A method for establishing and applying a fingerprint spectrum of Huatan Jieyu mixture

Abstract

The present invention provides a method for establishing and applying a fingerprint spectrum of a phlegm-relieving and depression-removing mixture, and relates to the technical field of fingerprint spectrum analysis. The present invention prepares a test solution and performs detection under specific high-performance liquid chromatography conditions, imports the obtained chromatogram of the test solution into a traditional Chinese medicine fingerprint spectrum similarity evaluation system, and performs data import, multi-point correction and data matching on the chromatogram of the test solution to obtain a fingerprint spectrum, and fingerprint spectrum detection can be achieved through similarity analysis. The fingerprint spectrum detection method established by the present invention is stable, and at the same time, the characteristic information of each medicinal material in the prescription is reflected in the fingerprint spectrum, which can comprehensively, objectively and scientifically evaluate and control the quality of the product, and provide technical support and evaluation methods for the quality control and industrial production of the phlegm-relieving and depression-removing mixture product.

Description

Technical Field

[0001] The present invention relates to the technical field of fingerprint analysis, and in particular to a method for establishing a fingerprint of a phlegm-relieving and depression-relieving mixture and its application. Background Art

[0002] Huatan Jieyu Mixture is a modified version of Wendan Decoction from the "Qianjin Essential Prescriptions for Emergencies." It is composed of Pinellia ternata, Citrus aurantium immaturus, Tangerine peel, White Peony Root, Rhizoma Anemarrhenae, Licorice Root, Polygala tenuifolia, Cyperus rotundus, Poria cocos, and Curcuma aromatica. It has the functions of regulating qi and resolving phlegm, opening the orifices and relieving depression, and is used for epilepsy caused by internal phlegm-dampness. This decoction has been used clinically for many years with proven efficacy, but a comprehensive quality control method currently lacks. Currently, quality control of Chinese patent medicines typically follows quality standards, quantitatively testing a single component or several ingredients, combined with thin-layer chromatography for qualitative identification of the ingredients. This method is not only complex but also provides scant chemical information for quality control, failing to fully characterize product quality and characteristics. Furthermore, it falls short of achieving comprehensive product quality control requirements. Furthermore, simultaneous separation and qualitative and quantitative detection of multiple components is difficult under conventional chromatographic conditions.

[0003] Therefore, the present invention provides a method for establishing a fingerprint spectrum of a phlegm-relieving and depression-relieving mixture and its fingerprint spectrum for a new phlegm-relieving and depression-relieving preparation, establishes a fingerprint spectrum method and fingerprint spectrum with a large number of characteristic peaks and a large amount of information, can comprehensively, accurately and quickly monitor its quality, realize quality control of the phlegm-relieving and depression-relieving mixture, meet the requirements of product quality control, and provide a safe, effective and quality-controlled product for patients. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for establishing a fingerprint spectrum of Huatan Jieyu mixture and its application, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for establishing a fingerprint spectrum of Huatan Jieyu mixture, comprising the following steps:

[0007] Preparation of the test solution: dilute the Huatan Jieyu mixture with water, filter through a microporous membrane, and take the filtrate to obtain the test solution;

[0008] High performance liquid chromatography detection: The test solution is detected by high performance liquid chromatography to obtain a chromatogram of the test solution;

[0009] Fingerprint establishment: the chromatogram of the test solution is imported into the traditional Chinese medicine fingerprint similarity evaluation system, and the chromatogram of the test solution is subjected to data import, multi-point correction and data matching to obtain a fingerprint;

[0010] The chromatographic conditions of the HPLC detection are:

[0011] The method adopts a C18 packed reverse phase chromatography column, with acetonitrile as mobile phase A and 0.08-0.12% phosphoric acid aqueous solution as mobile phase B, and performs gradient elution; the gradient elution program is: 0-8 min, 5%-9% acetonitrile; 8-70 min, 9%-30% acetonitrile; 70-90 min, 30%-45% acetonitrile; 90-95 min, 45%-80% acetonitrile; 95-100 min, 80% acetonitrile; 100-105 min, 80%-5% acetonitrile; 105-110 min, 5% acetonitrile; the flow rate is 0.8-1.2 mL / min, the detection wavelength is 225-235 nm, the injection volume is 5-15 μL, and the column temperature is 25-35°C.

