Fingerprint spectrum of Linggui curcuma zedoary and liquorice decoction and quantitative analysis of multi-components by single marker

Abstract

The invention provides a detection method of a fingerprint spectrum of Linggui curcuma zedoary and liquorice decoction. The invention also provides a construction method and a quality detection method of the standard fingerprint spectrum of the Linggui Zhugan decoction. The invention also provides a detection method for quantitative analysis of multi-components by single marker of seven main active components in the Linggui Zhugan decoction. According to the fingerprint spectrum of the Linggui curcuma zedogan decoction and the method for quantitative analysis of multiple components by single marker, the high performance liquid fingerprint spectrum of the Linggui curcuma zedogan decoction can be established, the detection method for quantitative analysis of multiple components by single marker of seven main active components in the Linggui curcuma zedogan decoction is realized, a quality evaluation system of the Linggui curcuma zedogan decoction is further improved, and the quality of the Linggui curcuma zedogan decoction is improved. A basis is provided for subsequently proving the rationality of various medication forms in clinical wide application, and a basis is provided for evaluating the clinical medication quality.

Description

Technical Field

[0001] This invention belongs to the technical field of quality control and detection of traditional Chinese medicine components, and relates to a fingerprint spectrum of Linggui Zhugan Decoction and a method for multiple evaluation of its components. Specifically, it relates to a detection method for the fingerprint spectrum of Linggui Zhugan Decoction, a method for constructing a standard fingerprint spectrum of Linggui Zhugan Decoction, a quality detection method for the fingerprint spectrum of Linggui Zhugan Decoction, and a method for multiple evaluation of seven main active components in Linggui Zhugan Decoction. Background Technology

[0002] Linggui Zhugan Decoction is the 19th formula in the "List of Famous Ancient Prescriptions (First Batch)" compiled and published by the State Administration of Traditional Chinese Medicine and the State Drug Administration. It was first recorded in Zhang Zhongjing's *Synopsis of Prescriptions of the Golden Chamber* during the Han Dynasty. It has the effects of warming yang, resolving phlegm, strengthening the spleen, and promoting diuresis. Literature indicates that Linggui Zhugan Decoction has advantages in treating various systemic diseases, including cardiovascular diseases (chronic heart failure, arrhythmia, etc.), non-alcoholic fatty liver disease, metabolic diseases (dyslipidemia, insulin resistance, etc.), kidney disease, peripheral vertigo, and Alzheimer's disease. It is widely used clinically and exists in various forms, including decoctions prepared at home by patients, decoctions prepared in hospitals or at decoction centers, and prescription preparations and approved compound preparations.

[0003] Traditional Chinese medicine (TCM) compound formulas are highly complex systems, and their efficacy is the result of the synergistic effect of multiple components. Therefore, establishing quality control methods that can comprehensively reflect their intrinsic quality is crucial for ensuring the stability, safety, and reliability of their clinical efficacy. Currently, although there has been some progress in the quality control research of Linggui Zhugan Decoction, the following bottlenecks and challenges still exist: ① Regarding fingerprint chromatograms: Chinese patent 202211617634.2 shows that the chromatographic peaks with good separation, large peak areas, and identifiable by reference standards in the HPLC fingerprint chromatogram of Linggui Zhugan Decoction are glycyrrhizin, cinnamic acid, cinnamaldehyde, and ammonium glycyrrhizate. Luo Shan enriched the components in the Linggui Zhugan Decoction reference sample through freeze-drying, ultrasonication, macroporous resin purification, rotary evaporation, and reconstitution. Within 150 minutes, she identified 14 components, including cinnamaldehyde, neoglycyrrhizin, glycyrrhizin, apigenin, glycyrrhizin, glycyrrhizin, glycyrrhizin B, isoglycyrrhizin, kaempferol, eugenol, isoglycyrrhizin, glycyrrhizic acid, glycyrrhetinic acid, atractylodes lactone III, and atractylodes lactone II. Overall, the existing fingerprint chromatograms show that most components belong to licorice and cinnamon twig. ② Regarding content determination: Hu Qianfeng et al. established an HPLC method for determining five components—cinnamic acid, cinnamaldehyde, glycyrrhizic acid, atractylodes lactone III, and pachymic acid—in a decoction containing four herbs: Poria cocos, cinnamon twig, atractylodes macrocephala, and licorice, within 95 minutes. Yang Fei et al. used a 150mm short column to simultaneously determine the contents of six components—glycyrrhizin, glycyrrhizic acid ammonium, cinnamic acid, cinnamaldehyde, pachymic acid, and atractylodes lactone—within 50 minutes, ranging from approximately 79.063 to 140.576 μg / mL, 38.397 to 110.463 μg / mL, 8.469 to 13.463 μg / mL, 159.935 to 230.458 μg / mL, 21.038 to 42.957 μg / mL, and 23.437 to 45.433 μg / mL, respectively. However, pachymic acid is mainly distributed in the skin of Poria cocos, with extremely low content in the flesh, and it has poor water solubility. The skin of Poria cocos is a non-medicinal part and should be thoroughly cleaned. Therefore, the status of pachymic acid as a quality control indicator component of Linggui Zhugan Decoction is controversial. The presence of poria cocos acid in the above study of Linggui Zhugan Decoction may be due to incomplete peeling of Poria cocos. Current research on the quality control of Linggui Zhugan Decoction mainly focuses on the quantitative analysis of single or a few indicator components (such as glycyrrhizic acid, cinnamaldehyde, and atractylodes lactone) in the formula. However, this "point-based" control model cannot fully reflect the overall effect of the compound formula with multiple components and multiple targets, making it difficult to scientifically and comprehensively evaluate the overall quality of Linggui Zhugan Decoction. This may lead to unstable efficacy between different batches due to differences in component ratios. Furthermore, as Linggui Zhugan Decoction falls under the category of Class 3.1 new drugs, the research cycle is long and the sample testing volume is large. However, its content determination often uses the traditional external standard method, which is highly dependent on reference standards and has high testing costs, severely limiting the implementation and supervision of the formula's quality standards.

[0004] In recent years, the Quantitative Analysis of Multi-components by Single-marker (QAMS) method (also known as the substitution reference method), which uses only one reference standard to simultaneously determine the content of multiple components, has emerged. This method leverages the inherent functional and proportional relationships among the effective components of traditional Chinese medicine (TCM) to determine the content of a representative component (easily available, low-cost, and effective) in TCM. The content is then calculated based on the relative correction factor f. sx The content of other components to be tested in the traditional Chinese medicine can be calculated. This method can calculate the content of multiple other components with only one reference standard (internal reference), so it is replacing the traditional method of multiple reference standards and is being widely promoted in the content determination of chemical drugs, traditional Chinese medicine and their preparations.

[0005] In summary, this invention establishes a fingerprint spectrum for Linggui Zhugan Decoction in different dosage forms and a method for multi-dimensional evaluation. This multidimensional quality control analysis method closely reflects the actual situation of modern applications of Linggui Zhugan Decoction, and is of urgent need and great significance for improving the quality control level of Linggui Zhugan Decoction, reducing testing costs, and promoting the modernization and industrialization of this classic formula. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a fingerprint spectrum of Linggui Zhugan Decoction and a method for multi-evaluation of its components, to establish a fingerprint spectrum for evaluating the quality of Linggui Zhugan Decoction, and to realize a method for multi-evaluation of the seven main active components in Linggui Zhugan Decoction, thereby further improving the quality evaluation system of Linggui Zhugan Decoction and providing a basis for proving the rationality of its various forms of administration in clinical practice.

[0007] To achieve the above and other related objectives, the first aspect of this invention provides a method for detecting the fingerprint spectrum of Linggui Zhugan Decoction, comprising the following steps:

[0008] 1) Preparation of test solution: After adding the Linggui Zhugan Decoction sample to the solvent, sonicate, centrifuge, and take the supernatant to obtain the test solution;

[0009] 2) Preparation of reference solution: Add at least one of the following reference standards: protocatechuic acid, neoglycyrrhizin, apigenin, glycyrrhizin, glycyrrhizin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II to a solvent to dissolve and dilute to volume to obtain the reference solution.

[0010] 3) Determination: The test solution and the reference solution were determined by high performance liquid chromatography (HPLC) under the same chromatographic conditions to obtain the fingerprint spectra of the test solution and the reference solution. The fingerprint spectra of the test solution and the reference solution were compared to determine the main active ingredients in the fingerprint spectra of the test solution, thereby obtaining the fingerprint spectra of Linggui Zhugan Decoction.

[0011] The second aspect of this invention provides a method for constructing a standard fingerprint spectrum of Linggui Zhugan Decoction, comprising: detecting multiple batches of Linggui Zhugan Decoction samples using the aforementioned detection method for Linggui Zhugan Decoction fingerprint spectrum, obtaining fingerprint spectra of multiple batches of Linggui Zhugan Decoction samples to generate a common pattern control spectrum, using chromatographic peaks present in all spectra as common characteristic peaks, determining the relative retention time of the common characteristic peaks and the ratio of the area of ​​each common characteristic peak to the total peak area, and assigning and locating the index components in the Linggui Zhugan Decoction fingerprint spectrum according to the relative retention time, thereby establishing a standard fingerprint spectrum of Linggui Zhugan Decoction.

[0012] A third aspect of the present invention provides a quality detection method for the fingerprint spectrum of Linggui Zhugan Decoction, comprising: obtaining a fingerprint spectrum of Linggui Zhugan Decoction using the aforementioned detection method for the fingerprint spectrum of Linggui Zhugan Decoction, and comparing its similarity with a standard fingerprint spectrum of Linggui Zhugan Decoction obtained using the aforementioned construction method for the standard fingerprint spectrum of Linggui Zhugan Decoction.

[0013] The fourth aspect of this invention provides a method for detecting seven main active ingredients in Linggui Zhugan Decoction using a single method with multiple evaluations, comprising the following steps:

[0014] A) Preparation of the test solution: Same as step 1) of the detection method for the fingerprint spectrum of Linggui Zhugan Decoction;

[0015] B) Preparation of reference solution: Dissolve at least one of the following reference standards: glycyrrhizin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in a solvent and bring the volume to a final volume to obtain the reference solution.

[0016] C) External standard method: High performance liquid chromatography with the same chromatographic conditions as the detection method of Linggui Zhugan Decoction fingerprint spectrum was used to determine the test solution in step A) and the reference solution in step B), and the content of the seven main active ingredients in the test solution was calculated by external standard method.

[0017] D) Determination of relative correction factor: Using glycyrrhizin in the reference solution as an internal reference, the relative correction factor between glycyrrhizin and apigenin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the reference solution is calculated by the relative slope method or multi-point correction method. Then, based on the chromatographic peak areas of glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the test solution, the contents of glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the test solution are determined.

