Establishment method of traditional Chinese medicine fingerprint of postoperative ventilation decoction

Abstract

The application belongs to the field of pharmaceutical analysis, and particularly relates to a method for establishing a traditional Chinese medicine fingerprint of postoperative ventilation decoction. The method is realized through the following steps: firstly, preparing a control solution and a test solution, and then detecting by using high performance liquid chromatography to establish the fingerprint. The method provided by the application can effectively represent the change of the component quantity between samples by reasonably selecting the control component and adopting the method of fixed wavelength according to the maximum absorption wavelength of the component, so that the quality of the sample can be more accurately judged. The application further provides a preparation method screening and optimization method of the postoperative ventilation decoction. The prepared method has good repeatability and stability, and can more comprehensively monitor the quality of the postoperative ventilation decoction. The method provides a valuable reference for the quality evaluation and standardization of traditional Chinese medicine decoction.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of pharmaceutical analysis, and particularly relates to a method for establishing a traditional Chinese medicine (TCM) fingerprint of postoperative ventilation decoction. BACKGROUND

[0002] Postoperative ileus (POI) often occurs after surgery, and patients present the clinical phenomenon of being unable to take in food orally, which is characterized by impaired gastrointestinal peristalsis and can be manifested in postoperative symptoms such as nausea, vomiting, abdominal pain, and cessation of exhaust and defecation. Postoperative ventilation decoction is developed by pharmacists and clinical experts of Jiangsu Provincial Hospital of Traditional Chinese Medicine, and is composed of eight TCMs, including fried Semen Raphani, fried Semen Armeniacae Amarum, fried Perilla Fruit, Herba Taraxaci, charred Hoveniae Fructus, fried Semen Galli, Pericarpium Citri Reticulatae and Glycyrrhizae Radix. Fried Semen Raphani, fried Perilla Fruit and fried Semen Armeniacae Amarum are the monarch drugs, which are taken from the classic prescription “Sanzi Yangqin Decoction” and are derived from “Han's Medical Treatise”, and have the effects of descending qi, digesting food, warming lung and reducing sputum. Fried Semen Galli and charred Hoveniae Fructus digest food, strengthen stomach and promote digestion, and Pericarpium Citri Reticulatae regulates qi and strengthens spleen, while Herba Taraxaci clears heat, resolves toxins, dissipates stasis, promotes diuresis and relieves stranguria, and the four are the minister drugs, and Glycyrrhizae Radix is added to harmonize the drugs and tonify heart and spleen. The decoction treats lung, stomach and intestines together, and promotes recovery of gastrointestinal function by descending qi, reducing sputum, digesting food and promoting transportation. The whole decoction promotes recovery of postoperative gastrointestinal function by descending qi, reducing sputum, digesting food and promoting transportation.

[0003] The TCM fingerprint comprehensively reflects the types and quantities of chemical components contained in TCM and its preparations, and then describes and evaluates the overall quality of the drug. The HPLC fingerprint can effectively control the quality of TCM, and can provide sufficient and reliable information even if the chromatographic peaks are unknown compounds, and can be used to identify the quality of TCM.

[0004] The TCM compound has the characteristics of multiple components, multiple targets and multiple levels, which indicates that a single index cannot comprehensively and integrally reflect the characteristics of the compound, and the comprehensive evaluation of multiple indexes can comprehensively reflect multiple important indexes. At present, the postoperative ventilation decoction lacks relevant quality control methods, and the quality of the drug cannot be accurately judged. Therefore, it has become a problem to be solved to research a method for accurately judging the quality of the postoperative ventilation decoction. SUMMARY

[0005] In view of the technical blank and other problems in the prior art, the purpose of the present application is to provide a method for establishing a TCM fingerprint of postoperative ventilation decoction and a quality detection method of postoperative ventilation decoction, so as to solve the problem that the prior art cannot quantitatively determine the evaluation standard of the overall quality of the postoperative ventilation decoction.