[0012] Furthermore, the phlegm-resolving and depression-relieving mixture is composed of ten Chinese medicinal herbs: Pinellia ternata, Citrus aurantium immaturus, dried tangerine peel, white peony root, bamboo shavings, liquorice, Polygala tenuifolia, Cyperus rotundus, Poria cocos and Curcuma aromatica.

[0013] Furthermore, the chromatographic column uses Elite Supersil ODS2, acetonitrile as mobile phase A, and 0.1% phosphoric acid aqueous solution as mobile phase B, and gradient elution is performed; the gradient elution program is: 0-8 min, 5%-9% acetonitrile; 8-70 min, 9%-30% acetonitrile; 70-90 min, 30%-45% acetonitrile; 90-95 min, 45%-80% acetonitrile; 95-100 min, 80% acetonitrile; 100-105 min, 80%-5% acetonitrile; 105-110 min, 5% acetonitrile; the flow rate is 1.0 mL / min, the detection wavelength is 230 nm, the injection volume is 10 μL, and the column temperature is 30°C.

[0014] In the present invention, the chromatographic column stationary phase uses octadecylsilane bonded silica gel as filler.

[0015] Furthermore, the fingerprint has 20 common peaks, numbered 1 to 20, among which: chromatographic peak 2 is the characteristic peak of bamboo shavings; chromatographic peaks 3, 11, 13 to 16, 18, and 20 are the characteristic peaks of polygala tenuifolia; chromatographic peak 6 is the characteristic peak of white peony root; chromatographic peak 19 is the characteristic peak of dried tangerine peel; chromatographic peaks 1, 4, 7, 9, 10, and 12 are common to both immature bitter orange and dried tangerine peel; chromatographic peaks 8 and 17 are the characteristic peaks of The chromatographic peaks are shared by Pinellia ternata, Polygala tenuifolia, and Licorice; the chromatographic peak No. 5 is shared by Immature Citrus aurantium, Citrus reticulata, and White Peony Root; among the shared peaks: Peak No. 1 is synephrine, Peak No. 3 is Siberian Polygala sugar A6, Peak No. 6 is paeoniflorin, Peak No. 8 is liquiritin, Peak No. 11 is 3,6'-diesinapoylsucrose, Peak No. 12 is hesperidin, Peak No. 17 is ammonium glycyrrhizate, Peak No. 18 is Polygala tenuifolia saponin B, and Peak No. 20 is polygalic acid.

[0016] The present invention provides application of the fingerprint establishment method in quality detection and / or quality control of Huatan Jieyu mixture.

[0017] The present invention also provides a fingerprint detection method for Huatan Jieyu mixture, comprising the following steps:

[0018] Compare the HPLC detection spectrum of the sample to be tested with the above fingerprint spectrum, and perform similarity evaluation according to the traditional Chinese medicine fingerprint spectrum similarity evaluation system;

[0019] The HPLC detection spectrum of the sample to be tested is obtained by the following steps:

[0020] Preparation of the test solution: dilute the Huatan Jieyu mixture with water, filter through a microporous membrane, and take the filtrate to obtain the test solution;

[0021] High performance liquid chromatography detection: The test solution is detected by high performance liquid chromatography to obtain an HPLC detection spectrum of the sample to be tested;

[0022] The chromatographic conditions of the HPLC detection are:

[0023] The method adopts a C18 packed reverse phase chromatography column, with acetonitrile as mobile phase A and 0.08-0.12% phosphoric acid aqueous solution as mobile phase B, and performs gradient elution; the gradient elution program is: 0-8 min, 5%-9% acetonitrile; 8-70 min, 9%-30% acetonitrile; 70-90 min, 30%-45% acetonitrile; 90-95 min, 45%-80% acetonitrile; 95-100 min, 80% acetonitrile; 100-105 min, 80%-5% acetonitrile; 105-110 min, 5% acetonitrile; the flow rate is 0.8-1.2 mL / min, the detection wavelength is 225-235 nm, the injection volume is 5-15 μL, and the column temperature is 25-35°C.

