Compound basil particle characteristic chromatogram, construction method and application thereof, and quality detection method and multi-index component detection method of compound basil particles

Abstract

The invention provides a characteristic spectrum construction method and application of compound basil granules, and a quality detection method and a multi-index component detection method of the compound basil granules, and relates to the technical field of traditional Chinese medicine detection. The specific chromatogram of the compound basil granules provided by the invention comprises 13 known substances, namely the characteristic peaks of gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercitrin, rosmarinic acid, luteolin, apigenin, kaempferol, lysionotin and nardosinone. The method overcomes the defect that the quality of related dosage forms cannot be detected from multiple angles in all directions due to interference caused by complex chemical components of the compound basil granules, provides a basis for quality control of the compound basil granules, and is verified by methodology to be good in reproducibility and stability, reliable in recovery rate, low in detection cost, high in detection efficiency and suitable for popularization and application. The method plays a crucial role in the quality stability of the compound basil granule product.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for constructing a characteristic spectrum of compound basil granules and an application thereof, a quality detection method for compound basil granules and a multi-index component detection method. Background Art

[0002] Compound basil granules are a traditional Chinese medicine compound preparation made from seven traditional Chinese medicines, namely basil, ox tongue, lavender, nard, dodder, fennel and rose. It has the functions of promoting blood circulation, clearing obstructions, digesting food, nourishing blood, nourishing the heart and brain, and enhancing heart and brain functions. It is used to treat headaches, insomnia, forgetfulness, dreaminess, heart failure, arrhythmia, etc. caused by neurasthenia. Most of the medicinal materials in the prescription of compound basil granules are Uyghur medicines. There are few literature reports on the chemical composition of the above prescription Chinese medicinal materials, and there is no corresponding quality standard for compound basil granules. At present, the quality standards of Uyghur medicine preparations only include simple properties, microscopic identification, physical and chemical identification, thin layer identification, routine inspection, and determination of the content of a certain component. At present, there is no research on the establishment of fingerprint or characteristic spectrum methods for compound basil granules, which makes it impossible to conduct comprehensive quality control of compound basil granules. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a method for constructing a characteristic spectrum of compound basil granules and its application, a quality detection method of compound basil granules and a multi-index component detection method. The characteristic spectrum of compound basil granules provided by the present invention can perform quality control on compound basil granules from multiple angles and in all directions, providing a more powerful guarantee for the authenticity identification and large-scale production of compound basil granules.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides a characteristic spectrum of compound basil particles, comprising characteristic peaks of gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercetin, rosmarinic acid, luteolin, apigenin, kaempferol, chelidonin and naringenone.

[0006] Preferably, the characteristic spectrum of the compound basil particles also includes a characteristic peak whose relative retention time with rosmarinic acid is within the range of a specified value × (1 ± 5%); the specified value includes one or more of 0.037, 0.365, 0.379, 0.494, 0.547, 0.573, 0.602, 0.637, 0.644, 0.656, 0.847, 0.899, 0.924, 0.936, 0.973, 1.020, 1.034, 1.064, 1.195, 1.280, 1.358 and 1.398.

[0007] The present invention provides a method for constructing a characteristic spectrum of the compound basil granules described in the above technical solution, comprising the following steps:

[0008] Extracting the compound basil particles with an extractant to obtain a test solution; the extractant includes a lower alcohol and / or water;

[0009] The test solution and the mixed reference solution are subjected to high performance liquid chromatography to obtain a test liquid chromatogram and a reference liquid chromatogram, respectively, and the retention time of the test liquid chromatogram is compared with the retention time of the reference liquid chromatogram to obtain a characteristic spectrum of the compound basil granules;

[0010] The reference substances in the mixed reference substance solution include reference substances of gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercetin, rosmarinic acid, luteolin, apigenin, kaempferol, chelidonol and galangin;

[0011] The conditions for the high performance liquid chromatography detection include: the chromatographic column filler includes octadecylsilane bonded silica gel; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1-0.4 v / v% phosphoric acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes: 0-10 min, the volume fraction of the mobile phase A is 2-5%; 10-58 min, the volume fraction of the mobile phase A increases from 2-5% to 16-18%; 58-60 min, the volume fraction of the mobile phase A decreases from 16-18% to 12-15%; 60-68 min, the volume fraction of the mobile phase A decreases from 16-18% to 12-15% The integral fraction is 12-15%; from 68 to 110 min, the volume fraction of the mobile phase A increases from 12-15% to 28-31%; from 110 to 112 min, the volume fraction of the mobile phase A increases from 28-31% to 32-40%; from 112 to 130 min, the volume fraction of the mobile phase A increases from 32-40% to 45-60%; from 130 to 138 min, the volume fraction of the mobile phase A is 45-60%; from 138 to 145 min, the volume fraction of the mobile phase A decreases from 45-60% to 2-5%; from 145 to 150 min, the volume fraction of the mobile phase A is 2-5%.

[0012] Preferably, the solid-liquid ratio of the compound basil particles to the extractant is 0.5-2 g:100 mL;

[0013] When the extractant contains lower alcohol, the volume fraction of the lower alcohol in the extractant is 50 to 100%;

[0014] The lower alcohol includes methanol and / or ethanol.

[0015] Preferably, the extraction comprises ultrasonic extraction or reflux extraction; the power of the ultrasonic extraction is 100-300 W, the frequency is 30-50 kHz, and the time is 15-60 min; the time of the reflux extraction is 15-60 min.

[0016] Preferably, the concentration of gallic acid in the mixed reference solution is 302.04-369.16 μg / mL, the concentration of neochlorogenic acid is 8.457-10.337 μg / mL, the concentration of chlorogenic acid is 6.073-7.423 μg / mL, the concentration of caffeic acid is 1.433-1.751 μg / mL, the concentration of rutin is 25.056-30.624 μg / mL, the concentration of hyperoside is 8.114-9.917 μg / mL, the concentration of isoquercetin ... isoquercetin is 1.433-1.751 μg / mL, the concentration of rutin is 25.056-30.624 μg / mL, the concentration of The concentration of rosmarinic acid was 13.176-16.104 μg / mL, the concentration of rosmarinic acid was 5.62-6.906 μg / mL, the concentration of luteolin was 8.892-10.868 μg / mL, the concentration of apigenin was 5.832-7.128 μg / mL, the concentration of kaempferol was 11.945-14.599 μg / mL, the concentration of chelidonol was 6.781-8.287 μg / mL, and the concentration of naringenone was 347.76-425.04 μg / mL;

[0017] The solvent in the mixed reference substance solution includes lower alcohol.

[0018] Preferably, the mobile phase flow rate of the HPLC detection is 0.8-1.2 mL / min, the detection wavelength is 330 nm, the column temperature is 30-35° C., the theoretical plate number is ≥5000 calculated based on the rosmarinic acid peak, and the injection volume is 10-20 μL.

[0019] The present invention also provides the application of the characteristic spectrum of the compound basil granules described in the above technical scheme or the characteristic spectrum of the compound basil granules constructed by the construction method described in the above technical scheme in the detection of the compound basil granules.

[0020] The present invention also provides a quality detection method for the compound basil granules, comprising the following steps:

[0021] Extracting the compound basil particles to be tested with an extractant to obtain a sample solution to be tested; the extractant includes lower alcohol and / or water;

[0022] The sample liquid to be tested is subjected to high performance liquid chromatography to obtain a liquid chromatogram of the sample to be tested;

[0023] A similarity evaluation system for Chinese medicine chromatographic fingerprints is used to evaluate the similarity between the liquid chromatogram of the sample to be tested and the characteristic spectrum of the compound basil granules. If the similarity is ≥0.9, the quality of the compound basil granules to be tested is qualified; if the similarity is <0.9, the quality of the compound basil granules to be tested is unqualified; the characteristic spectrum of the compound basil granules is the characteristic spectrum of the compound basil granules described in the above technical solution or the characteristic spectrum of the compound basil granules constructed by the construction method described in the above technical solution, and is used in the detection of compound basil granules;

[0024] The conditions for the high performance liquid chromatography detection include: the chromatographic column filler includes octadecylsilane bonded silica gel; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1-0.4 v / v% phosphoric acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes: 0-15 min, the volume fraction of the mobile phase A is 2%; 15-20 min, the volume fraction of the mobile phase A increases from 2% to 18%; 20-62 min, the volume fraction of the mobile phase A is 18%; 62-65 min, the volume fraction of the mobile phase A increases from 18% to 65%; 65-77 min, the volume fraction of the mobile phase A is 65%; 77-80 min, the volume fraction of the mobile phase A decreases from 65% to 2%; 80-85 min, the volume fraction of the mobile phase A is 2%.

[0025] The present invention also provides a multi-index component detection method for compound basil granules, comprising the following steps:

[0026] Extracting the compound basil particles to be tested with an extractant to obtain a sample solution to be tested; the extractant includes lower alcohol and / or water;

[0027] The sample solution to be tested is subjected to high performance liquid chromatography to obtain a multi-index component test result of the compound basil granules;

[0028] The test result includes a qualitative test result and / or a qualitative test result, wherein the qualitative test result is obtained by comparing the liquid chromatogram of the sample solution to be tested with the characteristic spectrum of the compound basil granules; the characteristic spectrum of the compound basil granules is the characteristic spectrum of the compound basil granules described in the above technical solution or the application of the characteristic spectrum of the compound basil granules constructed by the construction method described in the above technical solution in the detection of compound basil granules;

[0029] The conditions for the high performance liquid chromatography detection include: the chromatographic column filler includes octadecylsilane bonded silica gel; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1-0.4 v / v% phosphoric acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes: 0-15 min, the volume fraction of the mobile phase A is 2%; 15-20 min, the volume fraction of the mobile phase A increases from 2% to 18%; 20-62 min, the volume fraction of the mobile phase A is 18%; 62-65 min, the volume fraction of the mobile phase A increases from 18% to 65%; 65-77 min, the volume fraction of the mobile phase A is 65%; 77-80 min, the volume fraction of the mobile phase A decreases from 65% to 2%; 80-85 min, the volume fraction of the mobile phase A is 2%;

[0030] The multi-index components include gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercetin, rosmarinic acid, luteolin, apigenin, kaempferol, galangal, and at least two of the characteristic peaks whose relative retention time with rosmarinic acid is within the range of a specified value × (1 ± 5%); the specified value includes one or more of 0.037, 0.365, 0.379, 0.494, 0.547, 0.573, 0.602, 0.637, 0.644, 0.656, 0.847, 0.899, 0.924, 0.936, 0.973, 1.020, 1.034, 1.064, 1.195, 1.280, 1.358 and 1.398.

[0031] The characteristic spectrum of the compound basil granules provided by the present invention includes characteristic peaks of gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercetin, rosmarinic acid, luteolin, apigenin, kaempferol, chelidonol and naringenone, which overcomes the defect that the quality of the relevant dosage form cannot be fully detected from multiple angles due to the interference caused by the complex chemical composition of the compound basil granules, provides a basis for the quality control of the compound basil granules, and plays a vital role in the quality stability of the compound basil granules.

