is refined ginseng nourishing tablets. Eleven batches of the traditional Chinese medicine composition were provided by Hebei Taidu Biotechnology Co., Ltd., with batch numbers T231107-4, T231109-2, T240104, T240328, T240427, T240517, Y230626, Y231001, Y231218, Y240301, and Y240806 respectively.

[0082] Puerarin apigenin (Shanghai Yuanye Biotechnology Co., Ltd., J13HB173936).

[0083] Puerarin (batch number 110752-202217), daidzein (batch number 111738-202305), daidzein (batch number 111502-202404), and genistein (batch number 111704-202104) were all purchased from the National Institutes for Food and Drug Control.

[0084] Methanol and acetonitrile were of chromatographic grade and manufactured by Merck & Co., Ltd.

[0085] Phosphoric acid was of chromatographic purity and manufactured by Tianjin Baishi Chemical Co., Ltd.

[0086] Example 1

[0087] Fingerprint spectral detection method for traditional Chinese medicine compositions with hypoglycemic effects

[0088] 1. Chromatographic conditions

[0089] Chromatographic column: C18 reverse-phase chromatographic column (inner diameter 4.6 mm, length 250 mm, packing particle size 5 μm);

[0090] Flow rate: 1 mL·min -1 ;

[0091] Column temperature: 30℃;

[0092] Detection wavelength: 250nm;

[0093] Injection volume: 20 μL;

[0094] Mobile phase: Acetonitrile was used as mobile phase A, and 0.1% (v / v) aqueous solution of phosphoric acid was used as mobile phase B;

[0095] Gradient elution was used, and the specific procedure for gradient elution is shown in Table 1.

[0096] Table 1 Gradient elution program

[0097]

[0098] 2. Preparation of test solution: Take approximately 2.5g of each batch of traditional Chinese medicine composition sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 70% ethanol aqueous solution, weigh it, extract it by sonication (100Hz, 40W) for 30min, cool it, weigh it again, replenish the lost weight with solvent, shake it well, centrifuge it, filter the supernatant through a 0.22μm microporous membrane to obtain the test solution.

[0099] 3. Preparation of reference solutions: Accurately weigh specific amounts of puerarin, puerarin apigenin, daidzein, daidzein, and genistein reference standards, dissolve them in methanol, and dilute to 25 mL. Filter each solution through a 0.22 μm microporous membrane to obtain a concentration of 0.43 mg / mL. -1 Puerarin reference solution, concentration 0.41 mg / mL -1 Puerarin apigenin reference solution, concentration 0.481 mg / mL -1 The daidzein reference solution had a concentration of 0.66 mg / mL. -1 The daidzein reference solution had a concentration of 0.64 mg / mL. -1 A lignin-based standard solution;

[0100] 4. Blank solvent: 70% (v / v) aqueous ethanol solution.

[0101] 5. Determination: Under the chromatographic conditions described above, inject the test solution and reference solution into the liquid chromatograph, determine the chromatogram, and record the chromatogram after 135 minutes. The detection chromatograms of the blank solvent, reference solution, and traditional Chinese medicine composition sample are shown below. Figure 1 As shown.

[0102] By comparing the retention times of single medicinal materials and components, it was determined that peak 2 is puerarin, peak 4 is puerarin apigenin, peak 5 is daidzin, peak 13 is daidzinogen, and peak 14 is genistein.

[0103] 6. Methodological Validation

[0104] 6.1 Instrument Precision

[0105] This embodiment examines the precision of the methodology for establishing fingerprint spectra of traditional Chinese medicine compositions. The experimental method is as follows:

[0106] Take the above-mentioned test solution and inject it six times consecutively under the above chromatographic conditions, and determine the retention time and peak area of ​​each peak. Using daidzein peak 13 as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak, and calculate the RSD% value. The results are shown in Tables 2 and 3.

[0107] Table 2. Relative retention times of common peaks in precision tests

[0108]

[0109] Table 3. Relative peak areas of common peaks in precision tests

[0110]

[0111] The results showed that after six injections, the RSD values ​​of the relative retention time and relative peak area of ​​each common peak were all <3.0%, indicating that the instrument has good precision.

