Method for establishing traditional Chinese medicine fingerprint of gastrodia and uncaria decoction

Abstract

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

Description

TECHNICAL FIELD

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

[0002] The Decoction of Gastrodia and Uncaria is a clinical prescription of Hu Guangci, a famous doctor in Sichuan and Chongqing in recent times, and is first recorded in the New Meaning of TCM Internal Medicine Miscellaneous Disease Treatment. The Decoction of Gastrodia and Uncaria is composed of 11 TCMs, including Gastrodia, Uncaria, Concha Murex, Cyathula, Fructus Gardeniae, Radix Scutellariae, Eucommia ulmoides, Motherwort, Taxillus, Marsdenia tenacissima and Zhu Fushen, and has the effects of calming liver and extinguishing wind, clearing heat and activating blood, and tonifying liver and kidney. The Decoction of Gastrodia and Uncaria is widely used in clinical practice, and is mainly used for treating cardiovascular and cerebrovascular diseases such as hypertension, cerebral infarction, Parkinson's disease and epilepsy. In the Expert Consensus on TCM Diagnosis and Treatment of Hypertension published by the Cardiovascular Disease Branch of the Chinese Association of Traditional Chinese Medicine in 2019, the Decoction of Gastrodia and Uncaria is recommended for the treatment of hypertension of liver-yang hyperactivity type. In addition, research shows that the related research on the Decoction of Gastrodia and Uncaria has gradually increased in recent years, and it is increasingly valued by many researchers.

[0003] The TCM fingerprint comprehensively reflects the types and quantities of chemical components contained in TCMs and their preparations, and then makes overall description and evaluation of the quality of the drugs. The HPLC fingerprint can effectively control the quality of TCMs, and even if the chromatographic peaks are unknown compounds, the HPLC fingerprint can still provide sufficient and reliable information, and can be used for identifying the quality of TCMs.

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

[0005] In view of the technical blank and other problems in the prior art, the purpose of the present application is to provide a method for establishing a TCM fingerprint of the Decoction of Gastrodia and Uncaria and a quality detection method of the Decoction of Gastrodia and Uncaria, so as to solve the problem that the prior art cannot be quantitatively determined and cannot be used as an evaluation standard for the overall quality of the Decoction of Gastrodia and Uncaria.

[0006] To achieve the purpose, the technical scheme adopted by the present application is to provide a method for establishing a TCM fingerprint of the Decoction of Gastrodia and Uncaria, comprising the following steps:

[0007] (1) Preparation of reference solution: accurately weigh Gastrodin, Geniposide, Pinoresinol diglucoside, 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside, Baicalin and Hanbaikalide reference substances, and add methanol to prepare a mixed reference solution;

[0008] (2) Preparation of test solution: 1 mL of Tianma Gouteng Yin decoction was accurately measured and placed in a 2 mL volumetric flask, methanol was added to constant volume, and after mixing, ultrasonic extraction was performed for 1 min, followed by centrifugation at 14 000 r / min for 10 min, the supernatant was taken, and a 0.22 μm microporous filter membrane was used, to obtain the test solution;

[0009] (3) Detection by high performance liquid chromatography to establish the fingerprint spectrum; in step (3), the conditions of the high performance liquid chromatography are as follows: Waters ACQUITY UPLC HSS T3 column, specifications: 2.1 mm x 100 mm, 1.8 μm, flow rate 0.2 mL / min; column temperature: 35 ℃, sample size 2 μL. Mobile phase: acetonitrile (A) -0.1% formic acid aqueous solution (B) for gradient elution, gradient elution conditions: 0-4 min, 2% A→5% A; 4-5 min, 5% A→10% A; 5-7 min, 10% A→12% A; 7-21 min, 12% A→18% A; 21-22 min, 18% A→27% A; 22-30 min, 27% A→34% A; 30-33 min, 34% A→88% A; 33-34 min, 88% A→95% A; 34-38 min, 95% A→95% A; 38-39 min, 95% A→2% A; 39-40 min, 2% A→2% A. The detector is DAD, and variable wavelength determination is used: 0-19 min: 192 nm; 19-24 min: 220 nm; 24-32 min: 256 nm; 32-40 min: 276 nm.