[0032] Furthermore, the characteristic spectrum of the compound basil granules provided by the present invention also includes characteristic peaks whose relative retention time with rosmarinic acid is within the range of a specified value × (1 ± 5%); the specified values ​​include 0.037, 0.365, 0.379, 0.494, 0.547, 0.573, 0.602, 0.637, 0.644, 0.656, 0.847, 0.899, 0.924, 0.936, 0.973, 1.02 0, 1.034, 1.064, 1.195, 1.280, 1.358 and 1.398, a total of 35 characteristic peaks, further overcome the defect that the quality of the relevant dosage form cannot be detected from multiple angles and in an all-round manner due to the interference caused by the complex chemical composition of the compound basil granules, and the quality of the compound basil granules can be controlled from multiple angles and in an all-round manner, providing more powerful guarantees for the authenticity identification and large-scale production of the compound basil granules.

[0033] The method for constructing the characteristic spectrum of the compound basil granules provided by the present invention realizes the effective separation of characteristic peaks, and the peak shape is good and interference-free, thereby realizing the quality detection of the effectiveness of the compound basil granules from multiple angles and in all directions, and further realizes the quality control of the compound basil granules. The method is simple to operate, has good reproducibility and stability, reliable recovery rate, low detection cost, and high detection efficiency after methodological verification, and plays a vital role in the quality stability of the compound basil granules.

[0034] The quality detection method of the compound basil granules provided by the present invention is a rapid, comprehensive, highly specific, scientifically reasonable and quantifiable method for monitoring the comprehensive component quality of the compound basil granules. After methodological verification, the method has good reproducibility and stability, reliable recovery rate, low detection cost and high detection efficiency. The method plays a vital role in the product quality stability of the compound basil granules, can completely and accurately evaluate the effectiveness, safety and stability of the compound basil granules, control the product quality raw materials more accurately and comprehensively, and can ensure the quality and efficacy of the product.

[0035] The present invention establishes a multi-index component synchronous detection method for compound basil granules. After methodological verification, the method has good reproducibility and stability, reliable recovery rate, low detection cost and high detection efficiency, which plays a vital role in the quality stability of compound basil granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The reference solution of gallic acid and rosmarinic acid in Example 1 is scanned at a UV wavelength of 200 to 800 nm, wherein A: gallic acid; B: rosmarinic acid;

[0037] Figure 2 HPLC chromatogram of the test solution of method development-gradient 1 in Example 2;

[0038] Figure 3 HPLC chromatogram of the test sample of method development-gradient 2 in Example 2;

[0039] Figure 4 HPLC chromatogram of the test sample of method development-gradient 3 in Example 2;

[0040] Figure 5 HPLC chromatogram of the test sample of method development-gradient 4 in Example 2;

[0041] Figure 6 HPLC chromatogram of the method development-gradient 4 mixed reference substance in Example 2;

[0042] Figure 7 This is the HPLC superimposed chromatogram of the test sample solution for specificity investigation in Example 2, wherein A: test sample and single medicinal material, B: test sample and negative sample;

[0043] Figure 8 This is the HPLC chromatogram of the test solution for precision investigation in Example 2;

[0044] Fig. 9 This is an overlay chromatogram of the HPLC of the test solution for stability investigation in Example 2;

[0045] Fig.10 This is the HPLC chromatogram of the test solution for repeatability investigation in Example 2;

[0046] Fig.11 It is the HPLC chromatogram of the characteristic spectrum of the compound basil granules in Example 2, wherein 2: gallic acid, 3: neochlorogenic acid, 6: chlorogenic acid, 7: caffeic acid, 15: rutin, 16: hyperoside, 17: isoquercetin, 23: rosmarinic acid, 27: luteolin, 29: apigenin, 30: kaempferol, 34: chelidonol, 35: galangin;

[0047] Fig.12 This is the HPLC chromatogram of the test solution of method development-gradient 1 in Example 3, wherein A: gallic acid; B: rosmarinic acid;

[0048] Fig.13 This is the HPLC chromatogram of the test solution of method development-gradient 2 in Example 3, wherein A: gallic acid; B: rosmarinic acid;

[0049] Fig.14 This is the HPLC chromatogram of the sample for method development-gradient 3 in Example 3, wherein A: gallic acid; B: rosmarinic acid;

[0050] Fig.15HPLC chromatogram of the method development-gradient 3 mixed reference solution in Example 3, wherein A: gallic acid, B: rosmarinic acid;

[0051] Fig.16 This is an HPLC superimposed chromatogram of the test sample solution in the extraction method investigation in Example 3, wherein A: gallic acid, B: rosmarinic acid, 1: ultrasound, 2: reflux;

[0052] Fig.17 This is the HPLC superposition chromatogram of the test solution for the extraction time investigation in Example 3, wherein A: gallic acid, B: rosmarinic acid, 1: ultrasonic 15 min, 2: ultrasonic 30 min, 3: ultrasonic 45 min, 4: ultrasonic 60 min;

[0053] Fig.18 The HPLC superimposed chromatogram of the test sample solution for solvent extraction investigation in Example 3, wherein A: gallic acid, B: rosmarinic acid, 1: water, 2: methanol, 3: 70% methanol, 4: 70% ethanol;

[0054] Fig.19 The chromatograms of the HPLC test solution in Example 3 are superimposed, wherein A: gallic acid; B: rosmarinic acid, 1: 50 times, 2: 100 times, 3: 200 times;

[0055] Fig. 20 This is an HPLC superimposed chromatogram of the mixed reference solution for linearity investigation in Example 4, wherein A: gallic acid; B: rosmarinic acid, 1-5: linear solutions 1-5;

[0056] Fig.21 The superimposed chromatogram of the HPLC mixed reference solution for precision investigation in Example 4, wherein A: gallic acid, B: rosmarinic acid, 1-6: the 1st to 6th injections;

[0057] Fig. 22 This is an HPLC superposition chromatogram of the test solution for stability investigation in Example 4, wherein A: gallic acid, B: rosmarinic acid, 1: 0h, 2: 2h, 3: 4h, 4: 6h, 5: 8h, 6: 12h, 7: 24h;

[0058] Fig.23 The HPLC chromatogram of the test solution for the inter-day precision study in Example 4, wherein A: gallic acid; B: rosmarinic acid, 1: day 1, 2: day 2, 3: day 3, 4: day 4, 5: day 5;

[0059] Fig.24 This is an HPLC superimposed chromatogram of the test solution for repeatability investigation in Example 4, wherein A: gallic acid; B: rosmarinic acid, 1-6: test solution 1-6;

[0060] Fig.25 This is an HPLC superimposed chromatogram of the test solution for durability investigation in Example 4, wherein A: gallic acid; B: rosmarinic acid, 1: Agilent chromatographic column, 2: SHIMADZU chromatographic column, 3: Waters chromatographic column;

[0061] Fig.26 This is an HPLC superposition chromatogram of the spiked test solution for sample recovery investigation in Example 4, wherein A: gallic acid; B: rosmarinic acid, 1 to 6 are spiked test solutions 1 to 6;

[0062] Fig. 27 It is an HPLC superimposed chromatogram of 7 batches of samples of the compound basil granules in Example 4, wherein A: gallic acid; B: rosmarinic acid, 1: test sample 20231101, 2: test sample 20231102, 3: test sample 20231103, 4: test sample 20231104, 5: test sample 20231105, 6: test sample 20231106, 7: test sample 20231107;

[0063] Fig.28 The gallic acid standard curve diagram in Example 4;

[0064] Fig.29 This is the standard curve of rosmarinic acid in Example 4. DETAILED DESCRIPTION

[0065] The invention provides a characteristic spectrum of compound basil particles, comprising characteristic peaks of gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercetin, rosmarinic acid, luteolin, apigenin, kaempferol, chelidonin and naringenone.

[0066] In the present invention, the characteristic spectrum of the compound basil particles preferably also includes a characteristic peak whose relative retention time with rosmarinic acid is within the range of a specified value × (1 ± 5%). In a specific embodiment, the relative retention time includes a specified value × (1 ± 5%), a specified value × (1 ± 4.5%), a specified value × (1 ± 4%), a specified value × (1 ± 3.5%), a specified value × (1 ± 3%), a specified value × (1 ± 2.5%), a specified value × (1 ± 2%), a specified value × (1 ± 1.5%), a specified value × (1 ± 1 %), a characteristic peak within a specified value × (1 ± 0.5%) or a specified value range; the specified value preferably includes one or more of 0.037, 0.365, 0.379, 0.494, 0.547, 0.573, 0.602, 0.637, 0.644, 0.656, 0.847, 0.899, 0.924, 0.936, 0.973, 1.020, 1.034, 1.064, 1.195, 1.280, 1.358 and 1.398.

[0067] In the present invention, the number of characteristic peaks is preferably 35, and the retention times are preferably: 3.425±0.037min (peak 1 has a common characteristic peak), 10.237±0.109min (peak 2, gallic acid), 29.702±0.317min (peak 3, new chlorogenic acid), 34.225±0.365min (peak 4 has a common characteristic peak), 35.557±0.379min (peak 5, has a common characteristic peak), 39.814±0.424min (peak 6 chlorogenic acid), 42.214±0.450min (peak 7 caffeic acid), 46.321±0.494min (peak 8 has a common characteristic peak), 51.293±0.547min (peak 9 has a common characteristic peak). min (Peak 9 has common characteristic peaks), 53.758±0.573min (Peak 10, common characteristic peaks), 56.500±0.602min (Peak 11, common characteristic peaks), 59.731±0.637min (Peak 12, common characteristic peaks), 60.438±0.644min (Peak 13, common characteristic peaks), 61.515±0.656min (Peak 14, common characteristic peaks), 68.501±0.730min (Peak 15, rutin), 70.065±0.747min (Peak 16, hyperoside), 74.338±0.793min (Peak 17, isoquercitrin), 79.465±0.847m in (Peak 18, common characteristic peak), 84.330±0.899min (Peak 19, common characteristic peak), 86.679±0.924min (Peak 20, common characteristic peak), 87.840±0.936min (Peak 21, common characteristic peak), 91.237±0.937min (Peak 22, common characteristic peak), 93.799±1min (Peak 23, S peak, rosmarinic acid), 95.632±1.020min (Peak 24, common characteristic peak), 96.966±1.034min (Peak 25, common characteristic peak), 99.765±1.064min (Peak 26, common characteristic peak), 106.256± 1.133min (Peak 27, luteolin), 112.072±1.195min (Peak 28, common characteristic peak), 116.299±1.240min (Peak 29, apigenin), 118.054±1.259min (Peak 30, kaempferol), 120.066±1.280min (Peak 31, common characteristic peak), 127.333±1.358min (Peak 32, common characteristic peak), 131.138±1.398min (Peak 33, common characteristic peak), 134.187±1.431min (Peak 34, chelidonol), 137.091±1.462min (Peak 35, galangal).

[0068] The present invention uses peak 23 as the reference peak S of rosmarinic acid, and the relative retention time of each characteristic peak and the S peak is within the range of ±5% of the specified value; the specified values ​​of peaks 1 to 35 are: 0.037, 0.109, 0.317, 0.365, 0.379, 0.424, 0.450, 0.494, 0.547, 0.573, 0.602, 0.637, 0.644, 0 .656, 0.730, 0.747, 0.793, 0.847, 0.899, 0.924, 0.936, 0.973, 1 (peak 23, S peak, rosmarinic acid), 1.020, 1.034, 1.064, 1.133, 1.195, 1.240, 1.259, 1.280, 1.358, 1.398, 1.431 and 1.462.