[0006] In order to achieve the purpose, the technical scheme adopted by the present application is to provide a method for establishing a TCM fingerprint of postoperative ventilation decoction, which comprises the following steps:

[0007] (1) Preparation of the control solution: accurately weigh 5-HMF, chlorogenic acid, mustard thiocyanate, chicoric acid, naringin, 3,6'-di-glucorapeseed sucrose, hesperidin and rosmarinic acid controls, and prepare a mixed control solution by adding methanol;

[0008] (2) Preparation of the test solution: accurately weigh 5mL of the postoperative ventilation decoction, add methanol to the mark line in a 10mL volumetric flask, filter through a 0.22μm microporous filter, and take the filtrate to obtain the test solution;

[0009] (3) Detection by high performance liquid chromatography to establish the fingerprint spectrum; in step (3), the conditions of the high performance liquid chromatography are as follows: RD-C18 chromatographic column (4.6mm x 250mm, 5μm); mobile phase: acetonitrile (A)-methanol (B)-0.2% phosphoric acid (C), gradient elution (0-15min, 9%-10% A, 1%-5% B; 15-40min, 10%-15% B, 5%-13% B; 40-50min, 15%-20% A, 13%-20% B; 50-55min, 20%-10% A, 20%-0% B; 55-60min, 10% A, 0% B); volume flow rate 1.0min / mL; column temperature 30℃; detection wavelength 300nm, injection volume 10μL.

[0010] The concentration of 5-HMF in the mixed control solution is 39.200μg·mL -1 , the concentration of chlorogenic acid is 3.258μg·mL -1 , the concentration of mustard thiocyanate is 95.020μg·mL -1 , the concentration of chicoric acid is 14.500μg·mL -1 , the concentration of naringin is 13.950μg·mL -1 , the concentration of 3,6'-di-glucorapeseed sucrose is 38.290μg·mL -1 , the concentration of hesperidin is 36.040μg·mL -1 , and the concentration of rosmarinic acid is 11.830μg·mL -1 .

[0011] The preparation method of the postoperative ventilation decoction is as follows: weigh 30 parts by weight of fried Semen Raphani, 10 parts by weight of fried Semen Sinapis, 10 parts by weight of fried Perilla Fruit, 9 parts by weight of fried Endothelium Corneum Gigeriae Galli, 30 parts by weight of Herba Taraxaci, 10 parts by weight of Focchi Fructus, 6 parts by weight of Pericarpium Citri Reticulatae, and 3 parts by weight of Glycyrrhizae Radix et Rhizoma, put them into a pottery stove, decoct twice, add 12 times the amount of water to soak for 30min for the first decoction, decoct for 30min, add 6 times the amount of water to decoct for 30min for the second decoction, filter, combine the filtrates, and add water to make up to 1600 parts by weight.

[0012] A fingerprint spectrum of a postoperative ventilation formula decoction, which is constructed by the above-mentioned method for establishing fingerprint spectra of traditional Chinese medicine.

[0013] The fingerprint spectrum of the postoperative ventilation formula decoction contains the following peaks: 5-HMF (peak 2), chlorogenic acid (peak 4), sinigrin thiocyanate (peak 6), chicoric acid (peak 14), rutin (peak 15), 3,6'-di-squalyl sucrose (peak 17), hesperidin (peak 18), and rosmarinic acid (peak 19).

[0014] A method for quality testing of postoperative ventilation formula decoction involves testing a sample of postoperative ventilation formula decoction according to the above-described construction method to obtain a high-performance liquid chromatography (HPLC) chromatogram of the sample, constructing a fingerprint chromatogram of the postoperative ventilation formula decoction according to the above-described construction method, and comparing the HPLC chromatogram of the sample of postoperative ventilation formula decoction with the fingerprint chromatogram of the postoperative ventilation formula decoction.

[0015] Beneficial effects:

[0016] (1) This invention uses different gradient elution to perform high performance liquid chromatography detection on postoperative ventilation decoction, and the separation effect is good;

[0017] (2) The method provided by the present invention, by reasonably selecting the reference component and using the timed wavelength method according to the maximum absorption wavelength of the component, can effectively represent the change in the amount of component between samples, thereby more accurately judging the quality of the sample;

[0018] (3) The method provided by this invention screens and optimizes the preparation of reference standards and test solutions for the efficacy and structural characteristics of the ingredients of the postoperative ventilation decoction. The method has good repeatability and stability, and can more comprehensively monitor the quality of the postoperative ventilation decoction. This method provides a valuable reference for the quality evaluation and standardization of traditional Chinese medicine decoctions.