[0010] In the method for establishing the traditional Chinese medicine fingerprint spectrum of the Tianma Gouteng Yin decoction, the concentrations of gastrodin, gardenoside, pinoresinol diglucoside, 2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin, and wogonoside in the mixed reference solution are 0.075 9, 0.920 0, 0.054 1, 0.139 6, 2.125 5, and 0.955 5 mg / mL, respectively.

[0011] The method for establishing the herbal fingerprint spectrum of the Tianma Gouteng Decoction is as follows: The preparation method of the Tianma Gouteng Decoction is as follows: Weigh 9g each of Tianma, Zhizi, Huangqin, Duzhong, Yimucao, Sangjisheng, Shouwuteng, and Fushen, 12g each of Gouteng and Chuanniuxi, and 18g of Shijue Ming. Add water and decoct twice. Shijue Ming is decocted for 40 minutes first, and Gouteng is added 15 minutes before the end of the second decoction. The first decoction is soaked for 40 minutes, and the decoction time is 30 minutes after boiling, with 10 times the amount of water added. The second decoction is decocted with 4 times the amount of water, and the decoction time is 20 minutes after boiling. Combine the two decoction liquids and make up to equal volume.

[0012] A fingerprint spectrum of Gastrodia elata and Uncaria rhynchophylla decoction, which was constructed by the above-mentioned method.

[0013] The fingerprint spectrum of the Gastrodia and Uncaria Decoction contains six common peak components: 1. Gastrodin; 6. Gardenoside; 8. Pinoresinol diglucoside; 9. 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside; 11. Baicalin; and 14. Baicalin.

[0014] A method for quality testing of Gastrodia elata and Uncaria rhynchophylla decoction involves testing a sample of the decoction according to the aforementioned construction method to obtain a high-performance liquid chromatography (HPLC) chromatogram of the sample, constructing a fingerprint chromatogram of the decoction according to the aforementioned construction method, and comparing the HPLC chromatogram of the sample with the fingerprint chromatogram.

[0015] Beneficial effects:

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

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

[0018] (3) The method provided by the present invention has screened and optimized the preparation of reference standards and test solutions for the efficacy and structural characteristics of the components of Tianma Gouteng Decoction. The method has good repeatability and stability, and can more comprehensively monitor the quality of Tianma Gouteng Decoction. This method provides a valuable reference for the quality evaluation and standardization of traditional Chinese medicine decoctions.

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

[0020] (5) In this invention, the mass fraction and dry extract yield of gastrodin, geniposide, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin and wogonin were determined as evaluation indicators for the extraction process. The Box-Behnken design-response surface methodology experimental results were comprehensively scored using a multi-index comprehensive scoring method, thereby optimizing the extraction process. Attached Figure Description

[0021] Figure 1 The fingerprint chromatograms of 17 test samples show that peak 1 is gastrodin; peak 6 is geniposide; peak 8 is pinoresinol diglucoside; peak 9 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside; peak 11 is baicalin; and peak 14 is wogonin.

[0022] Figure 2 The graphs are for multivariate statistical analysis, where A is the PCA score graph and B is the OPLS-DA score graph.

[0023] Figure 3 Multivariate statistical analysis charts, where C is a cluster analysis chart and D is a VIP value ranking chart in OPLS-DA;

[0024] Figure 4 The images show the UPLC chromatograms of Gastrodia elata and Uncaria rhynchophylla decoction, where A is the mixed reference standard chromatogram and B is the test sample chromatogram (S1).

[0025] Figure 5 The impact of each factor on the overall score. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, but these equivalent forms also fall within the scope defined by the appended claims.

[0027] Example 1:

[0028] 1. Instruments and medicines

[0029] 1.1 Instruments

[0030] Agilent 1290 Infinity II ultra-high performance liquid chromatograph (Agilent Technologies), Eppendorf Centrifuge 5430R refrigerated centrifuge (Eppendorf GmbH, Germany), AR2140 0.0001 g balance (Shanghai Ohaus Instrument Co., Ltd.), 3260 mL ceramic medicine container (Kangshu), KQ-1000E medical ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0031] 1.2 Reagents and Reference Standards

[0032] Acetonitrile and methanol, both chromatographic grade, were purchased from MERCK GmbH, Germany. Formic acid, chromatographic grade, was purchased from Teco Strontium (Guangzhou) Technology Service Co., Ltd. Reference standards: gastrodin (batch number AFCB2851), geniposide (batch number AFCB1356), pinoresinol diglucoside (batch number AZCC1012), 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (batch number AFCI2156), baicalin (batch number AFCK2351), all purchased from Chengdu Abok Biotechnology Co., Ltd., and baicalin (batch number 180811) purchased from Beijing Century Aoke Biotechnology Co., Ltd. The purity of the above reference standards was ≥98%.