[0069] The present invention provides a method for constructing a characteristic spectrum of the compound basil granules described in the above technical solution, comprising the following steps:

[0070] Extracting the compound basil particles with an extractant to obtain a test solution; the extractant includes a lower alcohol and / or water;

[0071] The test solution and the mixed reference solution are subjected to high performance liquid chromatography to obtain a test liquid chromatogram and a reference liquid chromatogram, respectively, and the retention time of the test liquid chromatogram is compared with the retention time of the reference liquid chromatogram to obtain a characteristic spectrum of the compound basil granules;

[0072] The reference substances in the mixed reference substance solution include reference substances of gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercetin, rosmarinic acid, luteolin, apigenin, kaempferol, chelidonol and galangin;

[0073] The conditions for the high performance liquid chromatography detection include: the chromatographic column filler includes octadecylsilane bonded silica gel; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1-0.4 v / v% phosphoric acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes: 0-10 min, the volume fraction of the mobile phase A is 2-5%; 10-58 min, the volume fraction of the mobile phase A increases from 2-5% to 16-18%; 58-60 min, the volume fraction of the mobile phase A decreases from 16-18% to 12-15%; 60-68 min, the volume fraction of the mobile phase A decreases from 16-18% to 12-15% The integral fraction is 12-15%; from 68 to 110 min, the volume fraction of the mobile phase A increases from 12-15% to 28-31%; from 110 to 112 min, the volume fraction of the mobile phase A increases from 28-31% to 32-40%; from 112 to 130 min, the volume fraction of the mobile phase A increases from 32-40% to 45-60%; from 130 to 138 min, the volume fraction of the mobile phase A is 45-60%; from 138 to 145 min, the volume fraction of the mobile phase A decreases from 45-60% to 2-5%; from 145 to 150 min, the volume fraction of the mobile phase A is 2-5%.

[0074] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0075] In the present invention, the compound basil granules preferably include commercial products or homemade ones. The preparation method of the compound basil granules is described in detail below.

[0076] In the present invention, the preparation method of the compound basil granules preferably comprises the following steps: mixing the compound basil granule extract, auxiliary materials and adhesive, granulating, and obtaining the compound basil granules. In the present invention, the mass ratio of the compound basil extract to the auxiliary materials is preferably 1:1-2, and more preferably 1:1.5. In the present invention, the auxiliary materials preferably include one or more of lactose, soluble starch, dextrin and microcrystalline cellulose, and more preferably lactose. In the present invention, the adhesive preferably includes an alcohol solution or an alcohol-polyvinyl pyrrolidone (PVP) solution, and the alcohol in the adhesive preferably includes ethanol; the volume fraction of alcohol in the adhesive is preferably 80-98%, and in a specific embodiment, the volume fraction of alcohol in the adhesive can be 80%, 85%, 90%, 95% or 98%; the volume fraction of PVP in the adhesive is preferably 2-4%, and more preferably 3%. In the present invention, the mass ratio of the compound basil granule extract to the adhesive is preferably 1:0.2-0.6, and more preferably 1:0.3-0.5. In the present invention, the granulation preferably includes wet granulation. The present invention has no particular limitation on the specific operation of the wet granulation, and the wet granulation operation well known to those skilled in the art can be used. After the granulation is completed, the present invention preferably further includes drying the obtained granules to obtain compound basil granules. In the present invention, the drying temperature is preferably 40 to 65° C. The present invention has no particular limitation on the drying time, and drying to constant weight is sufficient.

[0077] In the present invention, the raw materials for preparing the compound basil granule extract preferably include, by weight: 100 to 300 parts of basil, more preferably 150 to 250 parts, and further preferably 200 parts; 12 to 36 parts of bugloss, more preferably 15 to 30 parts, and further preferably 20 to 25 parts; 6 to 18 parts of lavender, more preferably 8 to 15 parts, and further preferably 10 to 12 parts; 3 to 9 parts of nard, more preferably 4 to 8 parts, and further preferably 5 to 6 parts; 3 to 9 parts of rose, more preferably 4 to 8 parts, and further preferably 5 to 6 parts; 3 to 9 parts of fennel, more preferably 4 to 8 parts, and further preferably 5 to 6 parts; 3 to 9 parts of dodder, more preferably 4 to 8 parts, and further preferably 5 to 6 parts.

[0078] In the present invention, the preparation method of the compound basil granule extract comprises the following steps: decocting seven medicinal materials, namely, basil, ox tongue, lavender, nard, dodder, fennel and rose, concentrating the obtained water extract, and drying the obtained clear paste to obtain the compound basil granule extract. In the present invention, the mass ratio of the seven medicinal materials to the water for a single decoction is preferably 1:8-12, more preferably 1:9-11, and further preferably 1:10; the number of decoctions is preferably 2-4 times, more preferably 3 times; the time for a single decoction is preferably 1-3h, more preferably 1.5-2.5h, and further preferably 2h. In the present invention, the concentration is preferably reduced pressure concentration, and the pressure of the reduced pressure concentration is preferably 0.07-0.09MPa, more preferably -0.08MPa; the temperature of the reduced pressure concentration is preferably 70-75°C; the density of the clear paste at 60°C is preferably 1.15-1.25g / cm 3 , more preferably 1.2 g / cm 3 . In the present invention, the drying preferably includes vacuum drying, and the pressure of the vacuum drying is preferably 0.07 to 0.09 MPa, more preferably -0.08 MPa; the temperature of the vacuum drying is preferably 60 to 65°C. After completing the vacuum drying, the present invention preferably also includes crushing the extract obtained by drying to obtain a compound basil granule extract. In the present invention, the water content of the compound basil granule extract is preferably 0 to 6%, more preferably 1 to 5%, and more preferably 2 to 4%.

[0079] The invention uses an extractant to extract compound basil particles to obtain a test solution; the extractant comprises lower alcohol and / or water, more preferably water, a lower alcohol-water mixed solvent or a lower alcohol, and further preferably a lower alcohol-water mixed solvent; the lower alcohol preferably comprises methanol and / or ethanol, and more preferably methanol; when the extractant contains lower alcohol, the volume fraction of the lower alcohol in the extractant is preferably 50-100%, more preferably 60-90%, and further preferably 70-80%.

[0080] In the present invention, the solid-liquid ratio of the compound basil particles to the extractant is preferably 0.5-2 g:100 mL, more preferably 0.5-1.5 g:100 mL, further preferably 0.5-1 g:100 mL, and most preferably 0.5 g:100 mL.

[0081] In the present invention, the extraction preferably includes ultrasonic extraction or reflux extraction. In the present invention, the power of the ultrasonic extraction is preferably 100-300W, more preferably 150-250W, and more preferably 200W, the frequency is preferably 30-50kHz, more preferably 35-45kHz, and more preferably 40kHz, and the time is preferably 15-60min, more preferably 30-60min, and more preferably 50-60min. In the present invention, the time of the reflux extraction is preferably 15-60min, more preferably 15-50min, and more preferably 15-30min.

[0082] After the mixing, the present invention preferably further comprises filtering the obtained ultrasonic system to obtain a test solution. In the present invention, the filtration is preferably performed by 0.22-0.45 μm filter membrane filtration.

[0083] After obtaining the test solution, the present invention performs high performance liquid chromatography detection (recorded as the first high performance liquid chromatography detection) on the test solution and the mixed reference solution to obtain a test liquid chromatogram and a reference liquid chromatogram, respectively; the retention time of the test liquid chromatogram is compared with the retention time of the reference liquid chromatogram to obtain the characteristic spectrum of the compound basil granules;

[0084] The reference substances in the mixed reference substance solution include reference substances of gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercetin, rosmarinic acid, luteolin, apigenin, kaempferol, chelidonol and galangin.

[0085] In the present invention, the concentration of gallic acid in the mixed reference solution is preferably 302.04-369.16 μg / mL, more preferably 320-350 μg / mL, and further preferably 335.6 μg / mL; the concentration of neochlorogenic acid is preferably 8.457-10.337 μg / mL, more preferably 9-10 μg / mL, and further preferably 9.397 μg / mL; the concentration of chlorogenic acid is preferably 6.073-7.423 μg / mL, more preferably 6.5-7 μg / mL, and further preferably 6.748 μg / mL; The concentration of caffeic acid is preferably 1.433-1.751 μg / mL, more preferably 1.5-1.7 μg / mL, and further preferably 1.592 μg / mL; the concentration of rutin is preferably 25.056-30.624 μg / mL, more preferably 27-28 μg / mL, and further preferably 27.84 μg / mL; the concentration of hyperoside is preferably 8.114-9.917 μg / mL, more preferably 8.5-9.5 μg / mL, and further preferably 9.015 μg / mL; the concentration of isoquercetin is preferably 13.176-1 The concentration of rosmarinic acid is preferably 5.62-6.906 μg / mL, more preferably 6-6.5 μg / mL, and more preferably 6.278 μg / mL; the concentration of luteolin is preferably 8.892-10.868 μg / mL, more preferably 9-10 μg / mL, and more preferably 9.88 μg / mL; the concentration of apigenin is preferably 5.832-7.128 μg / mL, more preferably 6-7 μg / mL , more preferably 6.48μg / mL; the concentration of kaempferol is preferably 11.945-14.599μg / mL, more preferably 13-14μg / mL, more preferably 13.272μg / mL; the concentration of phytosyl ester is preferably 6.781-8.287μg / mL, more preferably 7-8μg / mL, more preferably 7.534μg / mL; the concentration of naringenone is preferably 347.76-425.04μg / mL, more preferably 370-400μg / mL, more preferably 386.4μg / mL. In the present invention, the solvent in the mixed reference solution preferably includes a lower alcohol, more preferably includes methanol and / or ethanol, and more preferably methanol.