[0019] (4) The traditional Chinese medicine compound preparations involved in this invention contain many Chinese herbs and have complex active ingredients. It is difficult to accurately evaluate the quality of traditional Chinese medicine preparations by only measuring the content of one or a few active ingredients. This invention constructs an HPLC fingerprint of the postoperative ventilation formula decoction, which can effectively perform qualitative analysis on the product quality of the postoperative ventilation formula decoction and provide a basis for comprehensively evaluating the quality standards of the postoperative ventilation formula decoction.

[0020] (5) In this invention, the mass fractions of sinigrin thiocyanate, chicoric acid, hesperidin, and rosmarinic acid, as well as the dry extract rate, are determined as evaluation indicators for the extraction process. The multi-index comprehensive scoring method is used to comprehensively score the results of single-factor experiments and orthogonal experiments, thereby optimizing the extraction process. Attached Figure Description

[0021] Figure 1 The HPLC fingerprints of 15 batches of samples show the following peaks: 5-HMF (peak 2), chlorogenic acid (peak 4), sinigrin thiocyanate (peak 6), chicoric acid (peak 14), rutin (peak 15), 3,6'-dieratosuccinate (peak 17), hesperidin (peak 18), and rosmarinic acid (peak 19).

[0022] Figure 2 The characteristic peak chromatograms of the postoperative ventilation decoction, mixed control, and each individual herb are as follows: peak 2 is 5-HMF, peak 4 is chlorogenic acid, peak 6 is sinigrin thiocyanate, peak 14 is chicoric acid, peak 15 is rutin, peak 17 is 3,6'-dieratosuccinate, peak 18 is hesperidin, and peak 19 is rosmarinic acid.

[0023] Figure 3 The characteristic peak chromatograms of the postoperative ventilation decoction, mixed control, and each negative decoction are as follows: peak 2 is 5-HMF, peak 4 is chlorogenic acid, peak 6 is sinigrin thiocyanate, peak 14 is chicoric acid, peak 15 is rutin, peak 17 is 3,6'-disqualyl sucrose, peak 18 is hesperidin, and peak 19 is rosmarinic acid.

[0024] Figure 4 Radar chart showing the comprehensive score of different influencing factors;

[0025] Figure 5 Principal component analysis plots for different influencing factors;

[0026] Figure 6 Partial least squares discriminant analysis score graphs for different influencing factors. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, but these equivalent forms also fall within the scope defined by the appended claims.

[0028] Example 1:

[0029] 1. Materials

[0030] 1.1 Instruments

[0031] Waters e2695 high-performance liquid chromatograph, equipped with a 2998PDA detector (Waters Corporation, USA); MS205DU 0.0001 g electronic balance (Mettler-Lidocaine Shanghai Co., Ltd.); KQ-300DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 98-I-BN electronic temperature-controlled heating mantle (2000 mL, Tianjin Test Instrument Co., Ltd.); electric ceramic stove; HH type constant temperature water bath (Jintan Zhongda Instrument Factory); SHZ-DⅢ type circulating water multi-purpose vacuum pump (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.).

[0032] 1.2 Reagents and Reference Standards

[0033] 5-Hydroxymethylfurfural (batch number AFBI2704, purity 98.0%), sinigrin thiocyanate (batch number AFBG1363, purity 98.0%), chicoric acid (batch number AFCF1203, purity 98.0%), rutin (batch number AZ22041908, purity 98.0%), and 3,6'-dieratosuccinate (batch number AF2202203, purity 98.0%) were all purchased from Chengdu Efa Biotechnology Co., Ltd.; chlorogenic acid (batch number 110753-202119, purity 96.3%), hesperidin (batch number 110721-202220, purity 97.2%), and rosmarinic acid (batch number 231222-240621, purity 98.1%) were all purchased from the China National Institutes for Food and Drug Control. Methanol, acetonitrile, and phosphoric acid were of chromatographic grade, all other reagents were of analytical grade, and water was ultrapure water.