[0024] Furthermore, the chromatographic column uses Elite Supersil ODS2, acetonitrile as mobile phase A, and 0.1% phosphoric acid aqueous solution as mobile phase B, and gradient elution is performed; the gradient elution program is: 0-8 min, 5%-9% acetonitrile; 8-70 min, 9%-30% acetonitrile; 70-90 min, 30%-45% acetonitrile; 90-95 min, 45%-80% acetonitrile; 95-100 min, 80% acetonitrile; 100-105 min, 80%-5% acetonitrile; 105-110 min, 5% acetonitrile; the flow rate is 1.0 mL / min, the detection wavelength is 230 nm, the injection volume is 10 μL, and the column temperature is 30°C.

[0025] Furthermore, the similarity evaluation process determines that the similarity is ≥ 0.90, preferably ≥ 0.92.

[0026] Furthermore, the sample to be tested should have 20 common peaks in the fingerprint spectrum, and the retention time of each peak in the fingerprint spectrum should be within 0.5%.

[0027] The present invention discloses the following technical effects:

[0028] The present invention establishes a fingerprint of Huatan Jieyu mixture and uses similarity software to evaluate the differences in product component groups to provide support for product quality control.

[0029] The fingerprint detection method established in the present invention is stable and has the characteristics of high detection precision and good reproducibility, etc., and provides a reliable method for the quality evaluation and quality control of Huatan Jieyu mixture products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 Fingerprints of 10 batches of compound decoction prepared in Example 2 of the present invention for test and control samples;

[0032] Figure 2 HPLC comparison chart of the single herbal test sample, the compound decoction reference fingerprint and the mixed reference prepared in Example 2 of the present invention;

[0033] Figure 3 HPLC fingerprints of the decoction, concentrate, centrifuge and mixture (preparation) in Example 9 of the present invention;

[0034] Figure 4 This is a cluster dendrogram of the pilot and pilot samples in Example 10 of the present invention;

[0035] Figure 5 The transfer rate (A) and the cream yield (B) of the three index components in Example 11 of the present invention are shown;

[0036] Figure 6 The changes in solid content and total mass of different samples in Example 12 of the present invention. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] The following examples further illustrate the technical solution of the present invention. The instruments and reagents used in the following examples are as follows:

[0043] instrument:

[0044] LC-2030C 3D high-performance liquid chromatograph and workstation (Shimadzu Corporation, Japan); AP125WD 1 / 100,000 balance (Shimadzu Corporation, Japan); BSA224S 1 / 10,000 balance (Sartorius); GENIUS 16K high-speed centrifuge (Changsha Xinao Instrument Co., Ltd.); SY-5000 rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); TC-15 jacketed thermostat (Haining Xinhua Medical Instrument Factory).

[0045] Reagents:

[0046] Synephrine (batch number 110727-201809, 99.8%); paeoniflorin (batch number 110736-201943, mass fraction 95.1%), hesperidin (batch number 110721-201818, mass fraction 96.2%), polygala tenuifolia root III (batch number 111850-201504, mass fraction 95.5%), liquiritin (batch number 111610-201607, mass fraction 93.1%), ammonium glycyrrhizate (batch number 11073 1-201720, mass fraction 97.7%), and 3,6'-diesinapoylsucrose (batch number 111848-201805, mass fraction 96.6%) were purchased from the China Food and Drug Administration; polygalic acid (batch number 151127, mass fraction ≥98%), Siberian polygala sugar A6 (batch number 151015, mass fraction ≥95%), and polygalaenoside B (batch number 150629, mass fraction ≥98%) were obtained from Chengdu Pufeide Biotechnology Co., Ltd. Acetonitrile (Merck, Germany) was chromatographic grade; all other reagents were of analytical grade.

[0047] The medicinal materials used in the pilot test of the Huatan Jieyu mixture of the present invention were collected from three production areas, and the product quality was satisfactory, as shown in Table 1. The medicinal materials used in the pilot production of Huatan Jieyu mixture were purchased from Guangzhou Zhixin Chinese Medicine Pieces Co., Ltd. and met the quality requirements of the 2020 edition of the Chinese Pharmacopoeia. The pilot production was conducted at a pharmaceutical company in Guangzhou that complies with GMP requirements. The pilot decoction, concentrate, centrifuge, and finished product were collected for quality control.