[0086] In the present invention, the conditions for the first high performance liquid chromatography detection include: the chromatographic column filler includes octadecylsilane bonded silica gel, and the chromatographic column is preferably a SHIMADZU Shim-packVP-ODS chromatographic column, Agilent EclipseXDB-C18 or Waters C18; the model of the chromatographic column preferably includes 4.6mm×250mm, and the filler particle size is preferably 5μm; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1-0.4v / v% phosphoric acid aqueous solution, preferably 0.1-0.3v / v% phosphoric acid aqueous solution, more preferably 0.1-0.2v / v% phosphoric acid aqueous solution; the mobile phase flow rate is preferably 0.8-1.2mL / min, more preferably 0.9-1.1mL / min, and further preferably 1mL / min; the detection wavelength is preferably 330nm; the column temperature is preferably 30-35℃, more preferably 30-35℃, and more preferably 30-32℃; the theoretical plate number is based on rosemary The aromatic acid is preferably calculated to be ≥5000; the injection volume is preferably 10-20 μL, more preferably 10-15 μL; the elution method is gradient elution, and the gradient elution procedure includes: 0-10 min, the volume fraction of the mobile phase A is 2-5%, preferably 3-4%, more preferably 3%; 10-58 min, the volume fraction of the mobile phase A increases from 2-5% (preferably 3-4%, more preferably 3%) to 16-18% (preferably 16-17.5%, more preferably 17%); 58-60 min, the volume fraction of the mobile phase A decreases from 16-18% (preferably 16.5-17.5%, more preferably 17%) to 12-15% (preferably 13-14.5%).5%, more preferably 14%); 60-68min, the volume fraction of the mobile phase A is 12-15%, preferably 13-15%, more preferably 14%; 68-110min, the volume fraction of the mobile phase A increases from 12-15% (preferably 13-15%, more preferably 14%) to 28-31% (preferably 39-31%, more preferably 30%); 110-112min, the volume fraction of the mobile phase A increases from 28-31% (preferably 29-31%, more preferably 30%) to 32-40% (preferably 32-34%, more preferably 32%); 112- At 130 min, the volume fraction of the mobile phase A increases from 32 to 40% (preferably 32 to 34%, more preferably 32%) to 45 to 60% (preferably 45 to 50%, more preferably 45%); at 130 to 138 min, the volume fraction of the mobile phase A is 45 to 60%; at 138 to 145 min, the volume fraction of the mobile phase A decreases from 45 to 60% (preferably 45 to 50%, more preferably 45%) to 2 to 5% (preferably 3 to 4%, more preferably 3%); at 145 to 150 min, the volume fraction of the mobile phase A is 2 to 5%, preferably 3 to 4%, more preferably 3%. .

[0087] The present invention provides application of the characteristic spectrum of the compound basil granules described in the above technical scheme or the characteristic spectrum of the compound basil granules constructed by the construction method described in the above technical scheme in the detection of the compound basil granules.

[0088] The present invention also provides a quality detection method for the compound basil granules, comprising the following steps:

[0089] Extracting the compound basil particles to be tested with an extractant to obtain a sample solution to be tested; the extractant includes lower alcohol and / or water;

[0090] The sample liquid to be tested is subjected to high performance liquid chromatography detection (referred to as the second high performance liquid chromatography detection) to obtain a liquid chromatogram of the sample to be tested;

[0091] A similarity evaluation system for Chinese medicine chromatographic fingerprint is used to evaluate the similarity between the liquid chromatogram of the sample to be tested and the characteristic spectrum of the compound basil granules. If the similarity is ≥0.9, the quality of the compound basil granules to be tested is qualified; if the similarity is <0.9, the quality of the compound basil granules to be tested is unqualified; the characteristic spectrum of the compound basil granules is the characteristic spectrum of the compound basil granules described in the above technical scheme or the characteristic spectrum of the compound basil granules constructed by the construction method described in the above technical scheme;

[0092] The conditions for the high performance liquid chromatography detection include: the chromatographic column filler includes octadecylsilane bonded silica gel; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1-0.4 v / v% phosphoric acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes: 0-15 min, the volume fraction of the mobile phase A is 2%; 15-20 min, the volume fraction of the mobile phase A increases from 2% to 18%; 20-62 min, the volume fraction of the mobile phase A is 18%; 62-65 min, the volume fraction of the mobile phase A increases from 18% to 65%; 65-77 min, the volume fraction of the mobile phase A is 65%; 77-80 min, the volume fraction of the mobile phase A decreases from 65% to 2%; 80-85 min, the volume fraction of the mobile phase A is 2%.

[0093] In the present invention, the preparation method of the sample solution to be tested is the same as the preparation method of the test solution, which will not be repeated here.

[0094] In the present invention, the wavelength of the second HPLC detection is preferably 274-330nm, more preferably 274nm or 330nm; the detection wavelength of the gallic acid is preferably 274nm, and the detection wavelength of the rosmarinic acid is preferably 330nm. In the present invention, the gradient elution program of the second HPLC detection includes: 0-15min, the volume fraction of the mobile phase A is 2%; 15-20min, the volume fraction of the mobile phase A increases from 2% to 18%; 20-62min, the volume fraction of the mobile phase A is 18%; 62-65min, the volume fraction of the mobile phase A increases from 18% to 65%; 65-77min, the volume fraction of the mobile phase A is 65%; 77-80min, the volume fraction of the mobile phase A decreases from 65% to 2%; 80-85min, the volume fraction of the mobile phase A is 2%. In the present invention, other conditions of the second HPLC detection are preferably the same as those of the first HPLC detection, and will not be described in detail herein.

[0095] In the present invention, the Chinese medicine chromatographic fingerprint similarity evaluation system is preferably carried out using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Software (2012 Edition)" formulated by the State Pharmacopoeia Committee.

[0096] The present invention also provides a multi-index component detection method for compound basil granules, comprising the following steps:

[0097] Extracting the compound basil particles to be tested with an extractant to obtain a sample solution to be tested; the extractant includes lower alcohol and / or water;

[0098] The sample liquid to be tested is subjected to high performance liquid chromatography detection (referred to as the third high performance liquid chromatography detection) to obtain a multi-index component detection result of the compound basil granules;

[0099] The test results include qualitative test results and / or qualitative test results, wherein the qualitative test results are obtained by comparing the liquid chromatogram of the sample solution to be tested with the characteristic spectrum of the compound basil granules described in the above technical solution or the characteristic spectrum of the compound basil granules constructed by the construction method described in the above technical solution;

[0100] The conditions for the high performance liquid chromatography detection include: the chromatographic column filler includes octadecylsilane bonded silica gel; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1-0.4 v / v% phosphoric acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes: 0-15 min, the volume fraction of the mobile phase A is 2%; 15-20 min, the volume fraction of the mobile phase A increases from 2% to 18%; 20-62 min, the volume fraction of the mobile phase A is 18%; 62-65 min, the volume fraction of the mobile phase A increases from 18% to 65%; 65-77 min, the volume fraction of the mobile phase A is 65%; 77-80 min, the volume fraction of the mobile phase A decreases from 65% to 2%; 80-85 min, the volume fraction of the mobile phase A is 2%;

[0101] The multi-index components include gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercetin, rosmarinic acid, luteolin, apigenin, kaempferol, galangal, and at least two of the characteristic peaks whose relative retention time with rosmarinic acid is within the range of a specified value × (1 ± 5%); the specified value includes one or more of 0.037, 0.365, 0.379, 0.494, 0.547, 0.573, 0.602, 0.637, 0.644, 0.656, 0.847, 0.899, 0.924, 0.936, 0.973, 1.020, 1.034, 1.064, 1.195, 1.280, 1.358 and 1.398.

[0102] In the present invention, the preparation method of the sample solution to be tested is the same as the preparation method of the test solution, which will not be repeated here.

[0103] In the present invention, the wavelength of the third HPLC detection is preferably 274-330nm, more preferably 274nm or 330nm; the detection wavelength of the gallic acid is preferably 274nm, and the detection wavelength of the rosmarinic acid is preferably 330nm. In the present invention, the gradient elution program of the second HPLC detection includes: 0-15min, the volume fraction of the mobile phase A is 2%; 15-20min, the volume fraction of the mobile phase A increases from 2% to 18%; 20-62min, the volume fraction of the mobile phase A is 18%; 62-65min, the volume fraction of the mobile phase A increases from 18% to 65%; 65-77min, the volume fraction of the mobile phase A is 65%; 77-80min, the volume fraction of the mobile phase A decreases from 65% to 2%; 80-85min, the volume fraction of the mobile phase A is 2%. In the present invention, other conditions of the second HPLC detection are preferably the same as those of the first HPLC detection, and will not be described in detail herein.

[0104] In the present invention, the quantitative detection result is preferably determined by an external standard control method. The present invention has no special limitation on the quantitative operation of the external standard control method, and the quantitative operation can be performed by an external standard control method well known to those skilled in the art.

[0105] In order to further illustrate the present invention, the construction method and application of the characteristic spectrum of the compound basil granules, the quality detection method and the multi-index component detection method of the compound basil granules are described in detail below in combination with the embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0106] In the following examples, the reference substances, samples, reagents, and instruments used are shown in Tables 1 to 4.

[0107] Table 1 Test reference substances

[0108] Reference batch number source gallic acid 110831-201906 China Food and Drug Inspection Institute Caffeic acid 110885-201703 China Food and Drug Inspection Institute Rutin 100080-202012 China Food and Drug Inspection Institute Hyperoside 111521-201507 China Food and Drug Inspection Institute Isoquercetin 111809-202205 China Food and Drug Inspection Institute Rosmarinic acid 111871-202007 China Food and Drug Inspection Institute Apigenin 111901-202205 China Food and Drug Inspection Institute Kaempferol 110861-201310 China Food and Drug Inspection Institute Stonewort 111555-200602 China Food and Drug Inspection Institute Nardone 111832-201603 China Food and Drug Inspection Institute Luteolin 111520-200504 China National Institute for the Control of Pharmaceutical and Biological Products Neochlorogenic Acid MUST-23040213 Chengdu Mansite Biotechnology Co., Ltd. Chlorogenic acid MUST-22111711 Chengdu Mansite Biotechnology Co., Ltd.

[0109] Table 2 Test samples

[0110] Product Name batch number source Basil Q30048109 Xinjiang Madison Pharmaceutical Co., Ltd. Piece of Medicine Branch Bugloss C30010406 Xinjiang Madison Pharmaceutical Co., Ltd. Piece of Medicine Branch Lavender M30048022 Xinjiang Madison Pharmaceutical Co., Ltd. Piece of Medicine Branch spikenard G30031508 Xinjiang Madison Pharmaceutical Co., Ltd. Piece of Medicine Branch Rose H30016415 Xinjiang Madison Pharmaceutical Co., Ltd. Piece of Medicine Branch Cuscuta australis C30059708 Xinjiang Madison Pharmaceutical Co., Ltd. Piece of Medicine Branch Cumin 22010602 Xinjiang Renyu Chinese Medicine Pieces Co., Ltd. Compound Basil Granules 20231101 Laboratory-made Compound Basil Granules 20231102 Laboratory-made Compound Basil Granules 20231103 Laboratory-made Compound Basil Granules 20231104 Laboratory-made Compound Basil Granules 20231105 Laboratory-made Compound Basil Granules 20231106 Laboratory-made Compound Basil Granules 20231107 Laboratory-made

[0111] Table 3 Test reagents

[0112]

[0113]

[0114] Table 4 Test equipment

[0115]

[0116] Among them, the compound basil granules are homemade in the laboratory: by mass, seven Chinese medicinal materials: 1500g of basil, 180g of ox tongue, 90g of lavender, 45g of nard, 45g of rose, 45g of fennel, and 45g of dodder; add basil, ox tongue, lavender, nard, rose, fennel and dodder, add 10 times the weight of the seven medicinal materials and boil them for 3 times, each time for 1 hour, combine the water extracts obtained each time to obtain a water extraction mixture, and reduce the pressure and concentrate the water extraction mixture under the conditions of -0.08MPa and 70-75℃ to a density of 1.22-1.25g / cm at 60℃ 3 The clear paste is then vacuum dried at -0.08MPa and 60-65°C to a constant weight moisture content of ≤6wt%, to obtain a compound basil granule extract; the compound basil granule extract is evenly mixed with an auxiliary material (lactose) at a mass ratio of 1:1.5, 98vol% ethanol aqueous solution is used as a binder, wet granulation is performed, and the mixture is dried at 45°C to a constant weight to obtain a compound basil granule. The mass ratio of the compound basil granule extract to the binder is 1:0.4.