[0034] 1.3 Medicinal slices

[0035] Fried radish seeds (batch number 240402, origin Jiangsu), fried perilla seeds (batch number 240402, origin Jiangsu), fried yellow mustard seeds (batch number 240402, origin Anhui), dandelion (batch number 24030105, origin Jiangsu), fried chicken gizzard lining (batch number A240404, origin Jiangsu), charred hawthorn (batch number 240301, origin Jiangsu), dried tangerine peel (batch number 24010803, origin Jiangsu), and licorice root (batch number 20240301-01, origin Inner Mongolia) were all purchased from Jiangsu Provincial Hospital of Traditional Chinese Medicine. A total of 15 batches of the postoperative ventilation formula clinical decoction were prepared, numbered S1-S15 (prepared using laboratory clinical decoction process, batch numbers 24060501, 24060502, 24060503, 24060504, 24060601, 24060602, 24060603, 24060604, 24060605, 24060701, 24060702, 24060703, 24060801, 24060802, 24060803). The decoction of each single herb and the negative preparation were all prepared in the laboratory according to the clinical decoction process.

[0036] 2. Methods and Results

[0037] 2.1 Chromatographic conditions

[0038] Zhongpu Technology RD-C 18 The chromatographic column was 4.6 mm × 250 mm, 5 μm; the mobile phase was acetonitrile (A)-methanol (B)-0.2% phosphoric acid (C), with gradient elution (0-15 min, 9%-10% A, 1%-5% B; 15-40 min, 10%-15% B, 5%-13% B; 40-50 min, 15%-20% A, 13%-20% B; 50-55 min, 20%-10% A, 20%-0% B; 55-60 min, 10% A, 0% B); the flow rate was 1.0 min / mL; the column temperature was 30℃; the detection wavelength was 300 nm; and the injection volume was 10 μL.

[0039] 2.2 Solution Preparation

[0040] 2.2.1 Preparation of Clinical Decoctions

[0041] In accordance with the regulations and requirements of the "Management Standards for Traditional Chinese Medicine Decoction Rooms in Medical Institutions," 30 parts by weight of stir-fried radish seeds, 10 parts by weight of stir-fried mustard seeds, 10 parts by weight of stir-fried perilla seeds, 9 parts by weight of stir-fried chicken gizzard lining, 30 parts by weight of dandelion, 10 parts by weight of charred hawthorn, 6 parts by weight of dried tangerine peel, and 3 parts by weight of licorice were weighed according to the daily prescription dosage. These were placed in a ceramic stove and decocted twice. For the first decoction, 12 times the amount of water was added for soaking for 30 minutes, followed by decocting for 30 minutes. For the second decoction, 6 times the amount of water was added for decocting for 30 minutes. The decoctions were filtered, and the filtrates were combined. Water was added to the filtrate to bring the volume to 1600 parts by weight, with each part by weight representing g. A total of 15 batches of samples were produced, numbered S1 to S15.

[0042] 2.2.2 Mixed reference solution

[0043] Accurately weigh appropriate amounts of each reference standard and add methanol to prepare solutions containing 39.200 μg·mL 5-HMF. -1 chlorogenic acid 3.258 μg·mL -1 Sinoprosine thiocyanate 95.020 μg·mL -1 Chicoric acid 14.500 μg·mL -1 Rutin 13.950 μg·mL -1 3,6'-Disorhoyloylsucrose 38.290 μg·mL -1 Hesperidin 36.040 μg·mL -1 Rosmarinic acid 11.830 μg·mL -1 The mixed solution is filtered through a 0.22μm microporous membrane to obtain the final product.

[0044] 2.2.3 Test solution

[0045] Accurately weigh 5 mL of the clinical decoction and 5 mL of each test sample into a 10 mL volumetric flask, add methanol to make up to the mark, filter through a 0.22 μm microporous membrane, and collect the filtrate.

[0046] 2.2.4 Preparation of single-herb medicinal slices and negative clinical decoctions

[0047] Prepare decoctions of single-herb medicinal slices (fried radish seed, fried yellow mustard seed, fried perilla seed, fried chicken gizzard lining, dandelion, charred hawthorn, tangerine peel, and licorice) according to the preparation process described in section "2.2.1". Weigh out the prescription amount of each of the following medicinal slices (excluding fried radish seed, fried yellow mustard seed, fried perilla seed, fried chicken gizzard lining, dandelion, charred hawthorn, tangerine peel, and licorice) according to the process specified in section "2.2.1" to prepare 8 negative solutions (excluding each of the following). Then prepare the solutions of each single-herb medicinal slice and the negative test sample according to section "2.2.3".