[0033] 1.3 Medicinal Materials

[0034] Gastrodia elata (originating from Guizhou, batch number 20240301-03), Uncaria rhynchophylla (originating from Hunan, batch number 20231202-01), Abalone shell (originating from Quanzhou, Fujian, batch number 23112414), Gardenia jasminoides (originating from Jiangxi, batch number 240401), Scutellaria baicalensis (originating from Shaanxi, batch number 240303), Achyranthes bidentata (originating from Sichuan, batch number 20240301-03), Eucommia ulmoides (originating from Guizhou, batch number 20240302-01), Leonurus japonicus (originating from Nanyang, Henan, batch number 240101), Taxillus chinensis (originating from Guangdong, batch number 240315), Polygonum multiflorum (originating from Guizhou, batch number 20240101-02), Poria cocos (originating from Yunnan, batch number 20240201-01). All the above processed medicinal materials were purchased from the pharmacy of Jiangsu Provincial Hospital of Traditional Chinese Medicine.

[0035] 2. Methods and Results

[0036] 2.1 Chromatographic conditions

[0037] A Waters ACQUITY UPLC HSS T3 column (size: 2.1 mm × 100 mm, 1.8 μm) was used, with a flow rate of 0.2 mL / min, a column temperature of 35 ℃, and an injection volume of 2 μL. The mobile phase was acetonitrile (A) and 0.1% formic acid aqueous solution (B) for gradient elution. The gradient elution conditions were as follows: 0–4 min, 2%A → 5%A; 4–5 min, 5%A → 10%A; 5–7 min, 10%A → 12%A; 7–21 min, 12%A → 18%A; 21–22 min, 18%A → 27%A; 22–30 min, 27%A → 34%A; 30–33 min, 34%A → 88%A; 33–34 min, 88%A → 95%A; 34–38 min, 95%A → 95%A; 38–39 min, 95%A → 2%A; 39–40 min, 2%A → 2%A. The detector was a DAD, and the measurement was performed using variable wavelengths: 0–19 min: 192 nm; 19–24 min: 220 nm; 24–32 min: 256 nm; 32–40 min: 276 nm.

[0038] 2.2 Preparation of standard solutions

[0039] Accurately weigh appropriate amounts of each reference standard, dissolve them in pure methanol solution, and prepare mixed reference standard solutions with concentrations of 0.0759, 0.9200, 0.0541, 0.1396, 2.1255, and 0.9555 mg / mL for gastrodin, geniposide, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin, and wogonin, respectively.

[0040] 2.3 Preparation of the test solution of Gastrodia and Uncaria Decoction

[0041] Weigh out 9 g each of Gastrodia elata, Gardenia jasminoides, Scutellaria baicalensis, Eucommia ulmoides, Leonurus japonicus, Taxillus chinensis, Polygonum multiflorum vine, and Poria cocos; 12 g each of Uncaria rhynchophylla and Achyranthes bidentata; and 18 g of Haliotis diversicolor. Add water and decoct twice. Haliotis diversicolor should be decocted for 40 minutes first, and Uncaria rhynchophylla should be added 15 minutes before the end of the second decoction. The soaking time (A), decoction time (B), and water volume (C) for the first decoction should be as specified in the test conditions in Table 1. For the second decoction, add 4 times the amount of water and decoct for 20 minutes after boiling. Combine the two decoctions and bring to a final volume equal.

[0042] Accurately measure 1 mL of Gastrodia elata and Uncaria rhynchophylla decoction into a 2 mL volumetric flask, add methanol to make up to volume, mix well, and then extract with ultrasonic assistance for 1 min. After that, centrifuge at 14,000 r / min for 10 min, take the supernatant, and filter it through a 0.22 μm microporous membrane to obtain the test solution.