[0117] The filtration is done through a 0.22-0.45 μm filter membrane.

[0118] Example 1

[0119] Preparation of Gallic Acid Reference Solution: Dissolve the Gallic Acid Reference in methanol to obtain a Gallic Acid stock solution at a concentration of 543 μg / mL.

[0120] Preparation of rosmarinic acid reference solution: Dissolve rosmarinic acid reference in methanol to obtain a rosmarinic acid stock solution with a concentration of 513 μg / mL.

[0121] Preparation of mixed reference solution: Accurately pipette 0.5 mL of gallic acid reference solution and 1 mL of rosmarinic acid reference solution into a 25 mL volumetric flask, add methanol to dilute to the mark to obtain a mixed reference solution with a gallic acid concentration of 10.86 μg / mL and a rosmarinic acid concentration of 20.52 μg / mL.

[0122] The gallic acid reference solution and the rosmarinic acid reference solution were respectively subjected to UV light at wavelengths of 200 to 800 nm. Figure 1 , wherein A is a gallic acid reference solution and B is a rosmarinic acid reference solution. It can be seen that the optimal detection wavelength for gallic acid is 274nm and the optimal detection wavelength for rosmarinic acid is 330nm.

[0123] Example 2

[0124] 1. Investigation of HPLC characteristic spectrum method - gradient elution procedure

[0125] Test sample: Compound basil granules batch number 20231105.

[0126] 1.1 Method development - Gradient 1

[0127] HPLC detection conditions: the chromatographic column uses octadecylsilane bonded silica gel as filler (SHIMADZU Shim-pack VP-ODS, column length 25 cm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile is used as mobile phase A, 0.1 vol% phosphoric acid aqueous solution is used as mobile phase B, the mobile phase flow rate is 1.0 mL / min, the column temperature is 30°C, and the detection wavelength is 330 nm. The theoretical plate number should be not less than 5000 calculated based on the rosmarinic acid peak, the injection volume is 10 μL, and the gradient elution conditions are shown in Table 5.

[0128] Table 5 HPLC characteristic spectrum - gradient 1

[0129] Time (min) Mobile phase A volume fraction / % Mobile phase B volume fraction / % 0 3 97 12 3 97 60 17 83 65 17 83 105 32 68 108 32 68 120 45 55 125 45 55 130 3 97 135 3 97

[0130] Preparation of the test solution: Take the compound basil particles, grind them into powder, accurately weigh 0.5 g, put them in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, ultrasonically treat at 200 W, 40 kHz for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol, shake well, filter, and the filtrate is the test solution.

[0131] Inject the test solution into the HPLC, detect with UV detector at 330nm, record the chromatogram, and see the results. Figure 2 The results showed that in the chromatogram of the test solution, some chromatographic peaks had poor separation and overlapped chromatographic peaks. The mobile phase gradient elution method needs to be further adjusted.

[0132] 1.2 Method development - gradient 2

[0133] The only difference from the present example “1.1 Method Development—Gradient 1” is that the gradient elution conditions are shown in Table 6.

[0134] Table 6 HPLC characteristic spectrum - gradient 2

[0135] Time (min) Mobile phase A volume fraction / % Mobile phase B volume fraction / % 0 3 97 12 3 97 60 17 83 65 17 83 108 30 70 110 35 65 120 45 55 125 45 55 130 3 97 135 3 97

[0136] Chromatogram of the test solution is shown in Figure 3 The results showed that the separation of some chromatographic peaks was poor and the chromatographic peaks were stacked. The mobile phase gradient elution method needed to be further adjusted.

[0137] 1.3 Method development - Gradient 3

[0138] The only difference from this example “1.1 Method Development—Gradient 1” is that the gradient elution conditions are shown in Table 7.

[0139] Table 7 HPLC characteristic spectrum - gradient 3

[0140]

[0141]

[0142] Chromatogram of the test solution is shown in Figure 4 The results showed that the separation of some chromatographic peaks was poor and the chromatographic peaks were stacked. The mobile phase gradient elution method needed to be further adjusted.

[0143] 1.4 Method Development - Gradient 4

[0144] HPLC detection conditions: The only difference from “1.1 Method Development - Gradient 1” is the gradient elution conditions (see Table 8).

[0145] Table 8 HPLC characteristic spectrum - gradient 4

[0146] Time (min) Mobile phase A volume fraction / % Mobile phase B volume fraction / % 0 3 97 10 3 97 58 17 83 60 14 86 68 14 86 110 30 70 112 32 68 130 45 55 138 45 55 145 3 97 150 3 97

[0147] Inject the test solution and the mixed reference solution into the high performance liquid chromatograph for determination and record the chromatogram. The results of the liquid chromatogram of the test solution are shown in Figure 5 , the liquid chromatography results of the mixed reference solution are shown in Figure 6 The results showed that in the chromatograms of the mixed reference solution and the test solution, the chromatographic peaks had good separation and obvious characteristics. This indicated that the elution procedure (Table 8) and HPLC detection conditions under "1.4 Method Development-Gradient 4" met the requirements for the characteristic spectrum of the compound basil granules.

[0148] 2. HPLC characteristic spectrum method validation-specificity investigation

[0149] Test sample: Compound basil granules batch number 20231105.

[0150] Preparation of mixed reference solution: Dissolve gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercetin, rosmarinic acid, luteolin, apigenin, kaempferol, chelidonol and naringenone reference substances in methanol to obtain mixed reference solutions with concentrations of 335.6 μg / mL, 9.397 μg / mL, 6.748 μg / mL, 1.592 μg / mL, 27.84 μg / mL, 9.015 μg / mL, 14.64 μg / mL, 6.278 μg / mL, 9.88 μg / mL, 6.48 μg / mL, 13.272 μg / mL, 7.534 μg / mL and 386.4 μg / mL, respectively.

[0151] Preparation of the test solution: Take the compound basil particles, grind them into powder, accurately weigh 0.5 g, put them in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, ultrasonically treat at 200 W, 40 kHz for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol, shake well, filter, and the filtrate is the test solution.

[0152] The HPLC detection conditions were the same as those in “1.4 Method Development—Gradient 4” of this example.

[0153] Preparation of single herbal medicine solution: seven Chinese medicinal herbs: 1500g basil, 180g oxalis, 90g lavender, 45g nard, 45g rose, 45g fennel, 45g dodder; take each single herbal medicine in the prescription, add 10 times the weight of the seven herbs in water and decoct for 3 times, each time for 1 hour, combine the water extracts obtained each time to obtain a water extraction mixture, and concentrate the water extraction mixture under reduced pressure at -0.08MPa and 70-75℃ to a density of 1.22-1.25g / cm at 60℃ 3 The clear paste is then vacuum dried at -0.08MPa and 60-65℃ to a constant weight moisture content of ≤6wt%, to obtain a single medicinal material extract; the single medicinal material extract is mixed with an auxiliary material (lactose) at a mass ratio of 1:1.5, and 98vol% ethanol aqueous solution is used as a binder for wet granulation, and dried at 45℃ to a constant weight to obtain a single medicinal material granule. The mass ratio of the single medicinal material extract to the binder is 1:0.4. Take the single medicinal material granules, grind them, accurately weigh 0.5g, place them in a ground-mouth conical flask, accurately add 100mL of 70vol% methanol aqueous solution, weigh the weight, ultrasonically treat for 60min at 200W and 40kHz, cool, weigh the weight, make up the lost weight with 70vol% methanol, shake well, filter, and the filtrate is a single medicinal material solution. A total of 7 single medicinal material solutions are prepared.

[0154] Preparation of negative sample solution: by weight, seven Chinese medicinal materials: 1500g of basil, 180g of oxalis, 90g of lavender, 45g of nard, 45g of rose, 45g of fennel and 45g of dodder; take the other medicinal materials that are missing from each single medicinal material according to the prescription ratio, add 10 times the weight of the seven medicinal materials and boil for 3 times, each time for 1 hour, combine the water extracts obtained each time to obtain a water extraction mixture, and concentrate the water extraction mixture under reduced pressure at -0.08MPa and 70-75℃ to a density of 1.22-1.25g / cm at 60℃ 3The clear paste is then vacuum dried at -0.08MPa and 60-65℃ to a constant weight moisture content of ≤6wt%, and the compound basil granule extract lacking a single medicinal material is obtained; the compound basil granule extract lacking a single medicinal material is mixed evenly with the auxiliary material (lactose) at a mass ratio of 1:1.5, and 98vol% ethanol aqueous solution is used as a binder, wet granulated, and dried at 45℃ to a constant weight to obtain the compound basil granules lacking a single medicinal material. The mass ratio of the compound basil granule extract to the binder is 1:0.4. Take the compound basil granules lacking a single medicinal material, grind them, accurately weigh 0.5g, place them in a ground-mouth conical flask, accurately add 100mL of 70vol% methanol aqueous solution, weigh the weight, ultrasonically treat for 60min at 200W and 40kHz, cool, weigh the weight, make up the lost weight with 70vol% methanol, shake well, filter, and the filtrate is the negative sample solution. A total of 7 negative sample solutions are prepared.

[0155] Inject the mixed control solution, test solution, single herbal medicine solution and negative sample solution into the high performance liquid chromatograph respectively and measure them. The results are shown in Figure 7 and Fig.11 ,in Figure 7 In the figure, A is the liquid chromatogram of the test sample and the single medicinal material, and B is the liquid chromatogram of the test sample and the negative sample.

[0156] Fig.11The results showed that 35 characteristic peaks were detected. The retention times of peaks 1 to 35 were 3.425min, 10.237min, 29.702min, 34.225min, 35.557min, 39.814min, 42.214min, 46.321min, 51.293min, 53.758min, 56.500min, 59.731min, 60.438min, 61.51 5min, 68.501min, 70.065min, 74.338min, 79.465min, 84.330min, 86.679min, 87.840min, 91.237 min, 93.799min, 95.632min, 96.966min, 99.765min, 106.256min, 112.072min, 116.299min, 118.0 54min, 120.066min, 127.333min, 131.138min, 134.187min, 137.091min, peak 23 is the reference peak S of rosmarinic acid; the relative retention time of each characteristic peak and peak S is within ±5% of the specified value; the specified values ​​of peaks 1 to 35 are: 0.037, 0.109, 0.317, 0.365, 0.379, 0.424, 0.450, 0.49 4, 0.547, 0.573, 0.602, 0.637, 0.644, 0.656, 0.730, 0.747, 0.793, 0.847, 0.899, 0.924, 0.936, 0.973, 1 (peak S), 1.020, 1.034, 1.064, 1.133, 1.195, 1.240, 1.259, 1.280, 1.358, 1.398, 1.431, 1.462.

[0157] 3. HPLC characteristic spectrum method validation-instrument precision inspection

[0158] The HPLC detection conditions were the same as those in “1.4 Method Development—Gradient 4” of this example.