[0048] 2.3 Drying rate

[0049] Accurately pipette 15 mL of the extract into a pre-weighed evaporating dish, evaporate to dryness in a water bath, and dry in a 105℃ oven for 3 hours until constant weight. Cool in a desiccator for 30 minutes, accurately weigh, and calculate the dry extract yield using the formula: Dry extract yield = [Extract weight (g) × Total sample volume (mL)] / [Weight of medicinal slices (g) × Sample volume (mL)] × 100%

[0050] 2.4 Establishment of HPLC fingerprint and content determination

[0051] 2.4.1 Precision Test

[0052] Take an appropriate amount of this product (S1) sample and inject it under the chromatographic conditions of "2.1" for 6 determinations. The peak area RSDs of 5-HMF, chlorogenic acid, sinigrin thiocyanate, chicoric acid, rutin, 3,6'-dieratosuccinyl sucrose, hesperidin and rosmarinic acid were 1.01%, 1.12%, 1.46%, 1.65%, 2.82%, 1.62%, 1.55% and 1.50%, respectively, indicating that the instrument has good precision.

[0053] 2.4.2 Repeatability Test

[0054] Take an appropriate amount of this product (S1) and prepare 6 test solutions in parallel according to the method in "2.2.3". Inject and determine the peak areas under the spectral conditions in "2.1". The RSDs of the peak areas of 5-HMF, chlorogenic acid, sinigrin thiocyanate, chicoric acid, rutin, 3,6'-dieratosuccinyl sucrose, hesperidin and rosmarinic acid were 0.46%, 0.48%, 0.52%, 0.62%, 1.31%, 0.45%, 0.49% and 0.40%, respectively, indicating that the method has good repeatability.

[0055] 2.4.3 Stability Test

[0056] Take an appropriate amount of this product (S1) and inject it at room temperature for 0, 2, 4, 8, 12, and 24 hours under the chromatographic conditions specified in section “2.1”. The peak area RSDs of 5-HMF, chlorogenic acid, sinigrin thiocyanate, chicoric acid, rutin, 3,6'-dieratosuccinyl sucrose, hesperidin, and rosmarinic acid were 1.93%, 2.53%, 2.72%, 2.83%, 1.95%, 2.83%, 2.65%, and 2.87%, respectively, indicating that the solution has good stability within 24 hours.

[0057] 2.4.4 Examination of Linear Relationships

[0058] Accurately pipette 2.5, 5, 7.5, 10, 12.5, and 15 μL of the reference solution from section "2.2.2" and inject them into the liquid chromatograph. Determine the chromatographic conditions according to section "2.1". Plot a standard curve with peak area as the ordinate (Y) and injection volume (μg) as the abscissa (X). The results are shown in Table 1, indicating that each component exhibits good linearity within its respective range.

[0059] Table 1 Linear Relationships of Each Component

[0060]

[0061] 2.4.5 Recovery Test

[0062] Take 6 portions (5 mL each) of this product (S1) with known content of each component, accurately add an appropriate amount of reference solution, and prepare the test solution according to the method in section "2.2.3". Inject and determine the sample under the chromatographic conditions in section "2.1", and calculate the recovery rate. The average recoveries of 5-HMF, chlorogenic acid, sinigrin thiocyanate, chicoric acid, rutin, 3,6'-dieratosucrose, hesperidin, and rosmarinic acid were 93.17%, 95.81%, 105.31%, 93.20%, 105.14%, 98.42%, 100.08%, and 93.24%, respectively, with RSDs of 1.51%, 1.96%, 0.66%, 2.05%, 1.96%, 0.52%, 1.99%, and 2.36%, respectively.

[0063] 2.4.6 Atlas Generation

[0064] Fifteen batches of samples (S1-S15) were taken, and test solutions were prepared according to the method in section "2.2.3". The samples were then analyzed under the chromatographic conditions in section "2.1". The relevant data were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". Using S1 as a reference, the mean method was employed, with a time width set to 0.1 min. Multi-point correction and Mark peak matching were performed to generate HPLC fingerprint chromatograms and reference chromatograms (R). A total of 19 common peaks were identified. (See attached table). Figure 1 The similarity scores are shown in Table 2, and all are above 0.991, indicating a high degree of similarity between different batches. The established fingerprint spectrum can comprehensively reflect the overall composition.