[0018] As described above, the fingerprint spectrum and component analysis method of Linggui Zhugan Decoction provided by this invention have the following beneficial effects:

[0019] (1) This invention uses the external standard method (ESM) (standard curve method, single-point calibration method) and the quality assessment method (QAMS) to determine multiple components in Linggui Zhugan Decoction. There is no significant difference in the content of the measured components. Principal component analysis (PCA) and cluster analysis were performed to compare the distribution attributes of the material basis of the four Linggui Zhugan Decoction dosage forms. The results showed that the material basis distribution attributes of the traditional pressure cooker decoction sample (CP-LGZG) were the most different from the reference sample, while the modern preparation (MP-LGZG) and the intelligent decoction sample (I-LGZG) had a high degree of reduction to the reference sample (TP-LGZG), which was consistent with the similarity evaluation results, thus verifying the feasibility and applicability of the QAMS method.

[0020] (2) The fingerprint spectrum detection method of the present invention is a highly specific method for detecting the fingerprint spectrum of Linggui Zhugan Decoction. The measured spectrum is stable and reliable, and applicable to various forms of Linggui Zhugan Decoction on the market, thus possessing universality. Moreover, the fingerprint spectrum similarity evaluation results show that there are certain differences in the composition and relative proportions of the material basis of Linggui Zhugan Decoction in different forms of medication. Using TP-LGZG as a reference for fingerprint spectrum similarity evaluation, the results show that the similarity between CP-LGZG and TP-LGZG is 0.873-0.895, indicating that process conditions such as high pressure and heating degree may have a significant impact on component dissolution and efficacy. Therefore, it is necessary to establish a multi-dimensional quality control system covering the entire process of medicinal materials, processed medicinal materials, intermediates, and preparations for classic prescriptions, which is conducive to fully presenting the overall chemical fingerprint of the reference sample of classic prescriptions.

[0021] (3) The QAMS quantitative results of this invention show that the content levels of licorice components such as apigenin, glycyrrhizin, and glycyrrhizic acid ammonium are basically consistent in samples with different dosage forms, indicating that licorice components have good water solubility and can be stably transferred in the decoction. However, the content of cinnamon phenylpropanoids and atractylodes terpenoids varies greatly. Cinnamaldehyde and its transformation product cinnamic acid, which are volatile oil components of cinnamon twig, and atractylodes lipid-soluble components such as atractylodes lactones II-III, play an important role in the quality of Linggui Zhugan Decoction. Both PCA and cluster analysis results showed that the content distribution of each indicator component differed significantly between CP-LGZG and TP-LGZG, while MP-LGZG and I-LGZG had relatively similar material properties to TP-LGZG, consistent with the fingerprint spectrum similarity evaluation results. This indicates that the selection of QAMS indicator components is representative and can basically reflect the overall material basis of Linggui Zhugan Decoction. It also verified that the degree of reduction of the reference sample by different forms of medication was MP-LGZG and I-LGZG > CP-LGZG, demonstrating that content determination is another key to the quality control of clinical medication forms of Linggui Zhugan Decoction.

[0022] (4) Based on the decoctions (reference samples) used in long-term clinical practice, modern preparations, and the decoction liquid obtained from two main decoction methods in the rapidly increasing demand for decoction services—traditional pressure cookers and intelligent decoction production lines—this invention establishes a fingerprint spectrum and a quality assessment and evaluation method (QAMS) for Linggui Zhugan Decoction. It evaluates the quality of four forms of Linggui Zhugan Decoction, thereby maximally restoring the material distribution of the reference sample, revealing that the intelligent decoction liquid has the highest similarity to the reference sample and the closest content level of each component. It can maximally restore the chemical properties of this classic formula and further improve the quality evaluation system of Linggui Zhugan Decoction. This provides a basis for proving the rationality of its multiple forms of use in clinical practice and provides a basis for its clinical drug quality evaluation.

[0023] (5) The QAMS method established in this invention eliminates the need to prepare reference solutions of various components for each test for the research and development of Class 3.1 new drugs such as Linggui Zhugan Decoction, thereby reducing the use of standard products, significantly saving research and development costs, improving research efficiency, and simplifying the quality standards of related compound prescriptions, thus forming a virtuous cycle for subsequent compound prescription production and supervision.

[0024] (6) This invention suggests that the clinical use of Linggui Zhugan Decoction should also be based on the benchmark sample. In principle, the distribution of the material basis and compatibility significance of classic prescriptions should be respected, so as to achieve the goal of using key information as the basis for quality control, so as to better inherit and develop classic prescriptions such as Linggui Zhugan Decoction in the process of upholding the correctness and innovation. Attached Figure Description

[0025] Figure 1 The image shown is a comparative fingerprint of Linggui Zhugan Decoction in this invention.

[0026] Figure 2 The fingerprint spectra of Linggui Zhugan Decoction prepared by different processes in this invention are shown. Among them, S1 to S3 are fingerprint spectra of TP-LGZG samples; S4 to S6 are fingerprint spectra of MP-LGZG samples; S7 to S9 are fingerprint spectra of CP-LGZG samples; and S10 to S12 are fingerprint spectra of I-LGZG samples.

[0027] Figure 3 The chart shows a bar graph comparing the content (mg / g, n=3) of seven main active ingredients in Linggui Zhugan Decoction prepared using different processes in this invention.

[0028] Figure 4A The figure shows the PCA score of Linggui Zhugan Decoction prepared by different processes in this invention through chemometric analysis. Among them, S1-3 are TP-LGZG samples; S4-6 are MP-LGZG samples; S7-9 are CP-LGZG samples; and S10-12 are I-LGZG samples.

[0029] Figure 4B The diagram shows the clustering relationship of Linggui Zhugan Decoction prepared by different processes in this invention through chemometric analysis. Among them, S1-3 are TP-LGZG samples; S4-6 are MP-LGZG samples; S7-9 are CP-LGZG samples; and S10-12 are I-LGZG samples. Detailed Implementation

[0030] The inventors of this application have developed a fingerprint spectrum and a method for multiple evaluations of components of Linggui Zhugan Decoction, which is described in detail below.

[0031] The first aspect of this invention provides a method for detecting the fingerprint spectrum of Linggui Zhugan Decoction, comprising the following steps:

[0032] 1) Preparation of test solution: After adding the Linggui Zhugan Decoction sample to the solvent, sonicate, centrifuge, and take the supernatant to obtain the test solution;

[0033] 2) Preparation of reference solution: Add at least one of the following reference standards: protocatechuic acid, neoglycyrrhizin, apigenin, glycyrrhizin, glycyrrhizin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II to a solvent to dissolve and dilute to volume to obtain the reference solution.

[0034] 3) Determination: The test solution and the reference solution were determined by high performance liquid chromatography (HPLC) under the same chromatographic conditions to obtain the fingerprint spectra of the test solution and the reference solution. The fingerprint spectra of the test solution and the reference solution were compared to determine the main active ingredients in the fingerprint spectra of the test solution, thereby obtaining the fingerprint spectra of Linggui Zhugan Decoction.

[0035] In step 1), the Linggui Zhugan Decoction sample is selected from at least one of the following: a reference sample (Traditional Process of Linggui Zhugan Decoction, TP-LGZG), a modern preparation sample (Modern Process of Linggui Zhugan Decoction, MP-LGZG), a traditional pressure cooker decoction sample (Traditional Pressure Cooking Process of Linggui Zhugan Decoction, CP-LGZG), or an intelligent decoction sample (Intelligent Process of Linggui Zhugan Decoction, I-LGZG).

[0036] In one embodiment, the preparation of the reference sample includes: soaking Poria cocos, cinnamon twig, Atractylodes macrocephala and licorice in water, boiling them over high heat in a decoction pot, then simmering them over low heat, filtering them, and obtaining the aqueous extract.

[0037] In a preferred embodiment, the mass ratio of Poria cocos, cinnamon twig, Atractylodes macrocephala, and licorice is 4:3:3:2; and the ingredients are prepared according to a one-day prescription dosage.

[0038] In a preferred embodiment, the ratio of the mass of Poria cocos added (g) to the volume of water added (mL) is 55-56:1200, preferably 55.20:1200.

[0039] In a preferred embodiment, the soaking time is 25-35 minutes, preferably 30 minutes.

[0040] In a preferred embodiment, the simmering time after boiling over high heat is 25-35 minutes, preferably 30 minutes.

[0041] In a preferred embodiment, the simmering time is 15-25 minutes, preferably 20 minutes.

[0042] In a preferred embodiment, the aqueous solution is concentrated to 550-650 mL after simmering, preferably 600 mL.

[0043] In a preferred embodiment, the filtration is a sieve filtration, wherein the mesh size of the sieve is no greater than 200 mesh, preferably 200 mesh.

[0044] In a preferred embodiment, the reference sample is freeze-dried and then stored under vacuum and light-protected conditions.

[0045] In a further preferred embodiment, after the freeze-drying process involves pre-freezing at -20°C to -80°C until complete solidification, the freeze-drying is carried out at a temperature of -40°C to -60°C and a vacuum degree of less than 12 Pa for 0.5 to 7 days.

[0046] In one embodiment, the preparation of the modern formulation sample includes the following steps:

[0047] A1) Soak Poria cocos, Cinnamomum cassia, Atractylodes macrocephala and Glycyrrhiza uralensis in water, reflux to extract, filter, and obtain the first filtrate, the first aromatic water and the first dregs;

[0048] A2) Add water to the first residue, reflux to extract, filter, and obtain the second filtrate and the second aromatic water;

[0049] A3) Combine the first and second filtrates, concentrate under reduced pressure, dry and pulverize to obtain the extract powder;

[0050] A4) Combine the first and second aromatic waters, add β-cyclodextrin for inclusion, dry and pulverize to obtain the inclusion complex;

[0051] A5) Mix the extract powder and inclusion complex thoroughly to obtain the modern formulation sample (MP-LGZG).

[0052] In a preferred embodiment, the modern formulation sample is an MP-LGZG sample with batch number Z201101 produced by Jiangsu Kangyuan Pharmaceutical Co., Ltd. It is obtained by scaling up the preparation process of the aforementioned modern formulation sample and is used for methodological investigation.

[0053] In step A1), the mass ratio of Poria cocos, cinnamon twig, Atractylodes macrocephala, and licorice is 4:3:3:2. The ingredients are prepared according to a one-day prescription dosage.

[0054] In step A1), the ratio of the mass of Poria cocos added (g) to the volume of water added (mL) is 1:23-25, preferably 1:24. This is equivalent to adding 8 times the amount of water.

[0055] In step A1), the soaking time is 25-35 minutes, preferably 30 minutes.