[0112] 6.2 Sample Stability

[0113] Take the above-mentioned test solution and determine it at 0, 4, 8, 12, 18 and 24 h according to the above chromatographic conditions. Take daidzein peak 13 as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak, and calculate the RSD value. The results are shown in Tables 4 and 5.

[0114] Table 4. Relative retention times of common peaks in stability tests

[0115]

[0116] Table 5. Relative peak areas of common peaks in stability tests

[0117]

[0118] The results showed that the RSD values ​​of the relative retention time and relative peak area of ​​each common peak were all <3.0% after measurements were performed at 0, 4, 8, 12, 18 and 24 h, indicating that the components of the test solution were stable within 24 h.

[0119] 6.3 Method reproducibility

[0120] Take the same batch of traditional Chinese medicine composition as described above, and prepare 6 test solutions according to the "Preparation Method of Test Solution" described above. Determine the chromatographic properties according to the chromatographic conditions described above. Using daidzein peak 13 as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak, and calculate the RSD% value. The results are shown in Tables 6 and 7.

[0121] Table 6. Relative retention times of common peaks in reproducibility tests

[0122]

[0123] Table 7. Relative peak areas of common peaks in reproducibility tests

[0124]

[0125] The results showed that the RSD values ​​of the relative retention time and relative peak area of ​​each common peak after testing the six test solutions were all <3.0%, indicating that the method has good reproducibility.

[0126] 7. Establishment of fingerprint spectrum for traditional Chinese medicine compositions

[0127] 7.1. Fingerprint spectrum common peak calibration

[0128] Following the above detection method, 11 batches of traditional Chinese medicine composition samples were tested, and detection chromatograms were obtained. The 20 common peaks in the HPLC fingerprint chromatograms were identified by relative retention time.

[0129] 7.2 Determination of the reference peak in the fingerprint spectrum

[0130] Peak 13 (daidzein) has a moderate retention time and peak area, indicating good separation and stability. Therefore, peak 13 (daidzein) was selected as the reference peak.

[0131] 7.3 Relative retention time and relative peak area of ​​common peaks

[0132] The 20 common peaks were identified, with peak 13 (daidzein) as the reference. Its retention time and peak area were set to 1. The relative retention times and relative peak areas of the other common peaks were calculated, and the results are shown in Tables 8 and 9.

[0133] Table 8. Relative retention times of 11 batches of traditional Chinese medicine compositions

[0134]

[0135] Table 9. Relative peak areas of 11 batches of traditional Chinese medicine compositions

[0136]

[0137] The results showed that the RSD of the relative retention time of all 11 batches of traditional Chinese medicine composition test samples was <1%.

[0138] 7.4. Similarity evaluation of HPLC fingerprint spectra of 11 batches of traditional Chinese medicine compositions

[0139] The 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software was used to evaluate the similarity of fingerprint chromatograms of 11 batches of traditional Chinese medicine compositions. The chromatogram of sample S07 was used as the reference chromatogram. A control chromatogram R was generated using the median method with a time window width of 0.1. Multi-point correction was performed using the common peak at point 13 as the reference peak. The generated control chromatogram is shown below. Figure 2 The results are shown in Table 10.

[0140] Table 10 Similarity Evaluation Table

[0141]

[0142] The results showed that the similarity between the fingerprint spectra of the 11 batches of test samples and the control spectra was >0.97.

[0143] 7.5 Correlation between Chinese herbal medicine compositions and various single medicinal materials and attribution of characteristic peaks

[0144] Prepare test solutions for each medicinal material, negative sample, and reference standard in the prescription (the preparation process is the same as in 2. Preparation of test solution in Example 1). Measure the fingerprint chromatograms of each reagent at the corresponding dosage of the traditional Chinese medicine composition (chromatographic conditions are the same as in 1. Chromatographic conditions in Example 1). Import the chromatograms of the traditional Chinese medicine composition, single medicinal material, negative sample, and reference standard into the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software. By comparing the retention times of the substance peaks of the traditional Chinese medicine composition, single medicinal material, negative sample, and reference standard, the peaks with a retention time RSD value <1% are the characteristic peaks of the traditional Chinese medicine composition product, as shown in Table 11.