[0048] Table 1 Origin information of Chinese medicinal materials for Huatan Jieyu mixture

[0049]

[0050] Chromatographic conditions:

[0051] The chromatographic column was Elite Supersil ODS2 (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile-0.1% phosphoric acid solution, with a gradient elution: 0–8 min, 5%–9% acetonitrile; 8–70 min, 9%–30% acetonitrile; 70–90 min, 30%–45% acetonitrile; 90–95 min, 45%–80% acetonitrile; 95–100 min, 80% acetonitrile; 100–105 min, 80%–5% acetonitrile; 105–110 min, 5% acetonitrile; the volume flow rate was 1.0 mL min -1 ; Column temperature was 30℃; Injection volume was 10μL.

[0052] Example 1

[0053] Preparation of the compound decoction: Using the RANDBETWEEN function in Excel software to generate random numbers, the medicinal materials from different origins and batches listed in Table 1 were randomly combined into 10 batches of prescriptions (proportion: 9% of French Pinellia, 9% of Citrus aurantium, 5% of Tangerine Peel, 9% of White Peony Root, 9% of Rhizoma Anemarrhenae, 6% of Licorice Root, 9% of Polygala Tenuifolia, 9% of Cyperus Rhizoma, 9% of Poria, and 9% of Curcuma Radix). The medicinal materials from each batch were added to 15-fold water and decocted twice, each for 1.5 hours. The decoction was then filtered through 200 mesh. The filtrates were combined and mixed thoroughly to obtain the compound decoction.

[0054] Preparation of single herbal decoction: Take single herbal herbs from different origins in Table 1, decoct them twice using the same method, combine the decoctions, and mix well.

[0055] Preparation of the mixture: Take the compound decoction, concentrate it under reduced pressure to an appropriate volume to obtain a concentrated solution; centrifuge it at high speed, separate the supernatant and obtain the centrifuge liquid; add the auxiliary materials, stir to dissolve, add water, and mix well to obtain the mixture.

[0056] Preparation of negative samples: Take other medicinal materials that do not contain this medicinal material, and extract, concentrate, and high-speed centrifuge according to the extraction and preparation method of Huatan Jieyu Mixture, and take the supernatant to obtain the negative control solution.

[0057] Example 2

[0058] Preparation of test samples: Accurately weigh 10 mL of each of the compound decoction and single herbal decoction from Example 1, dilute to 25 mL with water, and filter through a 0.22 μm microporous membrane. The filtrate is collected to obtain the compound decoction test sample and the single herbal decoction test sample. Ten batches of compound decoction test samples are designated S1 to S10.

[0059] Example 3

[0060] Preparation of mixed reference solution: Take appropriate amounts of hesperidin, paeoniflorin, synephrine, 3,6'-dieserinoylsucrose, polygalic acid, polygalaenoside B, polygala sibirica A6, liquiritin, and ammonium glycyrrhizate, accurately weigh them, and add methanol to prepare a mixed reference solution containing 21.93, 32.87, 15.92, 9.00, 73.02, 33.63, 4.89, 18.77, and 50.09 μg per mL, respectively.

[0061] Example 4

[0062] Precision test: One portion of the compound decoction sample (S1) prepared in Example 2 was injected and analyzed using the above-described chromatographic conditions for six consecutive times. The chromatogram was recorded. Using hesperidin as the reference peak, the relative retention times and relative peak areas of the common peaks were calculated to have RSDs of 0-0.19% and 0-2.06%, respectively, indicating that the precision of the instrument was good and met the requirements.

[0063] Example 5

[0064] Solution stability test: One portion of the compound decoction sample (S1) prepared in Example 2 was injected and analyzed at 0, 3, 6, 9, 12, 24, and 36 h according to the above chromatographic conditions. The chromatograms were recorded. Using hesperidin as the reference peak, the relative retention times and relative peak areas of the common peaks were calculated to have RSDs of 0-0.27% and 0-2.92%, respectively. This indicated that the test solution had good stability within 36 h and met the requirements.

[0065] Example 6

[0066] Repeatability test: The compound decoction sample (S1) prepared in Example 2 was injected and analyzed according to the above chromatographic conditions. The chromatogram was recorded. Using hesperidin as the reference peak, the relative retention time and relative peak area of ​​each common peak were calculated. The RSDs were 0-0.48% and 0-2.22%, respectively, indicating that the method had good repeatability and met the requirements.