[0056] In step A1), the reflux extraction time is 50-70 min, preferably 60 min.

[0057] In step A1) or A2), the filtration is performed using a sieve or gauze. The mesh size of the sieve or gauze is not less than 200 mesh, preferably 200 mesh.

[0058] In step A1), the first filtrate and the first aromatic water are collected separately using a dual extraction method. The dual extraction method is a conventional extraction method that uses a conventional dual extraction apparatus and takes advantage of the different boiling points of the first filtrate and the first aromatic water to collect them separately.

[0059] In step A2), the ratio of the mass of Poria cocos added (g) to the volume of water added (mL) is 1:17-19, preferably 1:18. This is equivalent to adding 6 times the mass of the total medicinal slices in water.

[0060] In step A2), the reflux extraction time is 25-35 min, preferably 30 min.

[0061] In step A2), the second filtrate and the second aromatic water are collected separately using a dual extraction method. This dual extraction method is a conventional extraction method using a standard dual extraction apparatus, taking advantage of the different boiling points of the second filtrate and the second aromatic water for separate collection.

[0062] In step A3), the temperature for vacuum concentration is 55-65°C, preferably 60°C.

[0063] In step A4), the amount of β-cyclodextrin added is 18-50g per 1L of the volume of the first aromatic water and the second aromatic water, preferably 20g.

[0064] In step A4), the drying temperature is 55-65°C, preferably 60°C.

[0065] In one embodiment, the preparation of the traditional pressure cooker decoction sample includes: soaking Poria cocos, cinnamon twig, Atractylodes macrocephala and licorice in water, boiling them in a pressure cooker over high heat, decocting them, and obtaining the aqueous extract.

[0066] In a preferred embodiment, the mass ratio of Poria cocos, cinnamon twig, Atractylodes macrocephala, and licorice is 4:3:3:2. The mixture is prepared according to a 7-day prescription.

[0067] In a preferred embodiment, the ratio of the mass of Poria cocos added (g) to the volume of water added (mL) is 1:6-10, preferably 1:8.

[0068] In a preferred embodiment, the soaking time is 25-35 minutes, preferably 30 minutes.

[0069] In a preferred embodiment, the water is boiled over high heat and then simmered until the aqueous solution is concentrated to 1600-2000 mL, preferably 1800 mL.

[0070] In a preferred embodiment, the filtration is a sieve filtration, wherein the mesh size of the sieve is not less than 200 mesh, preferably 200 mesh.

[0071] In a preferred embodiment, the traditional pressure cooker sample is freeze-dried and then stored under vacuum and light-proof conditions.

[0072] In a further preferred embodiment, the traditional pressure cooker sample is packaged into bags and stored at 3-5°C, preferably 4°C, before being freeze-dried.

[0073] In a further preferred embodiment, after the freeze-drying process involves pre-freezing at -20°C to -80°C until complete solidification, the freeze-drying is carried out at a temperature of -40°C to -60°C and a vacuum degree of less than 12 Pa for 0.5 to 7 days.

[0074] In one embodiment, the preparation of the intelligent decoction sample includes: soaking Poria cocos, Cinnamomum cassia, Atractylodes macrocephala and Glycyrrhiza uralensis in water for the first time in an intelligent device, then boiling over high heat and simmering over low heat, then soaking in water for the second time, boiling over high heat and simmering over low heat, filtering, and obtaining an aqueous extract.

[0075] In a preferred embodiment, the mass ratio of Poria cocos, cinnamon twig, Atractylodes macrocephala, and licorice is 4:3:3:2. The mixture is prepared according to a 7-day prescription.

[0076] In a preferred embodiment, the intelligent device is an intelligent decoction system, such as the intelligent decoction system produced by Zhejiang Houda Company.

[0077] In a preferred embodiment, during the two soaking processes, the ratio of the mass of Poria cocos added (g) to the total volume of water added (mL) is 1:18-24, preferably 1:19.

[0078] The amount of water added in both soaking sessions was calculated automatically by the smart device.

[0079] In a preferred embodiment, the soaking time during the first soaking is 25-35 minutes, preferably 30 minutes.

[0080] In a preferred embodiment, after the first soaking in water, the boiling and simmering times are 20-40 minutes, preferably 30 minutes.

[0081] In a preferred embodiment, after the second soaking in water, the boiling and simmering times are 10-30 minutes, preferably 20 minutes.

[0082] In a preferred embodiment, the filtration is a sieve filtration, wherein the mesh size of the sieve is not less than 200 mesh, preferably 200 mesh.

[0083] In a preferred embodiment, the traditional pressure cooker sample is freeze-dried and then stored under vacuum and light-proof conditions.

[0084] In a further preferred embodiment, the traditional pressure cooker sample is packaged into bags and stored at 3-5°C, preferably 4°C, before being freeze-dried.

[0085] In a further preferred embodiment, after the freeze-drying process involves pre-freezing at -20°C to -80°C until complete solidification, the freeze-drying is carried out at a temperature of -40°C to -60°C and a vacuum degree of less than 12 Pa for 0.5 to 7 days.

[0086] In step 1), the solvent should be accurately weighed before being added.

[0087] In step 1) or 2), the solvent is a 65-75% aqueous ethanol solution, preferably a 70% aqueous ethanol solution.

[0088] In step 1), the ratio of the mass g of the Linggui Zhugan Decoction sample added to the volume mL of the solvent added is 1:20-30, preferably 1:25.

[0089] In step 1), the ultrasonic extraction time is 10-60 min, preferably 30 min.

[0090] In step 1), the ultrasonic extraction power is 100-800W, and the ultrasonic extraction frequency is 40-60kHz. In one embodiment, the ultrasonic extraction power is 500W, and the ultrasonic extraction frequency is 53kHz.

[0091] In step 1), the centrifugation speed is 10000-15000 rpm, preferably 12000 rpm.

[0092] In step 1), the centrifugation time is 5-30 minutes, preferably 10 minutes.

[0093] In step 2), the CAS number of protocatechuic acid is 99-50-3, the CAS number of neoglycyrrhizin is 5088-75-5, the CAS number of apigenin is 74639-14-8, the CAS number of glycyrrhizin is 551-15-5, the CAS number of glycyrrhizin is 578-86-9, the CAS number of cinnamic acid is 140-10-3, the CAS number of cinnamaldehyde is 14371-10-9, the CAS number of ammonium glycyrrhizate is 53956-04-0, the CAS number of atractylodes lactone III is 73030-71-4, and the CAS number of atractylodes lactone II is 73069-14-4.

[0094] In step 3), the fingerprint spectrum of the test solution is compared with the fingerprint spectrum of the reference solution. Based on the known characteristic peaks in the fingerprint spectrum of the reference solution, the corresponding characteristic peaks in the fingerprint spectrum of the test solution are identified by relative retention time, thereby assigning and locating the main active ingredient in the fingerprint spectrum of the test solution.

[0095] In step 3), the chromatographic column used in the high-performance liquid chromatography is a C18 column. 18 Chromatographic column.

[0096] In one embodiment, the chromatographic column is a Pntulips BP C. 18 Plus column (250mm × 4.6mm, 5μm) or Agilent Eclipse XDB-C 18 Chromatographic column (250 mm × 4.6 mm, 5 μm).

[0097] In step 3), the detector in the high performance liquid chromatography is a photodiode array detector (DAD) or an ultraviolet detector (UV).

[0098] In step 3), the column temperature in the high performance liquid chromatography is 25-35℃, preferably 30℃.

[0099] In step 3), the injection volume in the high performance liquid chromatography is 5-15 μL, preferably 10 μL.

[0100] In step 3), the flow rate in the high performance liquid chromatography method is 0.7-0.9 mL / min, preferably 0.8 mL / min.

[0101] In step 3), the detection wavelength in the high performance liquid chromatography method is 215-225nm, preferably 220nm.

[0102] In step 3), the mobile phase in the high-performance liquid chromatography (HPLC) method is a 0.05-0.2% phosphoric acid aqueous solution-acetonitrile; wherein phase A is a 0.05-0.2% phosphoric acid aqueous solution, preferably a 0.1% phosphoric acid aqueous solution; phase B is acetonitrile; the analysis time is 86 min; gradient elution is used. The 0.05-0.2% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.05-0.2%. The 0.1% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.1%.

[0103] In one embodiment, as shown in Table 1, the specific procedure for gradient elution is as follows:

[0104] From 0 to 62 minutes, the volume ratio of phase A to phase B was 94:6 to 58:42.

[0105] 62-65 min, the volume ratio of phase A: phase B is 58:42-42:58;

[0106] 65-75 min, the volume ratio of phase A to phase B is 42:58-0:100;

[0107] 75-81 min, the volume ratio of phase A to phase B is 0:100-94:6;

[0108] 81-86 min, the volume ratio of phase A to phase B is 94:6-94:6.

[0109] The second aspect of this invention provides a method for constructing a standard fingerprint spectrum of Linggui Zhugan Decoction, comprising: detecting multiple batches of Linggui Zhugan Decoction samples using the aforementioned detection method for Linggui Zhugan Decoction fingerprint spectrum, obtaining fingerprint spectra of multiple batches of Linggui Zhugan Decoction samples to generate a common pattern control spectrum, using chromatographic peaks present in all spectra as common characteristic peaks, determining the relative retention time of the common characteristic peaks and the ratio of the area of ​​each common characteristic peak to the total peak area, and assigning and locating the index components in the Linggui Zhugan Decoction fingerprint spectrum according to the relative retention time, thereby establishing a standard fingerprint spectrum of Linggui Zhugan Decoction.

[0110] In the above method, the number of batches of the Linggui Zhugan Decoction sample to be tested is 10 to 15, preferably 12.

[0111] In the above method, the common pattern comparison chromatogram is compared using the software version 2012.130723 of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" published by the National Pharmacopoeia Commission to compare the similarity of fingerprint chromatograms of multiple batches of Linggui Zhugan Decoction samples.

[0112] In one embodiment, the similarity of the fingerprint spectra of the multiple batches of Linggui Zhugan Decoction samples is >0.95.

[0113] In one embodiment, the fingerprint chromatograms of the multiple batches of Linggui Zhugan Decoction samples were matched with chromatographic peaks using a multi-point calibration method, with a time width set to 0.1 min.

[0114] In the above method, the resolution of each common characteristic peak in the standard fingerprint spectrum of Linggui Zhugan Decoction is greater than 1.2.