[0145] Table 11. Correlation and Characteristic Peak Assignment between Traditional Chinese Medicine Compositions and Various Single Herbs

[0146]

[0147] The results showed that the chemical components of each individual herb in the raw materials of the traditional Chinese medicine composition were basically present, and there was a certain correlation with the herbs. By identifying the characteristic compounds, the difficulty in quality control during the production of this product was avoided. It is possible to comprehensively control and identify the absence of certain components in the traditional Chinese medicine composition product, which facilitates quality control.

[0148] Examples 2-7

[0149] The only difference between Examples 2-7 and Example 1 is the extraction solvent used in the preparation of the test solution. The resulting chromatograms are as follows: Figure 3 As shown.

[0150] Table 12 Preparation of test solution using different extraction solvents

[0151]

[0152] The results showed that, by comparing the spectra of the extraction solvents of the test samples in Examples 1-7, the types of substance peaks were basically the same. However, for the same dosage of test sample, the response values ​​of the substance peaks were slightly different for different extraction solvents. After comparison, it was determined that 50%~75% ethanol can be used as the extraction solvent to extract the various substances of the traditional Chinese medicine composition. Therefore, an aqueous ethanol solution was determined as the extraction solvent for the traditional Chinese medicine composition sample, preferably a 50%~75% aqueous ethanol solution.

[0153] Example 8

[0154] The difference between this embodiment and Example 1 is that ultrasonic extraction is replaced with reflux extraction in the preparation of the test solution. The specific steps are as follows: Take a sample of traditional Chinese medicine composition, about 2.5g per batch, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 70% ethanol aqueous solution, weigh it, reflux extract for 30min, cool it, weigh it again, replenish the lost weight with solvent, shake well, centrifuge, filter the supernatant through a 0.22μm microporous membrane to obtain the test solution.

[0155] The spectra detected in Examples 1 and 8 are as follows: Figure 4 As shown.

[0156] The results showed that, for the same dose of test sample, the retention times of the substances under reflux and ultrasonic extraction methods were within the error range. However, the response values ​​of each component were slightly greater under ultrasonic extraction than under reflux extraction. Moreover, ultrasonic experiments are simple and easy to operate. Therefore, the ultrasonic extraction method used in the preparation of the test sample in Example 1 is more conducive to the fingerprint spectrum detection of the traditional Chinese medicine composition.

[0157] Example 9

[0158] The only difference between this embodiment and Embodiment 1 is that the mobile phase is acetonitrile (A) and a 0.15% (v / v) aqueous solution of phosphoric acid (B).

[0159] Example 10

[0160] The only difference between this embodiment and Embodiment 1 is that the mobile phase is acetonitrile (A) and a 0.05% (v / v) aqueous solution of phosphoric acid (B).

[0161] Comparative Example 1

[0162] The only difference between this comparative example and Example 1 is that the mobile phase is acetonitrile as mobile phase A and water as mobile phase B.

[0163] Comparative Example 2

[0164] The only difference between this comparative example and Example 1 is that the mobile phase is methanol as mobile phase A and a 0.1% volume fraction aqueous solution of phosphoric acid as mobile phase B.

[0165] Comparative Example 3

[0166] The only difference between this comparative example and Example 1 is that the mobile phase used is methanol as mobile phase A and water as mobile phase B.

[0167] Comparative Example 4

[0168] The only difference between this comparative example and Example 1 is that the mobile phase is acetonitrile as mobile phase A and 0.1% acetic acid aqueous solution as mobile phase B.

[0169] Comparative Example 5

[0170] The only difference between this comparative example and Example 1 is that the mobile phase is acetonitrile as mobile phase A and formic acid aqueous solution with a volume fraction of 0.1% as mobile phase B.

[0171] The detection spectra obtained from Examples 1, 9-10, and Comparative Examples 1-5 are as follows: Figures 5-7 As shown.