[0067] Example 7

[0068] Fingerprint establishment: Take 10 batches of compound decoction samples prepared in Example 2 (S1-S10), sample injection and measurement according to the above chromatographic conditions, and import the obtained spectra into the Chinese medicine fingerprint similarity evaluation system (2012 version). With S1 as the reference spectrum, the average method is adopted, the time window is 0.1min, multi-point correction and peak matching are set, and the control fingerprint spectrum (R) is generated. The results are shown in Figure 1 .

[0069] Using the control fingerprint as a reference, the similarity between each batch of decoction was greater than 0.920, reflecting minimal differences in the main substance groups within the different batches. Detailed data are shown in Table 2. Based on the screening principles of good reproducibility and resolution, a total of 20 common peaks were identified. In the fingerprints of the 10 batches of decoction, the ratio of the common peak area to the total chromatographic peak area was (59.52 ± 2.60)%.

[0070] Table 2 Similarity results of fingerprints of 10 batches of compound decoctions

[0071]

[0072]

[0073] Example 8

[0074] Identification and attribution of major chromatographic peaks:

[0075] Take the single herbal decoction prepared in Example 2 and the mixed reference solution prepared in Example 3, inject and measure according to the above chromatographic conditions, record the chromatogram, and compare it with the fingerprint of the compound decoction based on the retention time and spectrum of the chromatographic peak (see Figure 2Peak 2 was identified as the characteristic peak of Rhizoma Coptidis; peaks 3, 11, 13-16, 18, and 20 were identified as the characteristic peaks of Polygala tenuifolia; peak 6 was identified as the characteristic peak of Paeonia lactiflora; and peak 19 was identified as the characteristic peak of Pericarpium Citri Reticulatae; peaks 1, 4, 7, 9, 10, and 12 were shared by Immaturus Aurantii Immaturus and Pericarpium Citri Reticulatae; peaks 8 and 17 were shared by Pinellia ternatae, Polygala tenuifolia, and Glycyrrhiza uralensis; and peak 5 was shared by Immaturus Aurantii Immaturus, Pericarpium Citri Reticulatae, and Paeonia lactiflora. Nine components were identified from the shared peaks: peak 1 (synephrine), peak 3 (Siberian Polygala sugar A6), peak 6 (paeoniflorin), peak 8 (liquiritin), peak 11 (3,6'-dieserinoylsucrose), peak 12 (hesperidin), peak 17 (ammonium glycyrrhizate), peak 18 (Polygala tenuifolia saponin B), and peak 20 (polygalic acid). Figure 2 Middle: A-reference fingerprint of compound decoction, B-mixed reference substance, C-dried orange peel, D-fructus aurantii, E-white peony root, F-polygala tenuifolia, G-licorice root, H-bamboo shavings, I-cyperus rotundus, J-pillaria ternata, K-curcuma aromatica, L-poria.

[0076] The fingerprint of the present invention has 20 common peaks, and the retention time of each peak in the fingerprint and the standard deviation of the retention time of each common peak in 10 batches of products are all within 0.3%.

[0077] Table 3 Peak emergence time of each common peak

[0078] batch 01 02 03 04 05 06 07 08 09 10 RSD Common peak T / min T / min T / min T / min T / min T / min T / min T / min T / min T / min X％ Peak 1 3.69 3.65 3.66 3.66 3.67 3.65 3.64 3.67 3.66 3.64 0.01 Peak 2 7.31 7.29 7.30 7.29 7.30 7.30 7.27 7.18 7.21 7.18 0.05 Peak 3 21.73 21.69 21.68 21.67 21.69 21.71 21.56 21.29 21.37 21.32 0.17 Peak 4 24.55 24.53 24.52 24.51 24.46 24.52 24.40 24.15 24.23 24.19 0.16 Peak 5 27.28 27.25 27.24 27.22 27.23 27.26 27.10 26.80 26.90 26.85 0.19 Peak 6 30.35 30.30 30.29 30.28 30.29 30.32 30.16 29.87 29.96 29.91 0.19 Peak 7 36.31 36.27 36.26 36.25 36.25 36.30 36.12 35.83 35.92 35.88 0.19 Peak 8 38.53 38.53 38.52 38.51 38.50 38.55 38.36 38.09 38.16 38.16 0.18 Peak 9 42.38 42.34 42.33 42.33 42.33 42.37 42.24 41.98 42.05 42.02 0.16 Peak 10 44.75 44.71 44.69 44.68 44.68 44.74 44.58 44.28 44.35 44.32 0.19 Peak 11 48.26 48.21 48.20 48.19 48.19 48.25 48.07 47.77 47.84 47.82 0.19 Peak 12 49.04 49.00 48.99 48.98 48.96 49.03 48.86 48.57 48.63 48.62 0.19 Peak 13 54.95 54.89 54.88 54.87 54.88 54.93 54.76 54.47 54.52 54.52 0.19 Peak 14 68.12 68.05 68.03 68.02 68.05 68.11 67.96 67.66 67.69 67.76 0.17 Peak 15 74.14 74.07 74.06 74.04 74.09 74.14 73.97 73.68 73.68 73.81 0.18 Peak 16 75.35 75.30 75.28 75.27 75.31 75.36 75.20 74.93 74.94 75.06 0.16 Peak 17 87.68 87.65 87.63 87.63 87.67 87.69 87.60 87.35 87.34 87.44 0.14 Peak 18 90.90 90.37 90.36 90.35 90.39 90.41 90.32 90.07 90.04 90.14 0.24 Peak 19 92.83 92.80 92.78 92.77 92.82 92.84 92.74 92.51 92.49 92.59 0.14 Peak 20 96.11 96.10 96.09 96.09 96.11 96.12 96.08 95.99 95.98 96.01 0.05