[0115] In the above method, the standard fingerprint spectrum of Linggui Zhugan Decoction is as follows: Figure 1As shown, the relative retention time is defined as the ratio of the retention time of each common characteristic peak to the retention time of the reference peak. There are 26 common characteristic peaks, with peak 8 as the reference peak (S-peak) and a retention time of 1.000. The relative retention times of the other 25 common characteristic peaks are as follows: Peak 1: 0.211-0.233, preferably 0.211; Peak 2: 0.217-0.265, preferably 0.241; Peak 3: 0.384-0.470, preferably 0.427; Peak 4: 0.465-0.569, preferably... The value of peak 5 is 0.517; peak 6 is 0.561–0.686, preferably 0.624; peak 7 is 0.854–1.044, preferably 0.949; peak 8 is 0.882–1.078, preferably 0.980; peak 9 is 1.085–1.327, preferably 1.206; peak 10 is 1.165–1.424, preferably 1.294; peak 11 is 1.214–1.483, preferably 1.349; peak 12 is 1.349–1.648, preferably 1.49. Peak 9; Peak 13 is 1.460–1.785, preferably 1.622; Peak 14 is 1.616–1.975, preferably 1.796; Peak 15 is 1.872–2.288, preferably 2.080; Peak 16 is 2.007–2.453, preferably 2.230; Peak 17 is 2.015–2.463, preferably 2.239; Peak 18 is 2.023–2.472, preferably 2.248; Peak 19 is 2.026–2.477, preferably 2.251. Peak 20 is 2.033–2.485, preferably 2.259; peak 21 is 2.044–2.498, preferably 2.271; peak 22 is 2.062–2.520, preferably 2.291; peak 23 is 2.123–2.595, preferably 2.359; peak 24 is 2.153–2.631, preferably 2.392; peak 25 is 2.188–2.674, preferably 2.431; peak 26 is 2.278–2.784, preferably 2.531.

[0116] In one embodiment, the standard fingerprint spectrum of Linggui Zhugan Decoction identifies 10 fingerprint peaks: peak 3 is protocatechuic acid, peak 6 is neoglycyrrhizin, peak 7 is apigenin, peak 8 is glycyrrhizin, peak 12 is glycyrrhizin, peak 13 is cinnamic acid, peak 14 is cinnamaldehyde, peak 15 is ammonium glycyrrhizate, peak 21 is atractylodes lactone III, and peak 24 is atractylodes lactone II.

[0117] A third aspect of the present invention provides a quality detection method for the fingerprint spectrum of Linggui Zhugan Decoction, comprising: obtaining a fingerprint spectrum of Linggui Zhugan Decoction using the aforementioned detection method for the fingerprint spectrum of Linggui Zhugan Decoction, and comparing its similarity with a standard fingerprint spectrum of Linggui Zhugan Decoction obtained using the aforementioned construction method for the standard fingerprint spectrum of Linggui Zhugan Decoction.

[0118] In the above method, the similarity comparison is performed using the software version 2012.130723 of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" published by the National Pharmacopoeia Commission. Preferably, the similarity is ≥0.95.

[0119] The fourth aspect of this invention provides a method for detecting seven main active ingredients in Linggui Zhugan Decoction using a single method with multiple evaluations, comprising the following steps:

[0120] A) Preparation of the test solution: Same as step 1) of the detection method for the fingerprint spectrum of Linggui Zhugan Decoction;

[0121] B) Preparation of reference solution: Glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II reference standards are dissolved in solvent and then diluted to volume to obtain the reference solution.

[0122] C) External standard method: High performance liquid chromatography with the same chromatographic conditions as the detection method of Linggui Zhugan Decoction fingerprint spectrum was used to determine the test solution in step A) and the reference solution in step B), and the content of glycyrrhizin in the test solution was calculated by external standard method.

[0123] D) Determination of relative correction factor: Using glycyrrhizin in the reference solution as an internal reference, the relative correction factor between glycyrrhizin and apigenin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the reference solution is calculated by the relative slope method or multi-point correction method. Then, based on the chromatographic peak area measured for at least one of apigenin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the test solution, the content of at least one of apigenin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the test solution is determined.

[0124] In step B), the solvent is a 60-80% aqueous ethanol solution, preferably a 70% aqueous ethanol solution.

[0125] In step B), the reference solution is prepared by directly adding a solvent to prepare the reference solution or by gradually diluting a reference stock solution.

[0126] In one embodiment, the reference stock solution is prepared by mixing apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizinate, atractylodes lactone III, and atractylodes lactone II, wherein the concentrations of apigenin, glycyrrhizin, and atractylodes lactone are 502.907 μg / mL, glycyrrhizin, cinnamic acid, cinnamaldehyde, and ammonium glycyrrhizinate are 1366.392 μg / mL, atractylodes lactone III and atractylodes lactone II are 23.898 μg / mL.

[0127] In one embodiment, the reference solution is prepared by mixing apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II, wherein the concentration of apigenin is 3.929-502.907 μg / mL, the concentration of glycyrrhizin is 5.481-701.624 μg / mL, the concentration of cinnamic acid is 4.627-592.313 μg / mL, the concentration of cinnamaldehyde is 1.585-202.836 μg / mL, the concentration of ammonium glycyrrhizate is 34.637-1366.392 μg / mL, the concentration of atractylodes lactone III is 0.821-105.073 μg / mL, and the concentration of atractylodes lactone II is 0.747-23.898 μg / mL.

[0128] In step C), the external standard method refers to: taking a series of different volumes of the reference solution from step B), preparing a series of solutions with different concentrations, and analyzing them using high-performance liquid chromatography (HPLC). The linear relationship between the glycyrrhizin content and peak area in the reference solution is obtained. A standard working curve is plotted, with the peak area of ​​glycyrrhizin corresponding to its content, and the regression equation of the standard working curve is calculated. Then, the test solution is detected using HPLC. The obtained peak area of ​​glycyrrhizin in the test solution is substituted into the regression equation of the standard working curve to obtain the glycyrrhizin content.

[0129] In one embodiment, the standard working curve uses peak area as the vertical axis and the content of the reference solution as the horizontal axis.

[0130] In step D), glycyrrhizin, which is readily available, stable, has a high content, and has a legal source, is used as a reference.

[0131] In step D), the relative slope method is selected from at least one of the linear slope method passing through the origin or the linear slope method not passing through the origin.

[0132] In step D), the relative slope method calculates the relative correction factor according to formula (1).

[0133] The formula (1) is: f sx =K s / K x ,

[0134] In the formula, f sx K is the relative correction factor. s K represents the slope of the standard curve for glycyrrhizin, used as an internal reference in the reference solution; x The slope of the standard curve of the reference solution is the slope of the standard curve of at least one selected from apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II.

[0135] In step D), the multi-point correction method calculates the relative correction factor according to formula (2).

[0136] The formula (2) is: f sx =(A s / C s ) / (A x / C x ),

[0137] In the formula, f sx A is the relative correction factor. x C represents the peak area of ​​at least one selected from apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the reference solution; x The content of at least one selected from the following in the reference solution: apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II; A s C represents the peak area of ​​glycyrrhizin used as an internal reference in the standard solution; s This refers to the content of glycyrrhizin, used as an internal reference, in the standard solution. The multi-point calibration method is also known as the concentration method.

[0138] In one embodiment, the relative correction factor between glycyrrhizin and apigenin is 1.4151-1.4334, preferably 1.4271; the relative correction factor between glycyrrhizin and cinnamic acid is 0.6968-0.7116, preferably 0.7066; the relative correction factor between glycyrrhizin and cinnamaldehyde is 0.5996-0.6106, preferably 0.6063; the relative correction factor between glycyrrhizin and ammonium glycyrrhizate is 21.7515-21.9278, preferably 21.8553; the relative correction factor between glycyrrhizin and atractylodes lactone III is 1.1715-1.1913, preferably 1.1840; and the relative correction factor between glycyrrhizin and atractylodes lactone II is 0.7442-0.7625, preferably 0.7536.

[0139] In step D), the content of at least one of the following in the test solution: apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II, is calculated according to formula (3).

[0140] The formula (3) is: C' x =A' x V s C s f sx / A s V' x ,

[0141] In the formula, C' x The content of at least one selected from the group consisting of apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the test solution; A' x A represents the peak area of ​​at least one selected from apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the test solution; s C represents the peak area of ​​glycyrrhizin used as an internal reference in the standard solution; s V represents the content of glycyrrhizin used as an internal reference in the reference solution; s V' is the injection volume of glycyrrhizin, used as an internal control, in the reference solution; x f is the injection volume of at least one of the following in the test solution: apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II; sx This is the relative correction factor.

[0142] All water used in this invention is purified water.

[0143] 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 protection of the present invention. The following specific examples illustrate the implementation of the present invention, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0144] The reagents and instruments used in the following examples are as follows:

[0145] 1. Reagents

[0146] Poria cocos (Schw.) Wolf, a fungus of the Polyporaceae family, is a dried sclerotium (batch number 231001, origin: Sichuan); Cinnamomum cassia Presl, a plant of the Lauraceae family, is a dried young branch (batch number 230901-02, origin: Guangxi); Atractylodes macrocephala, a plant of the Asteraceae family, is a dried rhizome (batch number 231201, origin: Sichuan); Glycyrrhiza uralensis, a plant of the Fabaceae family, is a dried root and rhizome (batch number 231301-06, origin: Gansu). All ingredients were purchased from Shanghai Jiuzhoutong Pharmaceutical Co., Ltd., and were identified as genuine products by Shanghai University of Traditional Chinese Medicine. Information on samples S1-S12 of Linggui Zhugan Decoction is shown in Table 1 below. The MP-LGZG sample with batch number Z201101 was provided by Jiangsu Kangyuan Pharmaceutical Co., Ltd. and was used only for methodological investigation.

[0147] Table 1

[0148]

[0149] Anhydrous ethanol (AR grade, Sinopharm Chemical Reagent Co., Ltd.); acetonitrile (HPLC grade, Merck KGaA, Germany); phosphoric acid (AR grade, Sinopharm Chemical Reagent Co., Ltd.); purified water (Watsons Group Co., Ltd.). Reference standards cinnamaldehyde (batch number 110710-202223, purity 98.80%), glycyrrhizin (batch number 111610-202209, purity 95.20%), and cinnamic acid (batch number 110786-202305, purity 98.80%) were all purchased from the National Institutes for Food and Drug Control. Apigenin glycyrrhizin (batch number 170108-202112, purity 98.32%), atractylodes lactone III (batch number 020025-202311, purity 99.69%), and protocatechuic acid reference standard (batch number 250087-2) were also used. 02107 (batch number 99.07%) was purchased from Shanghai Hongyong Biotechnology Co., Ltd.; ammonium glycyrrhizate (batch number M04GB140062, purity 98.50%) and atractylodes lactone II (batch number J03IB219057, purity 99.78%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; neoglycyrrhizin (batch number AFCJ2702, purity 98.00%) was purchased from Chengdu Efa Biotechnology Co., Ltd., and glycyrrhizin (batch number wkq18030505, purity 98.00%) was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.