[0043] Table 1. Three-factor, three-level table for response surface optimization experiments

[0044]

[0045] 2.4 BBD-RSM Factor and Level Design

[0046] This study initially investigated several factors in the traditional decoction of Gastrodia elata and Uncaria rhynchophylla, including soaking time, decoction time, water volume, number of decoctions, the duration of pre-decoction of abalone shell, and the timing of adding Uncaria rhynchophylla. Under the premise of a fixed number of decoctions (2 times), a second decoction with 4 times the amount of water, a decoction time of 20 minutes after boiling, a 40-minute pre-decoction of abalone shell, and the addition of Uncaria rhynchophylla 15 minutes before the end of the second decoction, soaking time (A), decoction time (B), and water volume (C) were selected as factors in the BBD-RSM study.

[0047] 2.5 Fingerprint mapping and multivariate statistical analysis

[0048] 2.5.1 Precision

[0049] Take Tianma Gouteng Decoction S1, prepare the test solution according to section 2.2, and inject it continuously for 6 injections under the chromatographic conditions in section 2.1. Using geniposide as the reference peak, the relative peak area RSD of each chromatographic peak is less than 2.87%, and the relative retention time RSD is less than 0.26%, indicating that the instrument precision is good.

[0050] 2.5.2 Stability

[0051] Take Tianma Gouteng Decoction S1 and prepare the test solution according to section 2.2. Inject the sample 6 times at 0, 4, 8, 12, 16 and 24 h under the chromatographic conditions in section 2.1. Using geniposide as the reference peak, the RSD values ​​of the relative area of ​​each chromatographic peak were all less than 2.77% and the RSD values ​​of the relative retention time were all less than 1.13%, indicating that the sample has good stability at room temperature.

[0052] 2.5.3 Repeatability

[0053] Take Tianma Gouteng Decoction S1, and prepare 6 test solutions repeatedly according to section 2.2. Inject the samples under the chromatographic conditions in section 2.1. Using geniposide as the reference peak, the relative area RSD values ​​of each chromatographic peak are all less than 2.75%, and the relative retention times are all less than 0.39%, indicating that the method has good repeatability.

[0054] 2.5.4 Fingerprint Analysis

[0055] The data from 17 test samples were imported into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012). Fourteen peaks with good repeatability, stability, and distinct characteristics were selected as common peaks, and six components of the common peaks were identified. Figure 1 The chromatographic peaks were: 1. Gastrodin; 6. Geniposide; 8. Pinoresinol diglucoside; 9. 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside; 11. Baicalin; and 14. Baicalin. The relative areas of each chromatographic peak were recorded (Table 2).

[0056] Table 2 Relative peak areas of each chromatographic peak in the fingerprint chromatogram

[0057]

[0058] 2.5.5 Multivariate Statistical Analysis

[0059] The common peak data of the 17 test samples were imported into SIMCA 14.1 software for principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), and cluster analysis (CA). The results of the three analytical methods were basically consistent. The 17 test samples were divided into two major categories: test samples 1, 3, 4, 5, 14, and 15 were assigned to one category, and the remaining test samples were assigned to the other category. According to the variable importance inprojection (VIP) ranking, the peaks with VIP values ​​> 1 were peaks 11 (baicalin), 14 (wogonin), 4, 13, 12, 6 (geniposide), and 2 and 8 (pinenyl alcohol diglucoside). These components may be the differential components that divided the test samples into two categories. See the multivariate statistical analysis chart for details. Figure 2 and Figure 3 A is the PCA score chart; B is the OPLS-DA score chart; C is the cluster analysis chart; D is the VIP value ranking chart in OPLS-DA.

[0060] The common peak data were then imported into SPSS 27.0 for PCA analysis. Four principal components with eigenvalues ​​> 1 were extracted, with a cumulative contribution rate of 84.282% (see Table 3). The obtained eigenvectors were multiplied by the standardized data to obtain the principal component expression. The principal component composite model was then obtained by using the proportion of each principal component's eigenvalue to the sum of all extracted principal component eigenvalues ​​as weights.

[10] The sum of the scores of each principal component was calculated as the principal component comprehensive score (Table 4). Among them, the principal component comprehensive score of group 17 was the highest. The decoction process was: soaking time of 40 min, decoction time of 30 min after boiling, and water volume of 10 times.

[0061] Table 3. Eigenvalues ​​and Variance Contribution Rates of Principal Component Factors

[0062]

[0063] Table 4. Principal Component Composite Scores and Ranking

[0064]

[0065] 2.6 Comprehensive Evaluation of Multi-Indicator Component Content and Drying Rate

[0066] 2.6.1 System Suitability: The mixed reference solution and the test solution of Gastrodia and Uncaria Decoction were injected separately according to the chromatographic conditions described in section 2.1. The obtained peak resolution, theoretical plate number, and tailing factor were all good. See details below. Figure 3 A is the mixed control sample image, and B is the sample image of Tianma Gouteng Decoction (S1).