[0079] Example 9

[0080] Changes in fingerprints during pilot preparation:

[0081] Take 3 batches of decoction, concentrate, centrifuge and Huatan Jieyu mixture produced in the pilot test, accurately measure 1 mL respectively, prepare the test solution according to the method of Example 2, and inject and analyze according to the above chromatographic conditions, and record the chromatogram ( Figure 3 ). The fingerprints of the four samples were analyzed for similarity with the fingerprint established in Example 7. In the figure, A-mixture, B-centrifuge, C-concentrate, D-decoction, and R-fingerprint established in Example 7. Among them, the average similarities of the first three were 0.936, 0.886, and 0.865, respectively, and the number of chromatographic peaks was basically the same; two new chromatographic peaks appeared in the mixture's spectrum, and the similarity decreased. Peak 21 was an antibacterial agent, and peak 22 was a flavoring agent. 20 common peaks are stably present in the intermediates and finished products, which can be used to evaluate the quality changes of Huatan Jieyu Mixture in different production links.

[0082] Example 10

[0083] Cluster analysis:

[0084] The peak areas of 20 common peaks of 10 batches of small-scale tests (samples of Example 1) and 3 batches of pilot decoctions (samples of Example 9) were normalized and imported into SPSS 25.0 analysis software. The samples were classified by using the intergroup linkage method and the squared Euclidean distance as the measurement. The results are shown in Table 1. Figure 4 . Figure 4 A1~A10 are small test samples, B1~B3 are pilot samples.

[0085] There were varying degrees of differences among the decoctions. When d = 1, the three pilot batches were clustered into one category, indicating that the production process and the source of the medicinal materials could be distinguished in this fingerprint detection system. When d = 10, the three pilot batches and the eight small-scale decoction batches except A1 and A5 were clustered together, suggesting that the origin of the medicinal materials had a greater impact on the quality of the decoctions, followed by factors such as production batch size and equipment scale-up.

[0086] Example 11

[0087] Investigation on the transfer of measurement value in pilot production

[0088] Three batches each of the pilot-scale medicinal materials, decoction, concentrate, centrifuge, and Huatan Jieyu mixture were sampled and analyzed. The contents of synephrine, paeoniflorin, and hesperidin were determined, and the transfer rates were calculated. The contents and transfer rates of the different pilot-scale samples were calculated. Specific data are shown in Tables 4 and 5.

[0089] Table 4 Contents of synephrine, paeoniflorin and hesperidin in the medicinal material (A), decoction (B), concentrate (C), centrifuge (D) and mixture (E) (%)

[0090]

[0091] Table 5 Transfer rate of synephrine, paeoniflorin and hesperidin (%)

[0092]

[0093]

[0094] The transfer rates of the three index components in the pilot and small-scale tests of Huatan Jieyu Mixture were compared. Figure 5 .

[0095] Example 12

[0096] Investigation of paste yield, solid content and total mass

[0097] Accurately measure 25 mL each of the pilot decoction, concentrate, centrifuge liquid and Huatan Jieyu mixture, place them in an evaporating dish that has been dried to constant weight, evaporate to dryness on a water bath, dry to constant weight at 105°C, cool in a desiccator for 30 minutes, accurately weigh, and calculate the solid content, total mass and paste yield.