[0065] Table 2 Similarity of 15 batches of samples

[0066]

[0067] 2.4.7 Identification and Attribution of Common Peaks

[0068] By comparing the retention time and UV absorption spectrum of the reference fingerprint spectrum with those of the reference standard, eight components were identified: peak 2 (5-HMF), peak 4 (chlorogenic acid), peak 6 (sinosine thiocyanate), peak 14 (chicoric acid), peak 15 (rutin), peak 17 (3,6'-disqualyl sucrose), peak 18 (hesperidin), and peak 19 (rosmarinic acid). Further comparison of the spectra of single-herb medicinal slices and corresponding negative samples with those of the mixed reference standard and samples revealed 19 common peaks. Figure 2 , 3 Among them, peak 2 (5-HMF) belongs to charred hawthorn, peaks 5, 8, 15 (rutin), and 18 (hesperidin) belong to tangerine peel, peaks 7, 9, and 14 (chicoric acid) belong to dandelion, peak 10 belongs to roasted mustard seed, peaks 12 and 17 (3,6'-di-squalyl sucrose) belong to roasted radish seed, peak 13 (glycyrrhizin) belongs to licorice, peaks 16 and 19 (rosmarinic acid) belong to roasted perilla seed, peak 1 is a common component of tangerine peel and charred hawthorn, peak 3 is a common component of dandelion and tangerine peel, peak 4 (chlorogenic acid) is a common component of dandelion and charred hawthorn, and peaks 6 (sinosine thiocyanate) and 11 are common components of roasted radish seed and roasted mustard seed.

[0069] 2.4.8 Principal Component Analysis

[0070] The areas of 19 characteristic peaks from 15 batches of postoperative ventilation samples were input into SPSS 26.0 for dimensionality reduction factor analysis. Principal component analysis (PCA) was selected to calculate the eigenvalues ​​and cumulative variance contribution rates of the principal components. The results are shown in Table 3. Among them, the eigenvalues ​​of 5 principal components were >1, and the cumulative variance contribution rate was 90.831%, which can comprehensively characterize the information of common peaks in the samples. The rotated common peak factor loading matrix is ​​shown in Table 4. The main influencing factors of the principal components are peaks 2 (5-HMF), 3, 4 (chlorogenic acid), 5, 6 (sinosin thiocyanate), 7, 8, 10, 12, 13, 16, 17 (3,6'-dieratosucrose), 18 (hesperidin), and 19 (rosmarinic acid).

[0071] Table 3. PCA Eigenvalues ​​and Variance Contribution Rate

[0072]

[0073] Table 4 Principal component factor loading matrix

[0074]

[0075] 2.4.9 Sample Content Determination

[0076] Take 15 batches of samples, prepare test solutions according to the method in section “2.2.3”, accurately pipette 10 μL of each solution, and determine the mass fraction under the chromatographic conditions in section “2.1”. At the same time, calculate the dry extract rate according to the provisions in section “2.3”. The results are shown in Table 5.

[0077] Table 5. Results of component content determination (n=15)

[0078]

[0079] 2.5 Optimization of Extraction Process

[0080] 2.5.1 Single-factor experiment

[0081] This study followed the regulations and requirements of the "Management Standards for Traditional Chinese Medicine Decoction Rooms in Medical Institutions" and determined soaking time, water volume, extraction time, and number of extractions as factors affecting the extraction process (see Table 6). The test solution was prepared according to the provisions of section "2.2.3" and measured under the chromatographic conditions of "2.1". Based on the theory of traditional Chinese medicine compatibility and the efficacy of each effective component, the weight coefficients of the indicators were analyzed using the analytic hierarchy process (AHP). Combining the principal component analysis results of the clinical decoction fingerprint and referring to the main components of the principal and assistant herbs in the 20th edition of the Chinese Pharmacopoeia, sinapicine thiocyanate, chicoric acid, hesperidin, and rosmarinic acid were selected as indicator components. Since the similarity among the samples was greater than 0.995, the differences were small. Finally, sinapicine thiocyanate, rosmarinic acid, chicoric acid, hesperidin, and the dry extract rate were determined as indicators. The relative importance of these five evaluation indicators was compared, and pairwise comparison matrices were constructed. Scoring was assigned according to the literature method. Based on the judgment results, the data was normalized. The weighting coefficients for sinigrin thiocyanate, rosmarinic acid, chicoric acid, hesperidin, and extract yield were calculated to be 0.194, 0.194, 0.093, 0.093, and 0.425, respectively. The consistency ratio factor CR = 0.002 < 0.1, indicating that the judgment matrix meets the consistency requirements and the weighting coefficients are effective. Based on this, the comprehensive score was calculated using the following formula:

[0082] Among them W j This represents the weight coefficient of the j-th indicator. It is the ratio of the j-th index (index component content, dry paste rate) of the i-th (n = 1, 2, 3, 4) test sample to the maximum value of the j-th index.