[0150] 2. Instruments

[0151] AUD220W electronic analytical balance (Shimadzu Corporation, Japan), XS105 0.001 g / mL balance (Mettler AG, Switzerland), SK8210HP ultrasonic cleaner (Shanghai Keda Instrument Co., Ltd.), SHB-IIIA circulating water vacuum pump (Shanghai Huxi Industrial Co., Ltd.), 1260 Infinity II high-performance liquid chromatograph (Agilent Technologies, USA), BP C-18Plus column (250μm×4.6mm, 5μm, Shanghai Puning Analytical Technology Co., Ltd.)

[0152] Example 1

[0153] Prepare according to the prescription dosage for 1 day: Take 55.20g of Poria cocos, 41.40g of Cinnamomum cassia, 41.40g of Atractylodes macrocephala, and 27.60g of Glycyrrhiza uralensis, add 1200mL of water, soak for 30min, use a decoction pot, bring to a boil over high heat, decoct for 30min, then simmer over low heat for 20min to 600mL, filter through a 200-mesh sieve, obtain the water extract, record the volume, freeze-dry at -50℃ until completely solidified, freeze-dry at -50℃ and vacuum degree 12Pa for 1 day, store under vacuum and light-proof conditions, and obtain the reference (Traditional Process of Linggui Zhugan Decoction, TP-LGZG) sample.

[0154] Example 2

[0155] Prepare according to a one-day prescription: Take 55.20g of Poria cocos, 41.40g of Cinnamomum cassia, 41.40g of Atractylodes macrocephala, and 27.60g of Glycyrrhiza uralensis, add 8 times the amount of water, soak for 30 minutes, reflux extract for 1 hour, filter, and reserve the filtrate. Collect the aromatic water for later use. Add 6 times the amount of water to the residue, reflux extract for 0.5 hours, filter, and reserve the filtrate. Collect the aromatic water for later use. Combine the filtrates, concentrate under reduced pressure at 60℃, dry, and pulverize to obtain the extract powder. Combine the aromatic water, add 20g / L of β-cyclodextrin for inclusion complex, dry at 60℃, and pulverize to obtain the inclusion complex. Mix the extract powder with the inclusion complex to obtain the modern process of Linggui Zhugan Decoction (MP-LGZG) sample.

[0156] Example 3

[0157] Prepared according to a 7-day prescription: 386.40g of Poria cocos, 289.80g of Cinnamomum cassia, 289.80g of Atractylodes macrocephala, and 193.20g of Glycyrrhiza uralensis were combined and added to 3000mL of water. The mixture was soaked for 30 minutes, then boiled in a pressure cooker over high heat until reduced to 1800mL. The solution was filtered through a 200-mesh sieve to obtain the aqueous extract. The volume was recorded, and the extract was dispensed into individual bags and stored at 4℃ for later use. For freeze-drying, the product was pre-frozen at -50℃ until completely solidified, then freeze-dried at -50℃ and a vacuum of 12Pa for 1 day. It was then stored under vacuum and light-proof conditions to obtain the Traditional Pressure Cooking Process of Linggui Zhugan Decoction (CP-LGZG) sample.

[0158] Example 4

[0159] Prepared according to a 7-day prescription: 386.40g of Poria cocos, 289.80g of Cinnamomum cassia, 289.80g of Atractylodes macrocephala, and 193.20g of Glycyrrhiza uralensis were combined. The Zhejiang Houda Intelligent Decoction System was used. The system automatically added water twice based on an intelligent algorithm. The ratio of added Poria cocos (g) to total added water (mL) was 386.40:7450. After the first soaking for 30 minutes, the mixture was brought to a boil over high heat and then simmered over low heat for 30 minutes. The system then automatically added water again based on the intelligent algorithm, brought to a boil over high heat, and then simmered over low heat for 20 minutes. The extract was filtered through a 200-mesh sieve, the volume was recorded, and the extract was aliquoted and stored at 4℃ for later use. For freeze-drying, the extract was pre-frozen at -50℃ until completely solidified, then freeze-dried at -50℃ and a vacuum of 12Pa for 1 day. It was then stored under vacuum and light-proof conditions to obtain the Intelligent Process of Linggui Zhugan Decoction (I-LGZG) sample.

[0160] Example 5

[0161] The process was scaled up according to the method in Example 2 to obtain MP-LGZG sample 1# with batch number Z201101 provided by Jiangsu Kangyuan Pharmaceutical Co., Ltd.

[0162] Example 6

[0163] 1. Sample pretreatment

[0164] Accurately weigh 1 g of the reference sample prepared in Example 1, accurately add 25 mL of 70% ethanol aqueous solution, sonicate under running water (500 W power, 53 kHz frequency) for 30 min, centrifuge at 12000 rpm for 10 min, and collect the supernatant to obtain the test solution 1. # .

[0165] Accurately weigh the reference standards of protocatechuic acid, neoglycyrrhizin, apigenin, glycyrrhizin, glycyrrhizin, ginkgolide, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II, dissolve them in 70% ethanol aqueous solution, and make up to volume to prepare a mixed reference standard solution.

[0166] 2. Chromatographic conditions

[0167] The chromatographic conditions for high performance liquid chromatography are as follows: Column: BP C 18 Plus column (250 mm × 4.6 mm, 5 μm); detector: photodiode array detector (DAD); column temperature: 30 °C; injection volume: 10 μL; flow rate: 0.8 mL / min; detection wavelength: 220 nm; mobile phase: 0.1% phosphoric acid aqueous solution-acetonitrile, wherein phase A is 0.01% phosphoric acid aqueous solution and phase B is acetonitrile; analysis time: 86 min; gradient elution.

[0168] The specific procedure for gradient elution is as follows:

[0169] From 0 to 62 minutes, the volume ratio of phase A to phase B was 94:6 to 58:42.

[0170] 62-65 min, the volume ratio of phase A: phase B is 58:42-42:58;

[0171] 65-75 min, the volume ratio of phase A to phase B is 42:58-0:100;

[0172] 75-81 min, the volume ratio of phase A to phase B is 0:100-94:6;

[0173] 81-86 min, the volume ratio of phase A to phase B is 94:6-94:6.

[0174] 3. Measurement

[0175] High-performance liquid chromatography (HPLC) under the chromatographic conditions described in step 2 above was used to determine the test solution 1 in step 1 above. # 1. Reference solution, to obtain test solution 1 # The fingerprint chromatograms of the test solution and the reference solution were obtained. Among them, the test solution 1... # The fingerprint spectrum of the test solution should be compared with that of the reference solution. Based on the known characteristic peaks in the fingerprint spectrum of the reference solution, the relative retention time should be used to identify the test solution 1. # The corresponding characteristic peaks in the fingerprint spectrum, thereby identifying the test sample solution 1 # The index components in the fingerprint spectrum were assigned and located to obtain the fingerprint spectrum of Linggui Zhugan Decoction.

[0176] Example 7

[0177] 1. Sample pretreatment

[0178] Accurately weigh 1 g of the modern formulation sample prepared in Example 2, accurately add 23 mL of 68% ethanol aqueous solution, sonicate under running water (550 W power, 50 kHz frequency) for 28 min, centrifuge at 13000 rpm for 8 min, and collect the supernatant to obtain the test solution 2. # .

[0179] Accurately weigh the reference standards of protocatechuic acid, neoglycyrrhizin, apigenin, glycyrrhizin, glycyrrhizin, ginkgolide, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II, dissolve them in 68% ethanol aqueous solution, and make up to volume to prepare a mixed reference standard solution.

[0180] 2. Chromatographic conditions

[0181] The chromatographic conditions for high performance liquid chromatography are as follows: Column: BP C 18 Plus column (250 mm × 4.6 mm, 5 μm); detector: photodiode array detector (DAD); column temperature: 28 ℃; injection volume: 9 μL; flow rate: 0.6 mL / min; detection wavelength: 222 nm; mobile phase: 0.08% phosphoric acid aqueous solution-acetonitrile, wherein phase A is 0.08% phosphoric acid aqueous solution and phase B is acetonitrile; analysis time: 86 min; gradient elution.

[0182] The specific procedure for gradient elution is the same as step 2 in Example 6.

[0183] 3. Measurement

[0184] High-performance liquid chromatography (HPLC) under the chromatographic conditions described in step 2 above was used to determine the test solution 2 in step 1 above. # 1. Reference solution, to obtain test solution 2 # The fingerprint chromatograms of the test solution and the reference solution were obtained. Among them, the test solution 2... # The fingerprint spectrum of the test solution should be compared with that of the reference solution. Based on the known characteristic peaks in the fingerprint spectrum of the reference solution, the relative retention time should be used to identify the fingerprint of the test solution. # The corresponding characteristic peaks in the fingerprint spectrum, thereby identifying the test sample solution 2 # The index components in the fingerprint spectrum were assigned and located to obtain the fingerprint spectrum of Linggui Zhugan Decoction.

[0185] Example 8

[0186] 1. Sample pretreatment

[0187] Accurately weigh 1g of the traditional pressure cooker decoction sample prepared in Example 3, accurately add 25mL of 70% ethanol aqueous solution, sonicate under running water (500W power, 53kHz frequency) for 30min, centrifuge at 12000rpm for 10min, and collect the supernatant to obtain the test solution 3. # .

[0188] Accurately weigh the reference standards of protocatechuic acid, neoglycyrrhizin, apigenin, glycyrrhizin, glycyrrhizin, ginkgolide, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II, dissolve them in 70% ethanol aqueous solution, and make up to volume to prepare a mixed reference standard solution.

[0189] 2. Chromatographic conditions

[0190] The chromatographic conditions for high performance liquid chromatography are the same as in step 2 of Example 6.

[0191] 3. Measurement

[0192] High-performance liquid chromatography (HPLC) under the chromatographic conditions described in step 2 above was used to determine the test solution 3 in step 1 above. # 1. Reference solution, to obtain test solution 3 # The fingerprint chromatograms of the test solution and the reference solution were obtained. Among them, the test solution 3... # The fingerprint spectrum of the test solution should be compared with that of the reference solution. Based on the known characteristic peaks in the fingerprint spectrum of the reference solution, the relative retention time should be used to identify the fingerprint of the test solution. # The corresponding characteristic peaks in the fingerprint spectrum, thereby identifying the test sample solution 3 # The index components in the fingerprint spectrum were assigned and located to obtain the fingerprint spectrum of Linggui Zhugan Decoction.