[0067] 2.6.2 Examination of Linear Relationships

[0068] Take the standard solution prepared in section 2.2, add pure methanol solution, and serially dilute it by 2, 4, 8, 16, and 32 times. Inject the solutions according to the chromatographic conditions in section 2.1, record the peak areas, and perform linear regression with peak area as the ordinate (Y) and concentration as the abscissa (X). Calculate the regression equations and correlation coefficients (R²) for each index component. 2 The linear range is shown in Table 5.

[0069] Table 5. Regression equations and R values ​​for each indicative component. 2 Linear range

[0070]

[0071] 2.6.3 Precision

[0072] Take the standard solution prepared in section 2.2, and inject it six times consecutively under the chromatographic conditions in section 2.1. Record the peak area of ​​each chromatographic peak and calculate the RSD value. The RSD values ​​of the peak areas of gastrodin, geniposide, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin, and wogonin were 0.72%, 0.80%, 0.74%, 0.87%, 0.81%, and 0.82%, respectively, indicating good instrument precision.

[0073] 2.6.4 Stability

[0074] The test solution of Gastrodia and Uncaria Decoction S1 was injected at 0, 4, 8, 12, 16, and 24 h according to the chromatographic conditions in section 2.1, and the peak areas were recorded. The RSD values ​​of the peak areas of gastrodin, geniposide, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin, and wogonin were 1.30%, 0.78%, 1.79%, 1.97%, 0.98%, and 0.73%, respectively, indicating that the test solution was stable within 24 h at room temperature.

[0075] 2.6.5 Repeatability

[0076] Take the Tianma Gouteng Decoction S1 and prepare 6 test solutions repeatedly according to section 2.3. Inject the solutions under the chromatographic conditions in section 2.1 and record the peak areas. The RSD values ​​of the mass concentrations of gastrodin, geniposide, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin, and wogonin were 0.85%, 0.61%, 2.24%, 1.75%, 1.64%, and 0.72%, respectively, indicating that the method has good repeatability.

[0077] 2.6.7 Recovery rate

[0078] Six portions of a decoction containing known levels of each indicative component were prepared. Gastrodin, geniposide, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin, and wogonin were added at 100% level. The sample solutions were prepared according to section 2.3 and injected under the chromatographic conditions described in section 2.1 for content determination. The average recoveries of each indicative component were calculated to be 106.46%, 107.19%, 101.26%, 102.29%, 107.91%, and 106.72%, with RSD values ​​of 0.60%, 0.50%, 0.85%, 0.63%, 0.21%, and 0.35%, respectively, indicating that the recovery rate of this method meets the requirements.

[0079] 2.6.8 Drying rate

[0080] Accurately measure 20 mL of each of the 17 groups of Gastrodia elata and Uncaria rhynchophylla decoction solutions into a pre-weighed evaporating dish. Evaporate to dryness in a water bath at 100 ℃, then transfer to a 105 ℃ oven and dry for 3 hours until constant weight. Remove and cool in a desiccator, then weigh quickly.

[11] Calculate its dry paste rate.

[0081] Dry extract yield = (Total weight of dry extract / Weight of medicinal materials) × 100%

[0082] 2.6.9 Calculation of the overall score using the AHP weighting method

[0083] The weights were calculated using the AHP method (Table 6), with the total weight coefficient set to 1. The resulting weight coefficients were: gastrodin 0.1344, geniposide 0.1344, pinoresinol diglucoside 0.1344, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside 0.1344, baicalin 0.1344, wogonin 0.0700, and the dry extract yield was 0.2580. X1~X7 represent the above comprehensive scoring indicators, and the comprehensive score = (X1 / X...) / X7. 1max ×0.1344+X2 / X 2max ×0.1344+X3 / X 3max ×0.1344+X4 / X 4max ×0.1344+X5 / X 5max ×0.1344+X6 / X 6max ×0.0700+X7 / X 7max (×0.2580)×100. The comprehensive score results are shown in Table 7, showing that the highest score was for test number 17 (soaking for 40 min, boiling and simmering for 30 min, with 10 times the amount of water added), which is consistent with the comprehensive score of the principal components.