[0083] Table 6. Single-factor evaluation conditions of postoperative ventilation formula extraction process.

[0084]

[0085] The average mass fractions of sinigrin thiocyanate, chicoric acid, hesperidin, and rosmarinic acid, as well as the average dry extract rate, of 15 batches of clinical decoctions were used as the control (S). The combined scores of the control and the results of each test sample were placed on the same radar chart, as shown in the figure. Figure 4 Then, the components and dry matter percentage of the single-factor test samples were used as variables. Principal component analysis and partial least squares discriminant analysis were performed using SIMCA 14.1 software. (See below) Figure 5 , 6 .Depend on Figure 4 It can be seen that, compared with the clinical decoction group, the four factors all had different degrees of influence on the indicator components and the dry extract rate. Figure 5The results showed a large degree of dispersion among different extraction times, exceeding the 95% confidence interval. This indicates that the number of decoctions has a significant impact on the components and dry extract yield of each indicator, and the impact increases with the number of decoctions. However, extractions of 4 times increase production costs, so the extraction of 4 times is excluded. Figure 5 , 6 The data shows that the dispersion of components based on soaking time is relatively small, indicating that soaking time has a relatively small impact on each indicator component. This, combined with... Figure 4 The overall scores for soaking for 35 minutes and 50 minutes were quite similar, so 35 minutes was chosen to improve production efficiency. For the other two factors, the lower-scoring options were eliminated based on their overall scores.

[0086] 2.5.2 Orthogonal Experiment

[0087] Based on the results of the previous single-factor investigation, the following parameters were selected: water volume (8 times, 10 times, 12 times), decoction time (20 min, 40 min, 60 min), and number of decoctions (1, 2, 3). The parameters were then determined according to the three-factor, three-level L9 (3 3 An orthogonal experiment was conducted on the table, and the results were analyzed using a multi-index comprehensive scoring method to select the optimal extraction process. The results are shown in Tables 7 and 8.

[0088] Table 7 Experimental Design and Results

[0089]

[0090] Table 8 Results of Analysis of Variance

[0091]

[0092] Table 7 shows that the order of influence of each indicator on postoperative ventilation is C>B>A. Table 8's analysis of variance results show that the number of decoctions has a significant impact on the extraction process, consistent with the previous single-factor experiments. The comprehensive score range analysis shows that A3 is the optimal decoction time, but for sinigrin thiocyanate and chicoric acid, A2 is more suitable, and these two active ingredients have higher contents than other active ingredients. Extraction was carried out according to conditions A2B3C3. The third decoction and the total decoction from three decoctions were prepared as test solutions according to the conditions in section "2.2.3" and measured under the chromatographic conditions in "2.1". The results showed that the content of the third hydrolysate accounted for 12.388% (<15%) of the total decoction content. To improve production efficiency, A2B3C2 was ultimately determined as the optimal extraction condition.

[0093] 2.5.3 Verification of test results

[0094] Extraction was performed according to the determined process conditions, and three batches of test samples were obtained. The test solution was prepared according to the conditions under section “2.2.3” and measured under the chromatographic conditions under section “2.1”. The mass fraction of each index and the dry extract rate were calculated, as shown in Table 9.