[0159] Take the mixed control solution under the "Specificity Investigation" item and inject it into the HPLC instrument for 6 consecutive injections. Record the chromatogram. Figure 8 Taking rosmarinic acid as the S peak, the relative retention time and relative peak area of ​​each main chromatographic peak and the S peak were calculated. The results are shown in Tables 9 and 10.

[0160] Table 9 Instrument precision test results - relative retention time

[0161]

[0162]

[0163] Table 10 Instrument precision test results - relative peak area

[0164]

[0165] The results showed that after 6 consecutive injections of the mixed reference solution, the relative retention time RSD of each reference peak was less than 0.08%; the relative peak area RSD was less than 3.98%, indicating that the instrument precision of the method provided by the present invention was good.

[0166] 4. HPLC characteristic spectrum method validation-stability investigation

[0167] The HPLC detection conditions were the same as those in “1.4 Method Development - Gradient 4”.

[0168] Prepare the test solution according to the "Specificity Investigation", accurately pipette 10 μL of the test solution, place it for 0h, 3h, 6h, 12h, 18h, and 24h, inject it into the high performance liquid chromatograph, and measure it. Fig. 9 Taking rosmarinic acid as the S peak, the relative retention time and relative peak area of ​​each main chromatographic peak and the S peak were calculated. The results are shown in Tables 11 and 12.

[0169] Table 11 Stability test results - relative retention time

[0170]

[0171]

[0172] Table 12 Stability test results - relative peak area

[0173]

[0174]

[0175] The results showed that the relative retention time RSD of the main chromatographic peaks within 24 hours of sample determination was less than 1.18%, and the relative peak area RSD was less than 32.12%, indicating that the method provided by the present invention has good stability.

[0176] 5. HPLC characteristic spectrum method validation-repeatability investigation

[0177] The HPLC detection conditions were the same as those in “1.4 Method Development—Gradient 4” of this example.

[0178] According to the preparation method of the test solution under the "Specificity Investigation", 6 portions were prepared in parallel, and each test solution was injected into the high-level liquid chromatograph for determination. Fig.10Taking rosmarinic acid as the S peak, the relative retention time and relative peak area of ​​each main chromatographic peak and the S peak were calculated. The results are shown in Tables 13 and 14.

[0179] Table 13 Repeatability test results - relative retention time

[0180]

[0181]

[0182] Table 14 Repeatability test results - relative peak area

[0183]

[0184] The results showed that the relative retention time RSD of each main chromatographic peak of 6 samples was less than 0.32%, and the relative peak area RSD was less than 24.12%, indicating that the method had good repeatability.

[0185] 6. Peak attribution

[0186] In order to attribute the 35 chromatographic peaks marked in the test solution, the pieces of medicine lacking a single medicinal material were weighed according to the prescription ratio, and negative samples were prepared in the same way as the compound basil granules. Weigh the pieces of single medicinal materials, and prepare single medicinal material samples in the same way as the basil granules. Take the negative samples lacking basil, the negative samples lacking nardus, the negative samples lacking ox tongue, the negative samples lacking dodder, the negative samples lacking rose, the negative samples lacking lavender, the negative samples lacking fennel, and the 7 medicinal material samples of basil, nardus, ox tongue, dodder, rose, lavender, and fennel, respectively, and measure them. The preparation method is the same as the preparation method of the test solution under the "Specificity Investigation" in Example 2.

[0187] Preparation of basil-deficient negative sample solution: grind the basil-deficient negative sample, accurately weigh 0.5 g, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0188] Preparation of the negative sample solution lacking naringa: Grind the negative sample of naringa finely, accurately weigh 0.5 g, accurately weigh, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0189] Preparation of negative sample solution lacking Herba Glossinae: Grind the negative sample of Herba Glossinae into powder, accurately weigh 0.5 g, and place it in a ground-mouth conical flask. Accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0190] Preparation of negative sample solution lacking Cuscuta australis: Grind the Cuscuta australis negative sample, accurately weigh 0.5 g, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0191] Preparation of rose-negative sample solution: Grind the rose-negative sample, accurately weigh 0.5 g, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0192] Preparation of lavender-deficient negative sample solution: Grind the lavender-negative sample, accurately weigh 0.5 g, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0193] Preparation of negative sample solution lacking fennel: Grind the negative sample of fennel, accurately weigh 0.5 g, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0194] Preparation of basil medicinal material sample solution: Grind the basil medicinal material sample, accurately weigh 0.5 g, accurately weigh, place in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0195] Preparation of sample solution of nardostachys chinensis medicinal material: grind the sample of nardostachys chinensis medicinal material into powder, accurately weigh 0.5 g, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool it, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake it well, filter it, and obtain it.

[0196] Preparation of Herba Oxtongue Herbs Sample Solution: Grind Herba Oxtongue Herbs Sample, accurately weigh 0.5 g, place in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0197] Preparation of dodder sample solution: Grind the dodder sample, accurately weigh 0.5 g, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool it, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake it well, filter it, and obtain the solution.

[0198] Preparation of rose medicinal material sample solution: Grind the rose medicinal material sample, accurately weigh 0.5 g, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool it, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake it well, filter it, and obtain it.

[0199] Preparation of lavender sample solution: Grind lavender sample, accurately weigh 0.5 g, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0200] Preparation of fennel medicinal material sample solution: Grind the fennel medicinal material sample, accurately weigh 0.5 g, accurately weigh, place in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0201] The above solutions were injected into the HPLC, and the chromatograms were recorded and compared with the chromatogram of the sample 1 under the "Repeatability Study" of Example 2. The results are shown in Figure 7 .

[0202] The results showed that of the 35 characteristic peaks marked in the chromatogram of the test sample, 13 peaks were identified by comparison with the reference sample, peak 2 was gallic acid, peak 3 was neochlorogenic acid, peak 6 was chlorogenic acid, peak 7 was caffeic acid, peak 15 was rutin, peak 16 was hyperoside, peak 17 was isoquercetin, peak 23 was rosmarinic acid, peak 27 was luteolin, peak 29 was apigenin, peak 30 was kaempferol, peak 34 was chelidonol, and peak 35 was gadolinium. By comparing the chromatograms of the negative and single medicinal material solutions with the test sample solutions, the chromatographic peaks were attributed. Peaks 7 and 23 were attributed to Herba Lycoris Radiatae, peak 13 was attributed to basil, lavender and rose, peak 16 was attributed to basil, lavender and rose, peak 21 was attributed to basil, fennel and Cuscuta australis, peaks 22, 25, 26 and 31 were attributed to basil, lavender and Cuscuta australis, peak 32 was attributed to basil, Herba Lycoris Radiatae and Nardostachys grandiflora, peak 34 was attributed to basil, peak 35 was attributed to Nardostachys grandiflora, and other chromatographic peaks were common peaks of multiple medicinal materials, indicating that the medicinal materials in the prescription all had characteristic peaks.

[0203] Example 3

[0204] 1. Investigation of HPLC content determination method - gradient elution procedure

[0205] 1.1 Method development - Gradient 1

[0206] HPLC detection conditions: The chromatographic column uses octadecylsilane bonded silica gel as filler (SHIMADZU Shim-packVP-ODS, column length 25 cm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile is used as mobile phase A, 0.1 vol% phosphoric acid aqueous solution is used as mobile phase B, the mobile phase flow rate is 1.0 mL / min, the column temperature is 30°C, the detection wavelength of gallic acid is 274 nm, and the detection wavelength of rosmarinic acid is 330 nm. The theoretical plate number calculated based on the rosmarinic acid peak should not be less than 5000. The gradient elution conditions are shown in Table 15.

[0207] Table 15 HPLC Assay - Gradient 1

[0208] Time (min) Mobile phase A volume fraction / % Mobile phase B volume fraction / % 0 2.5 97.5 12 2.5 97.5 12.01 18 82 60 18 82 60.01 2.5 97.5 65 2.5 97.5

[0209] Preparation of test solution: Grind compound basil granules (batch number 20231105), accurately weigh 0.5 g, put into a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0210] Inject the test solution into the HPLC, detect with UV detector at 274nm and 330nm, record the chromatogram, and see the results. Fig.12 , where A is gallic acid and B is rosmarinic acid. The results showed that in the chromatogram of the test solution, the separation of the gallic acid peak was poor, and the mobile phase gradient elution method needed to be further adjusted.

[0211] 1.2 Method development - gradient 2

[0212] The only difference from “1.1 Method Development—Gradient 1” in this example is that the gradient elution conditions are shown in Table 16.

[0213] Table 16 HPLC Assay - Gradient 2

[0214]

[0215]

[0216] Chromatogram of the test solution is shown in Fig.13 , where A is gallic acid and B is rosmarinic acid. The results show that in the chromatogram of the test solution, the separation of the gallic acid chromatographic peak is poor, and there is still a chromatographic peak after 70 minutes, so the mobile phase gradient elution method needs to be further adjusted.

[0217] 1.3 Method development - Gradient 3

[0218] The only difference from “1.1 Method Development—Gradient 1” in this example is that the gradient elution conditions are shown in Table 17.

[0219] Table 17 HPLC Assay - Gradient 3

[0220] Time (min) Mobile phase A volume fraction / % Mobile phase B volume fraction / % 0 2 98 15 2 98 20 18 82 62 18 82 65 65 35 77 65 35 80 2 98 85 2 98

[0221] Preparation of Gallic Acid Reference Solution: Dissolve Gallic Acid Reference in methanol to obtain a Gallic Acid Reference Stock Solution at a concentration of 543 μg / mL.

[0222] Preparation of rosmarinic acid reference solution: Dissolve rosmarinic acid reference in methanol to obtain a rosmarinic acid reference stock solution with a concentration of 513 μg / mL.

[0223] Preparation of mixed reference solution: Accurately pipette 0.5 mL of gallic acid reference solution and 1 mL of rosmarinic acid reference solution into a 25 mL volumetric flask, add methanol to dilute to the mark to obtain a mixed reference solution with a gallic acid concentration of 10.86 μg / mL and a rosmarinic acid concentration of 20.52 μg / mL.

[0224] The preparation of the test solution was the same as in “1.1 Method Development - Gradient 1” in this example.

[0225] Inject the above solutions into a high performance liquid chromatograph, detect with a UV detector at 274nm and 330nm, and record the chromatogram. The results of the test solution are shown in Fig.14 , mixed reference solution results are shown in Fig.15 , wherein A is gallic acid and B is rosmarinic acid. The results show that the chromatograms of the reference solution and the test solution have good chromatographic peak separation. This indicates that the method gradient elution procedure (Table 17) and HPLC detection conditions under the item "1.3 Method Development-Gradient 3" of this embodiment meet the requirements for the determination of the content of compound basil granules.

[0226] 2. Investigation of the preparation method of test solution

[0227] 2.1 Method development - sample extraction method investigation

[0228] Chromatographic conditions and system suitability: HPLC detection conditions were the same as those in “1.3 Method Development—Gradient 3” in this example.

[0229] The preparation of gallic acid reference solution and rosmarinic acid reference solution is the same as that in "1.3 Method Development - Gradient 3" in this example.

[0230] Preparation of test solution 1: Grind the compound basil granules (batch number 20231105), accurately weigh 1 g, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 30 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0231] Preparation of test solution 2: Grind the compound basil granules (batch number 20231105), accurately weigh 1 g, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, reflux for 30 min, cool, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake well, filter, and obtain.