[0098] Among them, solid content (%, g / mL) = M (g) / 25mL × 100%, total solid mass (kg) = solid content × V (L), paste yield (%) = total solid mass of decoction (kg) / medicinal material input amount (kg) × 100%, where M is the mass of solid in 25mL sample, and V is the total volume of the sample. Results are shown in Figure 5-6 .

[0099] Figure 6 The changes in solid content and total mass of different samples: changes in solid content (A) and total mass (B).

[0100] The decoction volume is large and the solid content is low. After concentration, the volume is significantly reduced, the solid content is increased, but the total mass is lost. After high-speed centrifugation, part of the solids are removed in the form of precipitation, resulting in a significant decrease in their content in the centrifuge and total mass. When auxiliary materials are added during the liquid preparation step, the solid content and total mass are increased. The average solid content and total mass of the mixture are (14.16±0.17)% and (16.89±0.24) kg. Figure 5 B shows that the average paste yield of the three batches of pilot samples is (21.77±0.56)%, which is consistent with the results of the small test.

[0101] Analysis of fingerprint patterns, transfer rates, and paste yields showed high similarity between the pilot decoction and the control fingerprint patterns, stable transfer rates for the three index components, and consistent paste yields compared to the small-scale test, suggesting a stable scale-up extraction process. Ignoring excipient peaks, the number, shape, and retention times of chromatographic peaks in the fingerprints from the decoction, concentrate, centrifuge, and mixture were essentially consistent. Transfer rates for each component were similar in the pilot and small-scale finished products, as were solid yields, reflecting a stable formulation process and demonstrating the feasibility of this fingerprint method for quality control. Regardless of the missing medicinal ingredient or significant process changes, the resulting fingerprints, solid yields, and transfer rates of the index components would be inconsistent. Comprehensive consideration of fingerprint patterns, transfer rates, and solids revealed high similarity between the small-scale and pilot-scale products. HCA analysis indicated uniform quality across the three pilot-scale batches, indicating minimal impact of process scale-up on the quality of the decoction, laying the foundation for the product's efficacy.

[0102] The fingerprint established by the present invention can reflect the overall chemical composition characteristics of the compound. The quality of the pilot decoction is consistent with that of the small-scale test, which shows that the extraction process is reliable, provides a reference for quality and process control, and can be used for the quality control of Huatan Jieyu mixture.

[0103] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for establishing a fingerprint spectrum of Huatan Jieyu mixture, characterized in that: The following steps are involved: Preparation of the test solution: dilute the Huatan Jieyu mixture with water, filter through a microporous membrane, and take the filtrate to obtain the test solution; High performance liquid chromatography detection: The test solution is detected by high performance liquid chromatography to obtain a chromatogram of the test solution; Fingerprint establishment: the chromatogram of the test solution is imported into the traditional Chinese medicine fingerprint similarity evaluation system, and the chromatogram of the test solution is subjected to data import, multi-point correction and data matching to obtain a fingerprint; The chromatographic conditions of the HPLC detection are: A C18 packed reverse phase chromatography column was used, acetonitrile was used as mobile phase A, and 0.08-0.12% phosphoric acid aqueous solution was used as mobile phase B for gradient elution; the gradient elution program was as follows: 0-8 min, 5%-9% acetonitrile; 8-70 min, 9%-30% acetonitrile; 70-90 min, 30%-45% acetonitrile; 90-95 min, 45%-80% acetonitrile; 95-100 min, 80% acetonitrile; 100-105 min, 80%-5% acetonitrile; 105-110 min, 5% acetonitrile; the flow rate was 0.8-1.2 mL / min, the detection wavelength was 225-235 nm, the injection volume was 5-15 μL, and the column temperature was 25-35°C; The phlegm-removing and depression-relieving mixture is composed of ten Chinese medicinal herbs: pinellia tuber, immature bitter orange, dried tangerine peel, white peony root, bamboo shavings, liquorice root, polygala root, cyperus rotundus, poria and curcuma.