[0193] Example 9

[0194] 1. Sample pretreatment

[0195] Accurately weigh 1g of the intelligent decoction sample prepared in Example 4, accurately add 25mL of 70% ethanol aqueous solution, sonicate under running water (500W power, 53kHz frequency) for 30min, centrifuge at 12000rpm for 10min, and collect the supernatant to obtain the test solution 4. # .

[0196] Accurately weigh the reference standards of protocatechuic acid, neoglycyrrhizin, apigenin, glycyrrhizin, glycyrrhizin, ginkgolide, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II, dissolve them in 70% ethanol aqueous solution, and make up to volume to prepare a mixed reference standard solution.

[0197] 2. Chromatographic conditions

[0198] The chromatographic conditions for high performance liquid chromatography are the same as in step 2 of Example 6.

[0199] 3. Measurement

[0200] High-performance liquid chromatography (HPLC) under the chromatographic conditions described in step 2 above was used to determine the test solution 4 in step 1 above. # 1. Reference solution, to obtain test solution 4 # The fingerprint chromatograms of the test solution and the reference solution were obtained. Among them, the test solution 4... # The fingerprint spectrum of the test solution should be compared with that of the reference solution. Based on the known characteristic peaks in the fingerprint spectrum of the reference solution, the relative retention time should be used to identify the test solution. # The corresponding characteristic peaks in the fingerprint spectrum, thereby identifying the test sample solution 4 # The index components in the fingerprint spectrum were assigned and located to obtain the fingerprint spectrum of Linggui Zhugan Decoction.

[0201] Example 10

[0202] The detection method for the Linggui Zhugan Decoction fingerprint spectrum established in Example 7 was used to detect three batches (S1-S12) of the reference sample prepared in Example 1, the modern preparation sample prepared in Example 2, the traditional pressure cooker decoction sample prepared in Example 3, and the intelligent decoction sample prepared in Example 4. The samples were then imported into the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine" (version 2012.130723) software published by the National Pharmacopoeia Commission to establish HPLC fingerprint spectra for 12 batches of Linggui Zhugan Decoction samples prepared using different processes. The fingerprint spectra of the test samples were compared with the control fingerprint spectra of Linggui Zhugan Decoction obtained under the same fingerprint spectrum detection conditions, and the similarity of the fingerprint spectra of each batch of Linggui Zhugan Decoction was calculated. The results are shown in […]. Figure 2 .

[0203] like Figure 2 As shown, the similarity between the fingerprint chromatograms of 12 batches of Linggui Zhugan Decoction samples and the control chromatograms were 0.976, 0.984, 0.983, 0.987, 0.992, 0.986, 0.966, 0.969, 0.951, 0.996, 0.991, and 0.987, respectively, indicating good similarity. Among the three batches compared for each process, the similarity was >0.98, with the intelligent decoction process showing a similarity >0.99, indicating stable batch quality across all processes and accurate and reliable testing methods. Compared to the reference sample, the similarity was as follows: intelligent decoction process, modern preparation, and traditional decoction process, with similarities >0.98, 0.95, and 0.87, respectively. This indicates that each sample reproduced the material basis of the reference sample to varying degrees, with the intelligent decoction process showing the highest reproduction rate.

[0204] In addition, the fingerprint chromatograms of the aforementioned 12 batches of Linggui Zhugan Decoction were compared with S1 as the reference chromatogram. Multi-point calibration was used to match chromatographic peaks, with a time width set to 0.1 min. A standard fingerprint chromatogram was established using the mean method, identifying 26 common peaks with good stability and peak shape. These 26 common peaks accounted for over 87% of the total peak area, and their resolution was greater than 1.2. Using peak 8 as the reference peak (S peak), the retention time of these 26 common characteristic peaks was 1.000. The relative retention times of the other 25 common characteristic peaks were as follows: peak 1: 0.211; peak 2: 0.241; peak 3: 0.427; peak 4: 0.517; peak 5: 0.624. Peak 6 was 0.949; Peak 7 was 0.980; Peak 9 was 1.206; Peak 10 was 1.294; Peak 11 was 1.349; Peak 12 was 1.499; Peak 13 was 1.622; Peak 14 was 1.796; Peak 15 was 2.080; Peak 16 was 2.230; Peak 17 was 2.239; Peak 18 was 2.248; Peak 19 was 2.251; Peak 20 was 2.259; Peak 21 was 2.271; Peak 22 was 2.291; Peak 23 was 2.359; Peak 24 was 2.392; Peak 25 was 2.431; Peak 26 was 2.531. See the detailed test results below. Figure 1 .

[0205] Example 11

[0206] According to the standard fingerprint spectrum of Linggui Zhugan Decoction obtained in Example 10, it was compared with the fingerprint spectrum of the reference solution. Based on the known characteristic peaks in the fingerprint spectrum of the reference solution, the corresponding characteristic peaks in the fingerprint spectrum of Linggui Zhugan Decoction were identified by relative retention time. Ten fingerprint peaks were located and determined: peak 3 is protocatechuic acid, peak 6 is neoglycyrrhizin, peak 7 is apigenin, peak 8 is glycyrrhizin, peak 12 is glycyrrhizin, peak 13 is cinnamic acid, peak 14 is cinnamaldehyde, peak 15 is ammonium glycyrrhizate, peak 21 is atractylodes lactone III, and peak 24 is atractylodes lactone II.

[0207] Example 12: Investigation of Fingerprint Mapping Methodology

[0208] 1. Precision

[0209] The same batch of Linggui Zhugan Decoction samples (MP-LGZG, Z201101) prepared in Example 5 were used to prepare test solutions according to the detection method of Linggui Zhugan Decoction fingerprint chromatogram in Example 6 above. The samples were injected 6 times consecutively, and the obtained chromatograms were entered into the software of "Similarity Evaluation System for Chromatographic Fingerprint Spectrum of Traditional Chinese Medicine" version 2012.130723 for similarity comparison. The similarity of fingerprint chromatograms of the 6 samples was all >0.99, indicating that the instrument precision of the method is good.

[0210] 2. Stability

[0211] The same batch of Linggui Zhugan Decoction samples (MP-LGZG, Z201101) prepared in Example 5 were used to prepare test solutions according to the detection method of Linggui Zhugan Decoction fingerprint chromatogram in Example 6. After being placed for 0, 3, 6, 9, 12 and 24 hours respectively, the chromatograms were entered into the software of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" version 2012.130723 for similarity comparison. The similarity of fingerprint chromatograms of the 6 samples was all >0.98, indicating that the test solution had good stability within 24 hours at room temperature.

[0212] 3. Repeatability

[0213] Six samples of Linggui Zhugan Decoction (MP-LGZG, Z201101) prepared in the same batch as in Example 5 were prepared in parallel according to the detection method of Linggui Zhugan Decoction fingerprint chromatogram in Example 6. The obtained chromatograms were entered into the software of "Similarity Evaluation System for Chromatographic Fingerprint Spectrum of Traditional Chinese Medicine" version 2012.130723 for similarity comparison. The similarity of fingerprint chromatograms of the six samples was all >0.90, indicating that the method has good repeatability.

[0214] Example 13

[0215] 1. Sample pretreatment

[0216] Preparation of test solution: The preparation process of test solution is the same as in step 1 of Example 6, to obtain test solution 1*.

[0217] Weigh appropriate amounts of apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II, accurately weigh them, dilute with 70% ethanol aqueous solution and make up to volume to prepare a reference solution.

[0218] The reference solution was prepared by serial dilution of the reference stock solution. The concentrations of glycyrrhizin, apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the reference stock solution were 502.907 μg / mL, 701.624 μg / mL, 592.313 μg / mL, 202.836 μg / mL, 1366.392 μg / mL, 105.073 μg / mL, and 23.898 μg / mL, respectively.

[0219] In the reference solution, the concentrations of glycyrrhizin were 3.929-502.907 μg / mL, glycyrrhizin was 5.481-701.624 μg / mL, cinnamic acid was 4.627-592.313 μg / mL, cinnamaldehyde was 1.585-202.836 μg / mL, ammonium glycyrrhizate was 34.637-1366.392 μg / mL, atractylodes lactone III was 0.821-105.073 μg / mL, and atractylodes lactone II was 0.747-23.898 μg / mL.

[0220] 2. Chromatographic conditions

[0221] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method described herein are the same as those for the HPLC method in step 2 of Example 6.

[0222] 3. Measurement

[0223] 3.1 Using the external standard method, a series of reference solutions of different volumes were transferred to prepare a series of solutions of different concentrations. These solutions were then analyzed using high-performance liquid chromatography (HPLC) to plot a standard curve for glycyrrhizin. The obtained test solution was then analyzed using HPLC, and the analytical results were substituted into the standard curve to obtain the glycyrrhizin content in the test solution.

[0224] Specifically, a series of reference solutions of different volumes were prepared to form a series of solutions of different concentrations. These solutions were then analyzed using high-performance liquid chromatography (HPLC) to obtain the linear relationship between the glycyrrhizin content and peak area in the reference solutions. Corresponding standard working curves were plotted, and the regression equations for these curves were calculated. The test solutions were then analyzed using HPLC. The peak areas of glycyrrhizin in the test solutions were substituted into the regression equations of the standard working curves to obtain the glycyrrhizin content.

[0225] 3.2 Using glycyrrhizin in the reference solution as an internal reference, the relative correction factor between glycyrrhizin and glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III and atractylodes lactone II in the reference solution was calculated by formula (1) using the relative slope method or by formula (2) using the multi-point correction method.

[0226] Then, based on the chromatographic peak areas of apigenin, cinnamic acid, cinnamaldehyde, glycyrrhizic acid, atractylodes lactone III, and atractylodes lactone II in the test solution, determine the contents of apigenin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the test solution according to formula (3).

[0227] Example 14

[0228] 1. Sample pretreatment

[0229] Preparation of test solution: The preparation process of test solution is the same as in step 1 of Example 7, to obtain test solution 2*.

[0230] Preparation of the reference solution: The preparation process is the same as that in step 1 of Example 13.

[0231] 2. Chromatographic conditions

[0232] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method described herein are the same as those for the HPLC method in step 2 of Example 7.

[0233] 3. Measurement

[0234] The measurement process is the same as step 3 in Example 13.

[0235] Example 15

[0236] Accurately measure the reference standard stock solution from step 1 of Example 13, dilute it with 70% ethanol aqueous solution to make a series of reference standard solutions with different mass concentrations, and inject and determine them according to the chromatographic conditions of step 2 of Example 13, recording the chromatograms. Plot a standard curve with the reference standard mass concentration as the abscissa (X) and the peak area as the ordinate (Y), and perform linear regression calculations to obtain the regression equation, correlation coefficient, and linear range. Specific results are shown in Table 2. Table 2 shows that glycyrrhizin exhibits good linearity within the specified range.