[0232] The above solutions were injected into the HPLC, detected by UV detector at 274nm and 330nm, and the chromatograms were recorded. Fig.16, where A is gallic acid, B is rosmarinic acid, 1 is ultrasound, and 2 is reflux. The contents of gallic acid and rosmarinic acid by different extraction methods were calculated, and the results are shown in Table 18. The results showed that the difference in content between ultrasonic extraction and reflux extraction was small, and ultrasonic extraction was simple to operate, so the ultrasonic extraction method was selected.

[0233] Table 18 Results of sample extraction method investigation (mg·g -1 )

[0234]

[0235] 2.2 Method development - sample extraction time investigation

[0236] The HPLC detection conditions were the same as those in “1.3 Method Development—Gradient 3” in this example.

[0237] The preparation of gallic acid reference solution and rosmarinic acid reference solution is the same as that in "1.3 Method Development - Gradient 3" in this example.

[0238] Preparation of test solution: Grind the compound basil granules (batch number 20231105), accurately weigh 1 g, put it in a ground-mouth conical flask, accurately add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 15 min, 30 min, 45 min and 60 min, cool it, weigh it, make up the lost weight with 70 vol% methanol aqueous solution, shake it well, filter it, and get 4 test solutions, which were respectively recorded as test solution 1 (15 min), test solution 2 (30 min), test solution 3 (45 min) and test solution 4 (60 min).

[0239] Each solution was injected into a high performance liquid chromatograph, detected with a UV detector at 274 nm and 330 nm, and the chromatogram was recorded. Fig.17 , where A is gallic acid, B is rosmarinic acid, 1 is ultrasonic 15min, 2 is ultrasonic 30min, 3 is ultrasonic 45min, and 4 is ultrasonic 60min. The contents of gallic acid and rosmarinic acid at different extraction times were calculated, and the results are shown in Table 19. The results showed that with the extension of ultrasonic extraction time, the contents of gallic acid and rosmarinic acid increased successively, and the extraction was sufficient at 60min, so the ultrasonic extraction time was selected as 60min.

[0240] Table 19 Sample extraction time investigation results (mg·g -1 )

[0241]

[0242] 2.3 Method development - sample extraction solvent investigation

[0243] The HPLC detection conditions were the same as those in “1.3 Method Development—Gradient 3” in this example.

[0244] The preparation of gallic acid reference solution and rosmarinic acid reference solution is the same as that in "1.3 Method Development - Gradient 3" in this example.

[0245] Preparation of test solution: Grind the compound basil granules (batch number 20231105), accurately weigh 1 g, put it in a ground-mouth conical flask, accurately add 100 mL of solvent (water, methanol, 70 vol% methanol aqueous solution or 70 vol% ethanol aqueous solution), weigh the weight, treat with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with the solvent, shake well, filter, and obtain 4 test solutions, which were respectively recorded as test solution 1 (water), test solution 2 (methanol), test solution 3 (70 vol% methanol aqueous solution) and test solution 4 (70 vol% ethanol aqueous solution).

[0246] The solutions were injected into the HPLC, detected by UV detector at 274nm and 330nm, and the chromatograms were recorded. Fig.18 , where A is gallic acid, B is rosmarinic acid, 1 is water, 2 is methanol, 3 is 70 vol% methanol aqueous solution, and 4 is 70 vol% ethanol aqueous solution. The contents of gallic acid and rosmarinic acid in different extraction solvents were calculated, and the results are shown in Table 20. The results showed that the content was the highest when 70 vol% methanol aqueous solution was used as the extraction solvent, so 70 vol% methanol aqueous solution was selected as the best extraction solvent.

[0247] Table 20 Results of sample extraction solvent investigation (mg·g -1 )

[0248]

[0249]

[0250] 2.4 Method development - investigation of sample extraction solvent dosage

[0251] The HPLC detection conditions were the same as those in “1.3 Method Development—Gradient 3” in this example.

[0252] The preparation of gallic acid reference solution and rosmarinic acid reference solution is the same as that in "1.3 Method Development - Gradient 3" in this example.

[0253] (3) Preparation of test solution

[0254] Preparation of test solutions 1 and 2: Grind the compound basil granules (batch number 20231105), accurately weigh 1 g, put it in a ground-mouth conical flask, accurately add 70 vol% methanol aqueous solution (50 mL and 100 mL, respectively), weigh the weight, treat with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool, weigh the weight, make up the lost weight with the solvent, shake well, filter, and obtain 2 test solutions, which were respectively recorded as test solution 1 (50 mL, 50 times) and test solution 2 (100 mL, 100 times).

[0255] Preparation of test solution 3 (200 times): the same as the test solution preparation method in "1.1 Method Development - Gradient 1" in this example.

[0256] Each solution was injected into a high performance liquid chromatograph, detected with a UV detector at 274 nm and 330 nm, and the chromatogram was recorded. The results are shown in Fig.19 , where A is gallic acid, B is rosmarinic acid, 1 is 50 times, 2 is 100 times, and 3 is 200 times. The contents of gallic acid and rosmarinic acid with different extraction solvent dosages were calculated, and the results are shown in Table 21. The results showed that the content was the highest when the solvent dosage was 200 times, so the solvent dosage was selected as 200 times (1g: 200mL).

[0257] In summary, the best preparation method for the test solution is: grind the compound basil particles, accurately weigh 0.5 g, put it in a ground-mouth conical flask, add 100 mL of 70 vol% methanol aqueous solution, weigh the weight, treat it with ultrasound (power 200 W, frequency 40 kHz) for 60 min, cool it, weigh the weight, make up the lost weight with 70 vol% methanol aqueous solution, shake it well, filter it, and obtain the solution.

[0258] Table 21 Results of investigation on the amount of solvent used for sample extraction (mg·g -1 )

[0259]

[0260] Example 4

[0261] HPLC assay method validation

[0262] The HPLC detection conditions were the same as those in “1.3 Method Development—Gradient 3” in this example.

[0263] 1. Linear relationship investigation

[0264] Take 3mL of gallic acid reference substance stock solution and 6mL of rosmarinic acid reference substance stock solution under "1.3 Method Development-Gradient 3" in Example 3, place in a 25mL volumetric flask, add methanol to dilute to the mark, and obtain a mixed reference solution with a gallic acid concentration of 65.16μg / mL and a rosmarinic acid concentration of 123.12μg / mL. Take 0.5mL, 1mL, 2mL, 3mL, and 5mL of the mixed reference substance solution, respectively, place in a 25mL volumetric flask, add methanol to dilute and prepare a series of mixed reference substance solutions with gallic acid concentrations of 1.3032, 2.6064, 5.2128, 7.8192, and 13.0320μg / mL, and rosmarinic acid concentrations of 2.4624, 4.9248, 9.8496, 14.7744, and 24.6240μg / mL, respectively, which are recorded as linear solutions 1 to 5. Inject each solution into a high performance liquid chromatograph and measure Fig. 20 , where A is gallic acid, B is rosmarinic acid, and 1 to 5 are linear solutions 1 to 5. With concentration (X) as the abscissa and peak area (Y) as the ordinate, a standard curve is drawn. See the standard curve diagram. Fig.28 , Fig.29 .

[0265] The results showed that gallic acid had a good linear relationship in the range of 1.3032-13.0320 μg / mL, and rosmarinic acid had a good linear relationship in the range of 2.4624-24.6240 μg / mL.

[0266] 2. Instrument precision inspection

[0267] The mixed reference solution under "1.3 Method Development - Gradient 3" in Example 3 was injected into the HPLC for 6 consecutive injections. Fig.21 , where A is gallic acid, B is rosmarinic acid, and 1 to 6 are injections 1 to 6. The RSD values ​​of the retention time and peak area of ​​gallic acid and rosmarinic acid were calculated. The results are shown in Table 22 and Table 23.

[0268] The standard stipulates: RSD of retention time of gallic acid and rosmarinic acid ≤3.0% (n=6), and RSD of peak area ≤3.0% (n=6).

[0269] Table 22 Instrument precision test results - retention time

[0270]

[0271] Table 23 Instrument precision test results - peak area

[0272]

[0273] The results showed that after 6 consecutive injections, the RSDs of the retention times of gallic acid and rosmarinic acid were 0.09% and 0.06%, respectively; the RSDs of the peak areas of gallic acid and rosmarinic acid were 1.56% and 1.35%, respectively, indicating that the instrument precision of the method provided by the present invention is good.

[0274] 3. Stability inspection

[0275] Prepare 1 sample solution according to the method for preparing the sample solution in "1.1 Method Development - Gradient 1" in Example 3. Accurately pipette 10 μL of the sample solution and place it for 0 h, 3 h, 6 h, 12 h, 12 h, and 24 h, respectively. Inject it into the liquid chromatograph and measure it. Fig. 22 , where A is gallic acid, B is rosmarinic acid, and 1 to 7 are 0h, 3h, 6h, 12h, 12h, and 24h, respectively. The RSD values ​​of gallic acid and rosmarinic acid contents were calculated, and the results are shown in Table 24.

[0276] The standard stipulates that the RSD of the gallic acid and rosmarinic acid content of the test solution is ≤3.0% (n=7).

[0277] Table 24 Stability test results - content

[0278]

[0279] The results showed that the RSD of the gallic acid content of the sample measured within 24 hours was 2.55%, and the RSD of the rosmarinic acid content was 0.83%, indicating that the method provided by the present invention has good stability.

[0280] 4. Daily precision inspection

[0281] According to the test solution preparation method of "1.1 Method Development - Gradient 1" in Example 3, 5 portions of the test solution were prepared, and 1 portion was prepared every day and injected into the high performance liquid chromatograph for determination. Fig.23 , where A is gallic acid, B is rosmarinic acid, and 1 to 5 are days 1 to 5, respectively. The RSD values ​​of gallic acid and rosmarinic acid contents were calculated, and the results are shown in Table 25.

[0282] The standard stipulates that the RSD of the gallic acid and rosmarinic acid content of the test solution is ≤3.0% (n=5).

[0283] Table 25 Inter-day precision test results - content

[0284]

[0285] The results showed that the RSD of gallic acid content was 1.99%, and the RSD of rosmarinic acid content was 1.81%, indicating that the method provided by the present invention had good inter-day precision.

[0286] 5. Repeatability test

[0287] According to the preparation method of the test solution in "1.1 Method Development - Gradient 1" in Example 3, 6 portions were prepared in parallel, which were recorded as test solutions 1 to 6. Each solution was injected into a high performance liquid chromatograph and measured. Fig.24 , where A is gallic acid, B is rosmarinic acid, and 1 to 6 are test solutions 1 to 6. The RSD values ​​of gallic acid and rosmarinic acid contents were calculated, and the results are shown in Table 26.

[0288] The standard stipulates that the RSD of the gallic acid and rosmarinic acid content of the test solution is ≤3.0% (n=6).

[0289] Table 26 Repeatability test results - content

[0290]

[0291]

[0292] The results showed that the RSD of gallic acid content in 6 samples was 0.98%, and the RSD of rosmarinic acid content was 0.90%, indicating that the method had good repeatability.