[0172] The results show that: Figure 6 In the spectrum, the baseline was unstable when methanol was used as the organic phase in the gradient elution, and some peaks were not completely separated. Acetonitrile was better when it was used as the organic phase. Therefore, acetonitrile was chosen as the organic phase solvent for gradient elution. Figure 7 Spectral comparison showed that the peak separation and peak shape of the aqueous phase with added phosphoric acid were better than other spectra. Therefore, it was determined that phosphoric acid was added to the aqueous phase. Figure 5 During the spectral comparison, the separation and symmetry of the peaks of each substance were basically consistent. It was determined that the addition of 0.05% to 0.15% phosphoric acid to the mobile phase B for gradient elution was acceptable. Considering the effect of acid on the octadecylsilane-bonded silica gel packing of the chromatographic column, the mobile phase B was more preferably a low-concentration 0.05% phosphoric acid aqueous solution.

[0173] Comparative Examples 6-10

[0174] The only difference between this comparative example and Example 1 is the gradient elution procedure, which is shown in Table 13.

[0175] Table 13 Gradient elution procedures for Comparative Examples 6-10

[0176]

[0177] Example 1, the fingerprint spectra of the test samples obtained from Comparative Examples 6-10 are as follows: Figures 8-13 As shown.

[0178] Since the traditional Chinese medicine composition with hypoglycemic effect in this invention mainly contains proteins, vitamins, flavonoids, flavonols, isoflavones, alkaloids, etc., the individual medicinal materials such as kudzu root powder, astragalus powder, raspberry powder, ginseng (artificially cultivated), and polygonatum powder mainly contain components such as proteins, vitamins, flavonoids, flavonols, isoflavones, etc., this invention combines the components of each individual medicinal material in the composition and mainly focuses on establishing fingerprint spectra of flavonoids, isoflavones, flavonols, etc. Flavonoids, flavonols, isoflavones, etc., exist in the form of glycosides in legumes and are polar compounds. Based on the polarity and existence form of the substances, this invention designed its mobile phase elution organic phase (methanol / acetonitrile) and inorganic phase (water / acid solution of a certain concentration) volume ratio of 50:50 to begin the study. The types of mobile phases for comparative examples 1-5 and example 1 were first investigated.

[0179] Then, considering that all substances such as flavonoids, flavonols, and isoflavones in the traditional Chinese medicine composition for hypoglycemic effects are reflected in the fingerprint spectrum, and referring to the fingerprint spectra of individual herbs in the traditional Chinese medicine composition for hypoglycemic effects studied in our laboratory in the past, a series of experimental studies were conducted based on the elution scheme of Comparative Example 6, using the mobile phase elution gradient as the basis. The results showed that: in the chromatogram of Comparative Example 6, the interval between the peaks between 16 min and 46 min was too small; in the chromatogram of Comparative Example 7, the peaks at 18 min and 41 min were not completely separated; in the chromatogram of Comparative Example 8, the peak response values ​​within the first 20 min were too low; in the chromatogram of Comparative Example 9, the peaks at positions such as 38 min and 50 min were not completely separated; in the chromatogram of Comparative Example 10, the peaks at positions such as 20 min and 24 min were not completely separated; the chromatogram of Example 1 avoided the drawbacks of the first five schemes, and the peak separation degree and peak shape of the chromatogram of Example 1 were better than those of other schemes.

[0180] Therefore, when exploring fingerprint spectra, this invention considers factors such as good separation between substances, symmetrical peaks, and normal response values, and optimizes the mobile phase elution gradient. After a series of experimental studies, the gradient elution scheme of Example 1 was finally determined to be the optimal flow rate gradient elution scheme.

[0181] The test solution was prepared according to Example 1. A PDA detector was used to scan the test solution at wavelengths of 190–400 nm. The results were obtained through 3D views and full-wavelength scan spectra (e.g., [image of scan spectrum]). Figures 14-16 (As shown) Analyze;

[0182] The results showed that there was more chromatographic peak information and a stable baseline in the absorption wavelength range of 200-310 nm; in the wavelength range of 310-400 nm, the chromatographic peak response was significantly reduced and the number of peaks was less.