2. The method for establishing the fingerprint of Huatan Jieyu mixture according to claim 1, characterized in that: The chromatographic column used was Elite Supersil ODS2, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, for gradient elution; the gradient elution program was as follows: 0-8 min, 5%-9% acetonitrile; 8-70 min, 9%-30% acetonitrile; 70-90 min, 30%-45% acetonitrile; 90-95 min, 45%-80% acetonitrile; 95-100 min, 80% acetonitrile; 100-105 min, 80%-5% acetonitrile; 105-110 min, 5% acetonitrile; the flow rate was 1.0 mL / min, the detection wavelength was 230 nm, the injection volume was 10 μL, and the column temperature was 30°C.

3. The method for establishing the fingerprint of Huatan Jieyu mixture according to claim 1, characterized in that: The fingerprint spectrum has 20 common peaks, numbered 1 to 20, among which: chromatographic peak No. 2 is the characteristic peak of bamboo shavings; chromatographic peaks No. 3, 11, 13 to 16, 18, and 20 are characteristic peaks of Polygala tenuifolia; chromatographic peak No. 6 is the characteristic peak of white peony root; chromatographic peak No. 19 is the characteristic peak of dried tangerine peel; chromatographic peaks No. 1, 4, 7, 9, 10, and 12 are shared by Citrus aurantium immaturus and dried tangerine peel; chromatographic peaks No. 8 and 17 are shared by Pinellia ternata, Polygala tenuifolia, and Licorice; chromatographic peak No. 5 is shared by Citrus aurantium immaturus, dried tangerine peel, and white peony root; among the common peaks: Peak No. 1 is synephrine, Peak No. 3 is Siberian polygala sugar A6, Peak No. 6 is paeoniflorin, Peak No. 8 is liquiritin, Peak No. 11 is 3,6'-diesinapoylsucrose, Peak No. 12 is hesperidin, Peak No. 17 is ammonium glycyrrhizate, Peak No. 18 is Polygala tenuifolia saponin B, and Peak No. 20 is polygalic acid.

4. Application of the fingerprint establishment method according to any one of claims 1 to 3 in quality detection and / or quality control of Huatan Jieyu Mixture.

5. A fingerprint detection method for Huatan Jieyu mixture, characterized in that: The following steps are involved: Comparing the HPLC detection spectrum of the sample to be tested with the fingerprint spectrum according to any one of claims 1 to 4, and performing similarity evaluation according to the traditional Chinese medicine fingerprint spectrum similarity evaluation system; The HPLC detection spectrum of the sample to be tested is obtained by the following steps: Preparation of the test solution: dilute the Huatan Jieyu mixture with water, filter through a microporous membrane, and take the filtrate to obtain the test solution; High performance liquid chromatography detection: The test solution is detected by high performance liquid chromatography to obtain an HPLC detection spectrum of the sample to be tested; The chromatographic conditions of the HPLC detection are: The method adopts a C18 packed reverse phase chromatographic column, takes acetonitrile as mobile phase A, and takes 0.08-0.12% phosphoric acid aqueous solution as mobile phase B, and performs gradient elution; the gradient elution program is as follows: 0-8 min, 5%-9% acetonitrile; 8-70 min, 9%-30% acetonitrile; 70-90 min, 30%-45% acetonitrile; 90-95 min, 45%-80% acetonitrile; 95-100 min, 80% acetonitrile; 100-105 min, 80%-5% acetonitrile; 105-110 min, 5% acetonitrile; the flow rate is 0.8-1.2 mL / min, the detection wavelength is 225-235 nm, the injection volume is 5-15 μL, and the column temperature is 25-35°C.

6. The fingerprint detection method of Huatan Jieyu mixture according to claim 5, characterized in that: The chromatographic column used was Elite Supersil ODS2, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, for gradient elution; the gradient elution program was as follows: 0-8 min, 5%-9% acetonitrile; 8-70 min, 9%-30% acetonitrile; 70-90 min, 30%-45% acetonitrile; 90-95 min, 45%-80% acetonitrile; 95-100 min, 80% acetonitrile; 100-105 min, 80%-5% acetonitrile; 105-110 min, 5% acetonitrile; the flow rate was 1.0 mL / min, the detection wavelength was 230 nm, the injection volume was 10 μL, and the column temperature was 30°C.

7. The fingerprint detection method of Huatan Jieyu mixture according to claim 5, characterized in that: The similarity evaluation process determines that the similarity is ≥ 0.90.

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