[0293] 6. Durability inspection

[0294] According to the method for preparing the test solution in "1.1 Method Development - Gradient 1" in Example 3, three portions were prepared in parallel, and three chromatographic columns of different brands were selected. Each solution was injected into a high performance liquid chromatograph for determination. Fig.25 , where A is gallic acid, B is rosmarinic acid, 1 is an Agilent column, 2 is a SHIMADZU column, and 3 is a Waters column. The RSD values ​​of gallic acid and rosmarinic acid contents were calculated, and the results are shown in Table 27.

[0295] The standard stipulates that the RSD of the gallic acid and rosmarinic acid content of the test solution is ≤3.0% (n=3).

[0296] Table 27 Durability test results - content

[0297]

[0298] The results showed that the RSD of gallic acid content was 2.30%, and the RSD of rosmarinic acid content was 2.95%, indicating that the method had good durability.

[0299] 7. Sample recovery test

[0300] Dissolve the gallic acid reference substance and the rosmarinic acid reference substance in methanol to prepare a mixed reference substance solution with a gallic acid concentration of 0.2448 mg / mL and a rosmarinic acid concentration of 0.5033 mg / mL. Accurately weigh 6 portions of basil granules with known contents, 0.25 g each, and add 1 mL of the mixed reference substance solution to each portion. Prepare the test solution according to the test solution preparation method of "1.1 Method Development-Gradient 1" in Example 3, and record them as spiked test solutions 1 to 6. Inject each solution into a high performance liquid chromatograph and measure. See Fig.26 , where A is gallic acid, B is rosmarinic acid, and 1 to 6 are spiked test solutions 1 to 6. The sample recovery, average sample recovery, and RSD value of the sample recovery of gallic acid and rosmarinic acid were calculated respectively. The results are shown in Table 28.

[0301] The standard stipulates that the limit of sample recovery rate is 98-100%, and the RSD of sample recovery rate of gallic acid and rosmarinic acid in the test solution is ≤3.0% (n=6).

[0302] Table 28 Sample recovery test results

[0303]

[0304]

[0305] The results showed that the recovery rates of gallic acid and rosmarinic acid were 97.23%, 102.79%, the RSD of gallic acid and rosmarinic acid was 1.20%, and 1.10%, respectively, indicating that the recovery rate of this method met the requirements.

[0306] 8. Detection of different batches of compound basil granules

[0307] According to the test solution preparation method of "1.1 Method Development - Gradient 1" in Example 3, the test solutions of the 7 compound basil granules batch numbers in Table 2 were prepared, and each test solution was injected into a high performance liquid chromatograph for determination. The results are shown in Table 2. Fig. 27 , where A: gallic acid; B: rosmarinic acid, 1: sample 20231101, 2: sample 20231102, 3: sample 20231103, 4: sample 20231104, 5: sample 20231105, 6: sample 20231106, 7: sample 20231107. The fingerprint similarity evaluation software of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version formulated by the National Pharmacopoeia Committee was used for similarity evaluation. The corresponding 35 characteristic peaks (35 characteristic peaks in the characteristic spectrum of the compound basil granules) were detected in different batches of compound basil granules, and the similarity was not less than 0.9, indicating that the quality of the 7 batches of compound basil granules was qualified, the product quality was uniform and stable.

[0308] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A characteristic spectrum of a compound basil granule, comprising characteristic peaks of gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercetin, rosmarinic acid, luteolin, apigenin, kaempferol, chelidonin and galangin.

2. The characteristic spectrum according to claim 1, characterized in that: The characteristic spectrum of the compound basil particles also includes a characteristic peak whose relative retention time with rosmarinic acid is within the range of a specified value×(1±5%); the specified value includes one or more of 0.037, 0.365, 0.379, 0.494, 0.547, 0.573, 0.602, 0.637, 0.644, 0.656, 0.847, 0.899, 0.924, 0.936, 0.973, 1.020, 1.034, 1.064, 1.195, 1.280, 1.358 and 1.

398.

3. A method for constructing a characteristic spectrum of the compound basil granules according to claim 1 or 2, comprising the following steps: Extracting the compound basil particles with an extractant to obtain a test solution; the extractant includes a lower alcohol and / or water; The test solution and the mixed reference solution are subjected to high performance liquid chromatography to obtain a test liquid chromatogram and a reference liquid chromatogram, respectively, and the retention time of the test liquid chromatogram is compared with the retention time of the reference liquid chromatogram to obtain a characteristic spectrum of the compound basil granules; The reference substances in the mixed reference substance solution include gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercetin, rosmarinic acid, luteolin, apigenin, kaempferol, chelidonol and galangin; The conditions for the high performance liquid chromatography detection include: the chromatographic column filler includes octadecylsilane bonded silica gel; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1-0.4 v / v% phosphoric acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes: 0-10 min, the volume fraction of the mobile phase A is 2-5%; 10-58 min, the volume fraction of the mobile phase A increases from 2-5% to 16-18%; 58-60 min, the volume fraction of the mobile phase A decreases from 16-18% to 12-15%; 60-68 min, the volume fraction of the mobile phase A decreases from 16-18% to 12-15% The integral fraction is 12-15%; from 68 to 110 min, the volume fraction of the mobile phase A increases from 12-15% to 28-31%; from 110 to 112 min, the volume fraction of the mobile phase A increases from 28-31% to 32-40%; from 112 to 130 min, the volume fraction of the mobile phase A increases from 32-40% to 45-60%; from 130 to 138 min, the volume fraction of the mobile phase A is 45-60%; from 138 to 145 min, the volume fraction of the mobile phase A decreases from 45-60% to 2-5%; from 145 to 150 min, the volume fraction of the mobile phase A is 2-5%.

4. The construction method according to claim 3, characterized in that: The solid-liquid ratio of the compound basil particles to the extractant is 0.5-2 g:100 mL; When the extractant contains lower alcohol, the volume fraction of the lower alcohol in the extractant is 50 to 100%; The lower alcohol includes methanol and / or ethanol.

5. The construction method according to claim 3 or 4, characterized in that: The extraction includes ultrasonic extraction or reflux extraction; the power of the ultrasonic extraction is 100-300W, the frequency is 30-50kHz, and the time is 15-60min; the time of the reflux extraction is 15-60min.

6. The construction method according to claim 3, characterized in that: The concentration of gallic acid in the mixed reference solution is 302.04-369.16 μg / mL, the concentration of neochlorogenic acid is 8.457-10.337 μg / mL, the concentration of chlorogenic acid is 6.073-7.423 μg / mL, the concentration of caffeic acid is 1.433-1.751 μg / mL, the concentration of rutin is 25.056-30.624 μg / mL, the concentration of hyperoside is 8.114-9.917 μg / mL, and the concentration of isoquercetin is 13.176~16.104μg / mL, the concentration of rosmarinic acid was 5.62~6.906μg / mL, the concentration of luteolin was 8.892~10.868μg / mL, the concentration of apigenin was 5.832~7.128μg / mL, the concentration of kaempferol was 11.945~14.599μg / mL, the concentration of chelidonol was 6.781~8.287μg / mL, and the concentration of naringenone was 347.76~425.04μg / mL; The solvent in the mixed reference substance solution includes lower alcohol.

7. The construction method according to claim 3, characterized in that: The mobile phase flow rate of the high performance liquid chromatography detection is 0.8-1.2 mL / min, the detection wavelength is 330 nm, the column temperature is 30-35° C., the theoretical plate number is calculated based on the rosmarinic acid peak and is ≥5000, and the injection volume is 10-20 μL.

8. Use of the characteristic spectrum of the compound basil granules according to claim 1 or 2 or the characteristic spectrum of the compound basil granules constructed by the construction method according to any one of claims 3 to 7 in the detection of compound basil granules.

9. A quality detection method for compound basil granules, comprising the following steps: Extracting the compound basil particles to be tested with an extractant to obtain a sample solution to be tested; the extractant includes lower alcohol and / or water; The sample liquid to be tested is subjected to high performance liquid chromatography to obtain a liquid chromatogram of the sample to be tested; A similarity evaluation system for Chinese medicine chromatographic fingerprint is used to evaluate the similarity between the liquid chromatogram of the sample to be tested and the characteristic spectrum of the compound basil granules. If the similarity is ≥0.9, the quality of the compound basil granules to be tested is qualified; if the similarity is <0.9, the quality of the compound basil granules to be tested is unqualified; the characteristic spectrum of the compound basil granules is the characteristic spectrum of the compound basil granules according to any one of claims 1 to 2 or the characteristic spectrum of the compound basil granules constructed by the construction method according to any one of claims 3 to 7; The conditions for the high performance liquid chromatography detection include: the chromatographic column filler includes octadecylsilane bonded silica gel; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1-0.4 v / v% phosphoric acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes: 0-15 min, the volume fraction of the mobile phase A is 2%; 15-20 min, the volume fraction of the mobile phase A increases from 2% to 18%; 20-62 min, the volume fraction of the mobile phase A is 18%; 62-65 min, the volume fraction of the mobile phase A increases from 18% to 65%; 65-77 min, the volume fraction of the mobile phase A is 65%; 77-80 min, the volume fraction of the mobile phase A decreases from 65% to 2%; 80-85 min, the volume fraction of the mobile phase A is 2%.

10. A method for detecting multiple index components of compound basil granules, comprising the following steps: Extracting the compound basil particles to be tested with an extractant to obtain a sample solution to be tested; the extractant includes lower alcohol and / or water; The sample solution to be tested is subjected to high performance liquid chromatography to obtain a multi-index component test result of the compound basil granules; The test result includes a qualitative test result and / or a qualitative test result, wherein the qualitative test result is obtained by comparing the liquid chromatogram of the sample solution to be tested with the characteristic spectrum of the compound basil granules; the characteristic spectrum of the compound basil granules is the characteristic spectrum of the compound basil granules according to any one of claims 1 to 2 or the characteristic spectrum of the compound basil granules constructed by the construction method according to any one of claims 3 to 7; The conditions for the high performance liquid chromatography detection include: the chromatographic column filler includes octadecylsilane bonded silica gel; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1-0.4 v / v% phosphoric acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes: 0-15 min, the volume fraction of the mobile phase A is 2%; 15-20 min, the volume fraction of the mobile phase A increases from 2% to 18%; 20-62 min, the volume fraction of the mobile phase A is 18%; 62-65 min, the volume fraction of the mobile phase A increases from 18% to 65%; 65-77 min, the volume fraction of the mobile phase A is 65%; 77-80 min, the volume fraction of the mobile phase A decreases from 65% to 2%; 80-85 min, the volume fraction of the mobile phase A is 2%; The multi-index components include gallic acid, neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, hyperoside, isoquercetin, rosmarinic acid, luteolin, apigenin, kaempferol, galangal, and at least two of the characteristic peaks whose relative retention time with rosmarinic acid is within the range of a specified value × (1 ± 5%); the specified value includes one or more of 0.037, 0.365, 0.379, 0.494, 0.547, 0.573, 0.602, 0.637, 0.644, 0.656, 0.847, 0.899, 0.924, 0.936, 0.973, 1.020, 1.034, 1.064, 1.195, 1.280, 1.358 and 1.398.

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