[0095] Table 9. Experimental verification results (n=3)

[0096]

[0097] 3. Discussion and Conclusion

[0098] Traditional Chinese medicine (TCM) compound prescriptions are characterized by multiple components, multiple targets, and multiple levels. This indicates that a single indicator cannot comprehensively reflect the characteristics of a compound prescription. Multi-indicator comprehensive evaluation can integrate multiple important indicators, better grasp the intrinsic quality transfer laws in the transformation from ancient methods to modern extraction processes, and conduct a more scientific and rational evaluation of the decoction process, thereby optimizing the process. Through preliminary UPLC-Q-TOF-MS / MS full component analysis, literature review, and network pharmacology research, eight components in the postoperative ventilation formula were identified as being related to the inhibition of postoperative irritation index (POI). Their mechanism may involve promoting the recovery of gastrointestinal function by inhibiting intestinal inflammation. Therefore, these eight components were selected as the components for fingerprint analysis to comprehensively describe the composition of clinical decoction samples. Principal component analysis was used to select five major influencing components. Combined with the main components of the principal and assistant herbs in the 20th edition of the Chinese Pharmacopoeia, the following were finally identified: sinigrin thiocyanate, chicoric acid, hesperidin, and rosemary. The mass fraction of acid and the dry extract rate were used as evaluation indicators for the extraction process. A multi-index comprehensive scoring method was used to comprehensively score the results of single-factor experiments and orthogonal experiments. By measuring the components of traditional decoctions, the traditional decoctions and the extracted liquid were compared. Based on the comprehensive scoring results, this study determined the optimal extraction process as follows: Take the prescription amount of medicinal slices, add 12 times the amount of water and soak for 35 minutes, bring to a boil over high heat and maintain a simmer for 40 minutes, then filter; for the second decoction, add 6 times the amount of water, bring to a boil over high heat and maintain a simmer for 30 minutes, then filter and combine the filtrates.

[0099] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for establishing a traditional Chinese medicine fingerprint spectrum of a postoperative ventilation decoction, characterized in that, Includes the following steps: (1) Preparation of reference solution: Accurately weigh 5-HMF, chlorogenic acid, sinigrin thiocyanate, chicoric acid, rutin, 3,6'-disqualyl sucrose, hesperidin and rosmarinic acid reference standards, and add methanol to prepare a mixed reference solution; (2) Preparation of test solution: Accurately weigh 5 mL of postoperative ventilation decoction into a 10 mL volumetric flask, add methanol to make up to the mark, filter through a 0.22 μm microporous membrane, and take the filtrate to obtain the test solution; (3) Detection was performed using high performance liquid chromatography (HPLC) to establish a fingerprint spectrum; in step (3), the HPLC conditions were as follows: RD-C18 column from Zhongpu Technology, with specifications of 4.6 mm × 250 mm and 5 μm; mobile phase: acetonitrile A-methanol B-0.2% phosphoric acid C, gradient elution: 0-15 min, 9%-10% A, 1%-5% B; 15-40 min, 10%-15% A, 5%-13% B; 40-50 min, 15%-20% A, 13%-20% B; 50-55 min, 20%-10% A, 20%-0% B; 55-60 min, 10% A, 0% B; flow rate: 1.0 min / mL; column temperature: 30℃; detection wavelength: 300 nm; injection volume: 10 μL. The preparation method of the postoperative ventilation decoction is as follows: Weigh 30 parts by weight of stir-fried radish seeds, 10 parts by weight of stir-fried mustard seeds, 10 parts by weight of stir-fried perilla seeds, 9 parts by weight of stir-fried chicken gizzard lining, 30 parts by weight of dandelion, 10 parts by weight of charred hawthorn, 6 parts by weight of dried tangerine peel, and 3 parts by weight of licorice. Put them into a ceramic stove and decoct twice. For the first decoction, add 12 times the amount of water to soak for 30 minutes and decoct for 30 minutes. For the second decoction, add 6 times the amount of water and decoct for 30 minutes. Filter, combine the filtrates, and add water to make up to 1600 parts by weight. Each part by weight is g.

2. The method for establishing the herbal fingerprint spectrum of a postoperative ventilation decoction as described in claim 1, characterized in that, The concentration of 5-HMF in the mixed reference solution was 39.200 μg·mL. -1 The concentration of chlorogenic acid was 3.258 μg·mL. -1 The concentration of sinigrin thiocyanate was 95.020 μg·mL. -1 The concentration of chicoric acid was 14.500 μg·mL. -1 The concentration of rutin in naringin was 13.950 μg·mL. -1 The concentration of 3,6'-dierucyl sucrose was 38.290 μg·mL. -1 The concentration of hesperidin was 36.040 μg·mL. -1 The concentration of rosmarinic acid was 11.830 μg·mL. -1 .

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