[0237] Table 2

[0238]

[0239] Example 16

[0240] The relative slope method and multi-point correction method were used, with glycyrrhizin as the internal standard reference, to calculate the correction factors of six components relative to glycyrrhizin: apigenin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II. The relative slope method involved using the linear slopes of the standard curves of each component (both passing through the origin and not passing through the origin) and the slopes of glycyrrhizin / the other six analytes, and calculating the correction factor for each component according to formula (1). The multi-point correction method, also known as the concentration method, involved substituting the concentrations of each component in the mixed reference solution and their corresponding peak areas into formula (2) to obtain the relative correction factor. Specific test results are shown in Table 3 below. Table 3 shows that the correction factors obtained by each method exhibited small fluctuations, with RSD < 3%, indicating reliable values.

[0241] Table 3

[0242]

[0243] Example 17

[0244] 1. Precision

[0245] The same batch of Linggui Zhugan Decoction sample (MP-LGZG, Z201101) prepared in Example 5 was used to prepare the test solution according to the method in step 1 of Example 13. Simultaneously, the reference solution was prepared according to the method in step 1 of Example 13. 10 μL of both the reference solution and the test solution were precisely pipetted and injected six times consecutively within one day. The peak areas of the seven components were calculated. The results showed that the RSD of the peak areas of each component in both the reference and test solutions was <3%, indicating good precision.

[0246] 2. Stability

[0247] The same batch of Linggui Zhugan Decoction sample (MP-LGZG, Z201101) prepared in Example 5 was used to prepare test solutions according to the method in step 1 of Example 13. After standing for 0, 3, 6, 9, 12, and 24 hours, the solutions were injected and analyzed under the chromatographic conditions in step 2 of Example 13, and the RSDs of the seven components were calculated. The results showed that the RSDs of each component were <3%, and the test solutions had good stability at room temperature for 24 hours.

[0248] 3. Repeatability

[0249] Six test solutions were prepared in parallel using the same batch of Linggui Zhugan Decoction sample (MP-LGZG, Z201101) prepared in Example 5, following the method in step 1 of Example 13. The solutions were then analyzed according to step 2 of Example 13, and the contents of the seven components were calculated. The results showed that the contents of apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II were 5.4157, 4.6868, 0.9401, 0.8747, 12.2759, 0.1281, and 0.0354 mg / g, respectively, with RSD < 3%, indicating good repeatability.

[0250] 4. Recovery rate

[0251] Take 0.5 g of the same batch of Linggui Zhugan Decoction sample (MP-LGZG, Z201101) prepared in Example 5, and add reference standards equivalent to 50%, 100%, and 150% of the contents of apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the test sample, respectively. Accurately add 25 mL of 70% ethanol, sonicate (500 W power, 53 kHz frequency) for 30 min, centrifuge at 4℃ and 12000 rpm for 10 min, take the supernatant, prepare 3 parallel aliquots, and inject and determine according to the chromatographic conditions in step 2 of Example 13 above. Calculate the average recovery rate, and the specific results are shown in Table 4. As can be seen from Table 4, the recovery rates of each component are all within the limits required by the pharmacopoeia, and the method has good accuracy.

[0252] Table 4

[0253]

[0254] 5. Durability

[0255] Three batches (S4-S6) of the Linggui Zhugan Decoction prepared in Example 2 were analyzed using three chromatographic columns (Pntulips BP C-18Plus, Agilent Eclipse XDB-C18, and Welch Ultimate XB-C18) with specifications of 250mm × 4.6mm and 5μm, respectively, following step 2 in Example 13. The results showed that the content measured by the Welch Ultimate XB-C18 differed significantly from that measured by the other columns, while the other two columns showed that the RSD of seven compounds was <10%, indicating good robustness.

[0256] Example 18

[0257] Twelve batches of Linggui Zhugan Decoction samples (MP-LGZG, Z201101) prepared in Example 5 were used. Test solutions were prepared according to step 1 of Example 13 above, and the contents of the seven components were determined according to step 2 of Example 13 above. The contents of the seven components were calculated using the mean relative correction factor and the external standard method (standard curve, single-point correction). No significant differences were found in the results. (See attached table). Figure 3 .

[0258] Example 19

[0259] 1. PCA Analysis: Three batches (S1-S12) of each of the following samples were prepared: the reference sample prepared in Example 1, the modern formulation sample prepared in Example 2, the traditional pressure cooker decoction sample prepared in Example 3, and the intelligent decoction sample prepared in Example 4. Test solutions were prepared according to the method described in step 1 of Example 13 above. The samples were then analyzed according to step 2 of Example 13 above. The raw data were imported into the "SIMCA 14.1" software for unsupervised PCA analysis. Principal component analysis was performed using the content of seven components in the 12 batches of samples as variables. The results are shown in [Figure 1]. Figure 4A .Depend on Figure 4A The score plot results show that the reference sample, modern preparation, and intelligent decoction agent are all distributed on the same side, while the traditional pressure cooker decoction agent sample is distributed on the other side. This indicates that both modern and intelligent decoction agents can better reproduce the material basis of the reference sample, while the quality of the traditional pressure cooker decoction agent sample differs significantly from that of the reference sample.

[0260] 2. Cluster Analysis: The component content determination results of 12 batches (S1-S12) of the following samples were imported into IBM SPSS Statistics R26.0.0.0 (64-bit): the reference sample prepared in Example 1, the modern formulation sample prepared in Example 2, the traditional pressure cooker decoction sample prepared in Example 3, and the intelligent decoction sample prepared in Example 4. Using the component content as the variable, a hierarchical cluster analysis was performed using the inter-group linkage-cosine method. The results are shown in [Figure 1]. Figure 4B .Depend on Figure 4B According to the cluster analysis results, when the inter-cluster distance is 15, the 12 batches of Linggui Zhugan Decoction samples can be clustered into two categories: S17 to S9 are in one category, and the remaining batches are in another category. The results show that, except for the traditional pressure cooker decoction process, all other processes can better restore the material basis distribution of the reference sample, which is consistent with the PCA analysis results.

[0261] In summary, the fingerprint spectrum and multi-component evaluation method of Linggui Zhugan Decoction provided by this invention can establish a high-performance liquid chromatography fingerprint spectrum of Linggui Zhugan Decoction and realize a multi-component evaluation method for the one-test of seven major active ingredients in Linggui Zhugan Decoction. This further improves the quality evaluation system of Linggui Zhugan Decoction and provides a basis for proving the rationality of its various forms of use in clinical practice, as well as for its clinical drug quality evaluation. Therefore, this invention overcomes the various shortcomings of the prior art and has high industrial application value.

[0262] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for detecting the fingerprint spectrum of Linggui Zhugan Decoction, comprising the following steps: 1) Preparation of test solution: After adding the Linggui Zhugan Decoction sample to the solvent, sonicate, centrifuge, and take the supernatant to obtain the test solution; 2) Preparation of reference solution: Add at least one of the following reference standards: protocatechuic acid, neoglycyrrhizin, apigenin, glycyrrhizin, glycyrrhizin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II to a solvent to dissolve and dilute to volume to obtain the reference solution. 3) Determination: The test solution and the reference solution were determined by high performance liquid chromatography under the same chromatographic conditions to obtain the fingerprint spectra of the test solution and the reference solution. The fingerprint spectra of the test solution and the reference solution were compared to determine the main active ingredients in the fingerprint spectra of the test solution, thereby obtaining the fingerprint spectra of Linggui Zhugan Decoction.

2. The method for detecting the fingerprint spectrum of Linggui Zhugan Decoction according to claim 1, characterized in that, Includes one or more of the following conditions: S1) In step 1), the Linggui Zhugan Decoction sample is selected from at least one of the following: a reference sample, a modern preparation sample, a traditional pressure cooker decoction sample, or an intelligent decoction sample. S2) In step 1) or 2), the solvent is a 65-75% aqueous ethanol solution; S3) In step 1), the ratio of the mass of the Linggui Zhugan Decoction sample added to the volume of the solvent added is 1:20-30. g / mL; S4) In step 1), the ultrasonic extraction time is 10-60 min; S5) In step 1), the power of the ultrasonic extraction is 100-800W, and the frequency of the ultrasonic extraction is 40-60kHz; S6) In step 1), the centrifugation speed is 10000-15000rpm. S7) In step 1), the centrifugation time is 5-30 min; S8) In step 3), the chromatographic column in the high-performance liquid chromatography is C18. 18 Chromatographic column; S9) In step 3), the detector in the high-performance liquid chromatography is a photodiode array detector or an ultraviolet detector; S10) In step 3), the column temperature in the high-performance liquid chromatography is 25-35℃; S11) In step 3), the injection volume in the high-performance liquid chromatography is 5-15 μL; S12) In step 3), the flow rate in the high-performance liquid chromatography is 0.7-0.9 mL / min; S13) In step 3), the detection wavelength in the high-performance liquid chromatography is 215-225 nm; S14) In step 3), the mobile phase in the high performance liquid chromatography is 0.05-0.2% phosphoric acid aqueous solution-acetonitrile; wherein, phase A is 0.05-0.2% phosphoric acid aqueous solution; phase B is acetonitrile; the analysis time is 86 min; gradient elution.

3. The method for detecting the fingerprint spectrum of Linggui Zhugan Decoction according to claim 2, characterized in that, Includes one or more of the following conditions: B1) In item S1), the preparation of the reference sample includes: soaking Poria cocos, cinnamon twig, Atractylodes macrocephala and licorice in water, boiling them over high heat in a decoction pot, then simmering them over low heat, filtering them, and obtaining the aqueous extract. B2) In item S1), the preparation of the modern formulation sample includes the following steps: A1) Soak Poria cocos, Cinnamomum cassia, Atractylodes macrocephala and Glycyrrhiza uralensis in water, reflux to extract, filter, and obtain the first filtrate, the first aromatic water and the first dregs; A2) Add water to the first residue, reflux to extract, filter, and obtain the second filtrate and the second aromatic water; A3) Combine the first and second filtrates, concentrate under reduced pressure, dry and pulverize to obtain the extract powder; A4) Combine the first and second aromatic waters, add β-cyclodextrin for inclusion, dry and pulverize to obtain the inclusion complex; A5) Mix the extract powder and inclusion complex thoroughly to obtain a modern formulation sample; B3) In item S1), the preparation of the traditional pressure cooker decoction sample includes: soaking Poria cocos, cinnamon twig, Atractylodes macrocephala and licorice in water, boiling them in a pressure cooker over high heat, filtering them, and obtaining the water extract. B4) In item S1), the preparation of the intelligent decoction sample includes: soaking Poria cocos, Cinnamomum cassia, Atractylodes macrocephala and Glycyrrhiza uralensis in water for the first time in an intelligent device, then boiling over high heat and simmering over low heat, then soaking in water for the second time, then boiling over high heat and simmering over low heat, filtering, and obtaining the water extract. B5) In item S8), the chromatographic column is a Pntulips BP C. 18 Plus column or Agilent Eclipse XDB-C 18 Chromatographic column; B6) In item S14), the specific procedure for gradient elution is as follows: From 0 to 62 minutes, the volume ratio of phase A to phase B was 94:6 to 58:

42. 62-65 min, the volume ratio of phase A: phase B is 58:42-42:58; 65-75 min, the volume ratio of phase A to phase B is 42:58-0:100; 75-81 min, the volume ratio of phase A to phase B is 0:100-94:6; 81-86 min, the volume ratio of phase A to phase B is 94:6-94:

6.