[0183] By comparing different wavelengths of 203nm, 240nm, 250nm, 270nm, and 304nm, such as Figure 17 As shown, considering factors such as the proportion of each peak in the chromatogram and the response value, when 240~270nm is used as the detection wavelength, there is more information about each chromatographic peak, the separation of substance peaks is good, and the baseline is relatively stable.

[0184] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fingerprint spectral detection method for a traditional Chinese medicine composition with hypoglycemic effect, characterized in that, Includes the following steps: The reference solution and the test solution were subjected to high performance liquid chromatography to obtain the fingerprint spectrum of the traditional Chinese medicine composition; During the high-performance liquid chromatography detection, acetonitrile was used as mobile phase A and phosphoric acid aqueous solution was used as mobile phase B, with gradient elution employed. The specific procedure for gradient elution is as follows: From 0 min to 18 min, the volume fraction of mobile phase A changed from 2% to 5%, and the volume fraction of mobile phase B changed from 98% to 95%. From 18 min to 45 min, the volume fraction of mobile phase A changed from 5% to 16%, and the volume fraction of mobile phase B changed from 95% to 84%. From 45 min to 80 min, the volume fraction of mobile phase A changed from 16% to 45%, and the volume fraction of mobile phase B changed from 84% to 55%. From 80 to 100 minutes, the volume fraction of mobile phase A changed from 45% to 80%, and the volume fraction of mobile phase B changed from 55% to 20%. From 100 to 115 minutes, the volume fraction of mobile phase A changed from 80% to 2%, and the volume fraction of mobile phase B changed from 20% to 98%. From 115 min to 135 min, the volume fraction of mobile phase A was 2%, and the volume fraction of mobile phase B was 98%. The high-performance liquid chromatography (HPLC) detection was performed using a C18 reverse-phase column. During the high-performance liquid chromatography (HPLC) detection, the flow rate is 0.8–1.2 mL / min. -1 The column temperature is 25~35℃, and the injection volume is 10~30μL; The reference solutions include puerarin reference solution, puerarin apigenin reference solution, daidzein reference solution, daidzein reference solution, and genistein reference solution; The extraction solvent includes an aqueous solution of ethanol with a volume fraction of 50% to 75%; Extraction methods include ultrasonic extraction or reflux extraction.

2. The fingerprint spectrum detection method for a traditional Chinese medicine composition with hypoglycemic effect according to claim 1, characterized in that, The volume fraction of the phosphoric acid aqueous solution is 0.05% to 0.15%.

3. The fingerprint spectrum detection method for a traditional Chinese medicine composition with hypoglycemic effect according to claim 1, characterized in that, The detection wavelength for the high-performance liquid chromatography is 240~270nm.

4. The fingerprint spectrum detection method for a traditional Chinese medicine composition with hypoglycemic effect according to claim 1, characterized in that, The preparation method of the test solution includes the following steps: solvent extraction of the traditional Chinese medicine composition to obtain the test solution.

5. The fingerprint spectrum detection method for a traditional Chinese medicine composition with hypoglycemic effect according to claim 4, characterized in that, The components of the traditional Chinese medicine composition include kudzu root, yam, bitter melon peptide, astragalus, raspberry, bee flower, ginseng, oyster, polygonatum, and selenium-enriched black wheat peptide.

6. The fingerprint spectrum detection method for a traditional Chinese medicine composition with hypoglycemic effect according to claim 5, characterized in that, The extraction method is ultrasonic extraction.

7. The fingerprint spectrum detection method for a traditional Chinese medicine composition with hypoglycemic effect according to claim 1, characterized in that, The fingerprint spectrum of the traditional Chinese medicine composition contains 20 common peaks. Taking peak 13 as the reference peak, 5 common peaks were identified: peak 2 is puerarin, peak 4 is puerarin apigenin, peak 5 is daidzin, peak 13 is daidzinogen, and peak 14 is genistein.

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