[0084] Table 6 Weight Calculation by AHP Method

[0085]

[0086] Table 7 Weighted Comprehensive Score of 17 Groups of Samples

[0087]

[0088] 2.7 Establishment and Analysis of Variance of the BBD-RSM Regression Model

[0089] Design-Expert 13 software was used to optimize the decoction process by establishing a model using response surface methodology. Linear and nonlinear regressions were performed on each factor to establish a quadratic regression model equation for the comprehensive score (Y) on the three factors (A-soaking time; B-decoction time; C-water amount). Y = 86.10 + 3.55A + 2.81B + 2.06C - 3.60AB - 0.84AC + 4.44BC - 2.04A 2 -4.93B 2 -4.91C 2 The established model has a p-value of 0.0072 < 0.05, indicating significance; the p-value for the lack-of-fit term is 0.8499 > 0.05; and the correlation coefficient R0 is [missing value]. 2 =0.9063, Adjusted R value 2=0.7858, indicating that the model has a good fit and small experimental error, and can be used to analyze and predict the comprehensive score. The shape of the contour lines in the 3D response surface plot reflects the strength of the interaction between the two factors; ellipses indicate a stronger interaction, while circles indicate a weaker interaction. See details... Figure 4 The model-fitted decoction process consisted of a soaking time of 47.372 min, a decoction time of 31.556 min after boiling, and a water volume of 10.388 times the normal volume. Based on fingerprint analysis, multi-index content analysis, and actual operational conditions, the optimal process was ultimately determined to be a soaking time of 50 min, a decoction time of 30 min after boiling, and a water volume of 10 times the normal volume.

[0090] 2.8 Process Validation

[0091] Three batches of verification tests were conducted using the above-mentioned optimal process. The comprehensive scores obtained were 86.619, 87.420, and 87.766, which were basically consistent with the predicted value of 87.603, indicating that the optimized decoction method is relatively stable and feasible (Table 8).

[0092] Table 8. Verification Test Results

[0093]

[0094] 3. Discussion

[0095] Box-Behnken design—response surface methodology—describes the relationship between independent and dependent variables through mathematical modeling. By testing and optimizing the model, the optimal combination of independent variables is obtained. Compared to point analysis in orthogonal experiments, it offers a more comprehensive analysis and more accurate predictions. In this study, the Tianma Gouteng decoction samples were qualitatively and quantitatively evaluated from two perspectives: "Chinese herbal fingerprinting—multivariate statistical analysis" and "comprehensive evaluation of multi-index component content—dry extract rate." This approach is more scientifically sound than simple content determination.

[0096] In selecting indicative components, the 2020 edition of the Chinese Pharmacopoeia specifies two content detection items for Gastrodin and Baicalin in Gastrodia and Uncaria granules. Combining this with the content detection items for single medicinal materials in the Pharmacopoeia and preliminary experimental results, six detection indicators were ultimately determined: gastrodin, geniposide, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin, and wogonin. Gastrodin is one of the main active components of Gastrodia elata, possessing effects such as increasing cardiovascular blood flow, protecting the nervous system, enhancing immunity, and having sedative and hypnotic effects. Geniposide is the main pharmacologically active substance in Gardenia jasminoides, with effects such as hepatoprotective and choleretic properties, hypoglycemic effects, anti-atherosclerosis effects, improvement of neurodegenerative diseases, and antidepressant effects. Pinoresinol diglucoside is the main active component of Eucommia ulmoides, primarily possessing anti-inflammatory, antioxidant, and neuroprotective functions. 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside is a major component of Polygonum multiflorum (He Shou Wu), possessing anti-inflammatory, antioxidant, and protective effects against neurons, cardiomyocytes, and endothelial cells. It is helpful in treating diseases such as cerebral ischemia, Parkinson's disease, Alzheimer's disease, and atherosclerosis. Baicalin, a flavonoid glycoside, is the main active ingredient in Scutellaria baicalensis. It exerts various effects including anti-inflammatory, antiviral, antitumor, antithrombotic, immunomodulatory, and neuroprotective properties, and has therapeutic effects on diseases of the cardiovascular, respiratory, and digestive systems. Hanhuangqin glycoside is also a flavonoid component of Scutellaria baicalensis, primarily exerting anti-inflammatory, antitumor, antioxidant, neuroprotective, and cardiovascular protective effects.