4. The method for detecting the fingerprint spectrum of Linggui Zhugan Decoction according to claim 3, characterized in that, Includes one or more of the following conditions: C1) In item B1), the mass ratio of Poria cocos, Cinnamomum cassia, Atractylodes macrocephala, and Glycyrrhiza uralensis is 4:3:3:2; C2) In item B1), the ratio of the mass of Poria cocos added to the volume of water added is 55-56:1200, g / mL; C3) In item B1), the soaking time is 25-35 min; C4) In item B1), the simmering time after boiling over high heat is 25-35 minutes; C5) In item B1), the simmering time is 15-25 minutes; C6) In item B1), the aqueous solution after simmering is concentrated to 550-650 mL; C7) In item B1), the filtration is filtration through a screen, and the mesh size of the screen is no greater than 200 mesh. C8) In item B1), the reference sample is freeze-dried and then stored under vacuum and light-protected conditions; C9) In item B2), in step A1), the mass ratio of Poria cocos, Cinnamomum cassia, Atractylodes macrocephala, and Glycyrrhiza uralensis is 4:3:3:2; C10) In item B2), in step A1), the mass ratio of Poria cocos added to the volume of water added is 1:23-25, g / mL; C11) In item B2), in step A1), the soaking time is 25-35 min; C12) In item B2), in step A1), the reflux extraction time is 50-70 min; C13) In item B2), in step A1) or A2), the filtration is filtration through a screen or gauze; C14) In item B2), in step A2), the ratio of the mass of Poria cocos added to the volume of water added is 1:17-19, g / mL; C15) In item B2), in step A2), the reflux extraction time is 25-35 min; C16) In item B2), in step A3), the temperature of the vacuum concentration is 55-65°C; In item C17) B2), in step A4), the amount of β-cyclodextrin added is 18-50g per 1L of the volume of the first and second aromatic waters. C18) In item B2), in step A4), the drying temperature is 55-65°C; In item B3), C19) the mass ratio of Poria cocos, Cinnamomum cassia, Atractylodes macrocephala, and Glycyrrhiza uralensis is 4:3:3:

2. In item B3), C20) the ratio of the mass of Poria cocos added to the volume of water added is 1:6-10, g / mL; C21) In item B3), the soaking time is 25-35 min; C22) In item B3), the water is boiled over high heat and then simmered until the aqueous solution is concentrated to 1600-2000 mL; C23) In item B3), the filtration is filtration through a screen, and the mesh size of the screen is not less than 200 mesh; C24) In item B3), the traditional pressure cooker decoction sample is freeze-dried and then stored under vacuum and light-proof conditions; C25) In item B4), the mass ratio of Poria cocos, Cinnamomum cassia, Atractylodes macrocephala, and Glycyrrhiza uralensis is 4:3:3:

2. C26) In item B4), during the two soakings, the ratio of the mass of Poria cocos added to the total volume of water added is 1:18-24, g / mL; C27) In item B4), during the first soaking in water, the soaking time is 25-35 minutes; C28) In item B4), after the first soaking in water, the boiling time should be 20-40 minutes. C29) In item B4), after the second soaking in water, the boiling time should be 10-30 minutes. C30) In item B4), the filtration is filtration through a screen, and the mesh size of the screen is not less than 200 mesh; In item C31) B4), the traditional pressure cooker frying sample is freeze-dried and then stored under vacuum and light-proof conditions.

5. A method for constructing a standard fingerprint spectrum for Linggui Zhugan Decoction, comprising: By using the detection method of the fingerprint spectrum of Linggui Zhugan Decoction according to any one of claims 1-4, multiple batches of Linggui Zhugan Decoction samples were detected to obtain fingerprint spectrum of multiple batches of Linggui Zhugan Decoction samples to generate a common pattern control spectrum. The chromatographic peaks that are present in all the spectrum are taken as common characteristic peaks. The relative retention time of the common characteristic peaks and the ratio of the area of ​​each common characteristic peak to the total peak area are determined. Based on the relative retention time, the index components in the fingerprint spectrum of Linggui Zhugan Decoction are assigned and located to establish a standard fingerprint spectrum of Linggui Zhugan Decoction.

6. The method for constructing the standard fingerprint spectrum of Linggui Zhugan Decoction according to claim 5, characterized in that, The standard fingerprint spectrum of Linggui Zhugan Decoction uses the ratio of the retention time of each common characteristic peak to the retention time of the reference peak as the relative retention time. It includes 26 common characteristic peaks, with peak 8 as the reference peak (S peak) and a retention time of 1.

000. The relative retention times of the other 25 common characteristic peaks are as follows: Peak 1: 0.211–0.233; Peak 2: 0.217–0.265; Peak 3: 0.384–0.470; Peak 4: 0.465–0.569; Peak 5: 0.561–0.686; Peak 6: 0.854–1.044; Peak 7: 0.882–1.078; Peak 9: 1.085–1.327; Peak 10: 1.165–1.424; Peak 11: 1.214–1.

483. Peak 12: 1.349–1.648; Peak 13: 1.460–1.785; Peak 14: 1.616–1.975; Peak 15: 1.872–2.288; Peak 16: 2.007–2.453; Peak 17: 2.015–2.463; Peak 18: 2.023–2.472; Peak 19: 2.026–2.477; Peak 20: 2.033–2.485; Peak 21: 2.044–2.498; Peak 22: 2.062–2.

520. Peak 23 ranges from 2.123 to 2.595; Peak 24 ranges from 2.153 to 2.631; Peak 25 ranges from 2.188 to 2.674; and Peak 26 ranges from 2.278 to 2.

784.

7. The method for constructing the standard fingerprint spectrum of Linggui Zhugan Decoction according to claim 5, characterized in that, In the standard fingerprint spectrum of Linggui Zhugan Decoction, 10 fingerprint peaks were located and identified: peak 3 is protocatechuic acid, peak 6 is neoglycyrrhizin, peak 7 is apigenin, peak 8 is glycyrrhizin, peak 12 is glycyrrhizin, peak 13 is cinnamic acid, peak 14 is cinnamaldehyde, peak 15 is ammonium glycyrrhizate, peak 21 is atractylodes lactone III, and peak 24 is atractylodes lactone II.

8. A quality detection method for the fingerprint spectrum of Linggui Zhugan Decoction, comprising: The fingerprint spectrum of Linggui Zhugan Decoction is obtained by using the detection method of Linggui Zhugan Decoction according to any one of claims 1-4, and the similarity is compared with the standard fingerprint spectrum of Linggui Zhugan Decoction obtained by using the construction method of standard fingerprint spectrum of Linggui Zhugan Decoction according to any one of claims 5-7.

9. A method for detecting seven main active ingredients in Linggui Zhugan Decoction using a single-method-multiple-evaluation approach, comprising the following steps: A) Preparation of the test solution: Same as step 1) of the detection method of Linggui Zhugan Decoction fingerprint spectrum according to any one of claims 1-4; B) Preparation of reference solution: Glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II reference standards are dissolved in solvent and then diluted to volume to obtain the reference solution. C) External standard method: High performance liquid chromatography with the same chromatographic conditions as the detection method of Linggui Zhugan Decoction fingerprint spectrum according to any one of claims 1-4 was used to determine the test solution in step A) and the reference solution in step B), and the content of glycyrrhizin in the test solution was calculated by external standard method. D) Determination of relative correction factor: Using glycyrrhizin in the reference solution as an internal reference, the relative correction factor between glycyrrhizin and apigenin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the reference solution is calculated by the relative slope method or multi-point correction method. Then, based on the chromatographic peak area measured for at least one of apigenin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the test solution, the content of at least one of apigenin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the test solution is determined.

10. The method for detecting seven main active ingredients in Linggui Zhugan Decoction according to claim 9, characterized in that, Includes one or more of the following conditions: D1) In step B), the solvent is a 60-80% aqueous ethanol solution; D2) In step D), the relative slope method calculates the relative correction factor according to formula (1). The formula (1) is: f sx =K s / K x , In the formula, f sx K is the relative correction factor. s The slope of the standard curve for glycyrrhizin, which is used as an internal reference in the reference solution; K x The slope of the standard curve of the reference solution containing at least one of the following: glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II. D3) In step D), the multi-point correction method calculates the relative correction factor according to formula (2). The formula (2) is: f sx =(A s / C s ) / (A x / C x ), In the formula, f sx A is the relative correction factor. x C represents the peak area of ​​at least one selected from apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the reference solution; x The content of at least one selected from the following in the reference solution: apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II; A s C represents the peak area of ​​glycyrrhizin used as an internal reference in the standard solution; s This refers to the content of glycyrrhizin, which serves as an internal reference, in the standard solution. D4) In step D), the relative correction factor for glycyrrhizin and apigenin is 1.4151-1.4334; the relative correction factor for glycyrrhizin and cinnamic acid is 0.6968-0.7116; the relative correction factor for glycyrrhizin and cinnamaldehyde is 0.5996-0.6106; the relative correction factor for glycyrrhizin and ammonium glycyrrhizate is 21.7515-21.9278; the relative correction factor for glycyrrhizin and atractylodes lactone III is 1.1715-1.1913; and the relative correction factor for glycyrrhizin and atractylodes lactone II is 0.7442-0.7625. D5) In step D), the content of at least one of the following in the test solution: apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II is calculated according to formula (3). The formula (3) is: C' x =A' x V s C s f sx / A s V' x , In the formula, C' x The content of at least one selected from the group consisting of apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the test solution; A' x A represents the peak area of ​​at least one selected from apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II in the test solution; s C represents the peak area of ​​glycyrrhizin used as an internal reference in the standard solution; s V represents the content of glycyrrhizin used as an internal reference in the reference solution; s V' is the injection volume of glycyrrhizin, used as an internal control, in the reference solution; x f is the injection volume of at least one of the following in the test solution: apigenin, glycyrrhizin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, atractylodes lactone III, and atractylodes lactone II; sx This is the relative correction factor.

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