[0097] This experiment first analyzed and scored the fingerprint data of Gastrodia elata and Uncaria rhynchophylla decoction samples under different decoction processes using CA, OPLS-DA, and PCA methods. The optimal process (40 min soaking, 30 min decoction after boiling, and 10 times the water volume) was found in sample group 17. Secondly, the samples were scored using multi-index component content determination and dry extract yield, and the conclusions were consistent with the fingerprint data. A BBD-RSM design was used to fit the experimental process, resulting in a recommended process of 47.372 min soaking, 31.556 min decoction, and 10.388 times the water volume. Considering that Abalone shell is a shell-based medicinal material, and Gastrodia elata, Poria cocos, Achyranthes bidentata, and Polygonum multiflorum are all rhizomes with relatively hard textures, the soaking time can be appropriately extended. Therefore, the optimized decoction process was selected: add 10 times the amount of water to all the herbs and soak for 50 minutes; decoct the abalone shell for 40 minutes first; decoct the whole formula for 30 minutes after boiling; and add the uncaria until 15 minutes before the end of the decoction. After testing, the process was verified to be stable and feasible.

[0098] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for establishing a traditional Chinese medicine fingerprint spectrum of a decoction of Gastrodia elata and Uncaria rhynchophylla, characterized in that, Includes the following steps: (1) Preparation of reference solution: Accurately weigh gastrodin, geniposide, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin and wogonin reference standards, and add methanol to prepare a mixed reference solution; (2) Preparation of test solution: Accurately measure 1 mL of Gastrodia elata and Uncaria rhynchophylla decoction and place it in a 2 mL volumetric flask. Add methanol to make up to volume, mix well, and then extract with ultrasonic assistance for 1 min. After that, centrifuge at 14,000 r / min for 10 min, take the supernatant, and filter it through a 0.22 μm microporous membrane to obtain the test solution. (3) Detection was performed using high performance liquid chromatography (HPLC) to establish a fingerprint spectrum; in step (3), the HPLC conditions were as follows: a Waters ACQUITY UPLC HSS T3 column was used, with dimensions of 2.1 mm × 100 mm and a diameter of 1.8 μm, and a flow rate of 0.2 mL / min; the column temperature was 35 ℃, and the injection volume was 2 μL; the mobile phase was acetonitrile A-0.1% formic acid aqueous solution B for gradient elution, and the gradient elution conditions were as follows: 0–4 min, 2%A→5%A; 4–5 min, 5%A→10%A; 5–7 min, 10%A→12%A; 7–21 min, 12%A→18%A; 21–22 min, 18%A→27%A; 22–30 min, 27%A→34%A; 30–33 min, 34%A→88%A; 33–34 min, 34%A→88%A; 33–34 min, 34%A→88%A; 34%A→88%A; 35%A→10 ... min, 88%A→95%A; 34~38 min, 95%A→95%A; 38~39 min, 95%A→2%A; 39~40 min, 2%A→2%A; The detector is a DAD, and variable wavelength measurement is used: 0~19 min: 192 nm; 19~24 min: 220 nm; 24~32 min: 256 nm; 32~40 min: 276 nm.

2. The method for establishing a traditional Chinese medicine fingerprint spectrum of the Gastrodia and Uncaria decoction as described in claim 1, characterized in that, The concentrations of gastrodin, geniposide, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin, and wogonin in the mixed reference solution were 0.0759, 0.9200, 0.0541, 0.1396, 2.1255, and 0.9555 mg / mL, respectively.

3. The method for establishing a traditional Chinese medicine fingerprint spectrum of the Gastrodia and Uncaria decoction as described in claim 1, characterized in that, The preparation method of Tianma Gouteng Decoction is as follows: Weigh 9g each of Tianma, Zhizi, Huangqin, Duzhong, Yimucao, Sangjisheng, Shouwu Teng, and Fushen, 12g each of Gouteng and Chuanniuxi, and 18g of Shijue Ming. Add water and decoct twice. First decoct Shijue Ming for 40 minutes, and add Gouteng 15 minutes before the end of the second decoction. Soak for 40 minutes in the first decoction, decoct for 30 minutes after boiling, and add 10 times the amount of water. Add 4 times the amount of water in the second decoction and decoct for 20 minutes after boiling. Combine the two decoctions and make up to equal volume.

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