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

Abstract

The invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for establishing a traditional Chinese medicine fingerprint spectrum of a gastrodia elata and uncaria decoction. The method is realized by the following steps: firstly, preparing a reference substance solution and a test solution, then detecting by adopting a high performance liquid chromatography, and establishing a fingerprint spectrum. According to the method provided by the invention, by reasonably selecting the components of the reference substance and adopting a multi-wavelength method according to the maximum absorption wavelength of the components, the change of the component amount between the samples can be effectively represented, so that the quality of the samples is more accurately judged; the invention further provides a screening and optimizing method for the preparation method of the gastrodia elata and uncaria decoction, the prepared method is good in repeatability and stability, the quality of the gastrodia elata and uncaria decoction can be monitored more comprehensively, and the method provides valuable reference for quality evaluation and standardization of traditional Chinese medicine decoction.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and particularly relates to a method for establishing a traditional Chinese medicine fingerprint spectrum of a Gastrodia elata and Uncaria rhynchophylla decoction. Background Art

[0002] Tianma Gou Teng Yin is a clinical prescription of Hu Guangci, a famous doctor in Sichuan and Chongqing in modern times. It was first published in "New Interpretation of Syndrome and Treatment of Miscellaneous Diseases in Internal Medicine of Traditional Chinese Medicine". The whole prescription consists of 11 Chinese medicinal herbs: Tianma, Gou Teng, Semen Cassiae, Rhizoma Cynoglossi, Gardenia, Scutellaria, Eucommia, Leonurus, Viscum album, Polygonum multiflorum, and Poria cocos. It has the effects of calming the liver and extinguishing wind, clearing heat and activating blood circulation, and nourishing the liver and kidney. Tianma Gou Teng Yin has a wide range of clinical applications, mainly treating cardiovascular and cerebrovascular diseases such as hypertension, cerebral infarction, Parkinson's disease, and epilepsy. In the "Expert Consensus on Diagnosis and Treatment of Hypertension in Traditional Chinese Medicine" issued by the Cardiovascular Disease Branch of the Chinese Association of Traditional Chinese Medicine in 2019, Tianma Gou Teng Yin was recommended for the treatment of hypertension with hyperactivity of liver yang. In addition, a survey shows that the research related to Tianma Gou Teng Yin has gradually increased in recent years and has been increasingly valued by many researchers.

[0003] Traditional Chinese medicine fingerprints can comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicines and their preparations, and thus provide an overall description and evaluation of the quality of the medicines. HPLC fingerprints can effectively control the quality of traditional Chinese medicines. Even if the chromatographic peaks belong to unknown compounds, HPLC fingerprints can provide sufficient and reliable information and can be used to identify the quality of traditional Chinese medicines.

[0004] Traditional Chinese medicine compound has the characteristics of multiple components, multiple targets and multiple levels, which means that a single indicator cannot fully and holistically reflect the characteristics of the compound, and a comprehensive evaluation of multiple indicators can integrate multiple important indicators. At present, there is a lack of relevant quality control methods for Tianma Gou Teng Decoction, and it is impossible to accurately judge the quality of the drug. Therefore, studying a method that can accurately judge the quality of Tianma Gou Teng Decoction has become an urgent problem to be solved. Summary of the invention

[0005] In view of the technical gaps and other problems existing in the prior art, the purpose of the present invention is to provide a method for establishing a fingerprint of Gastrodia elata and Uncaria rhynchophylla decoction and a quality detection method for Gastrodia elata and Uncaria rhynchophylla decoction, so as to solve the problem that the prior art does not have quantitative determination and can hardly serve as an evaluation standard for the overall quality of Gastrodia elata and Uncaria rhynchophylla decoction.

[0006] The technical solution adopted by the present invention to achieve its purpose is: to provide a method for establishing a traditional Chinese medicine fingerprint of Tianma Uncaria decoction, comprising the following steps:

[0007] (1) Preparation of reference solution: accurately weigh gastrodin, gardenia glycoside, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin and wogonin reference substances, and add methanol to prepare a mixed reference solution;

[0008] (2) Preparation of test solution: accurately measure 1 mL of Tianma Gouteng decoction and place it in a 2 mL volumetric flask, add methanol to make up to volume, mix well and perform ultrasonic-assisted extraction for 1 min, then centrifuge at 14,000 r / min for 10 min, take the supernatant, and filter through a 0.22 μm microporous membrane to obtain the test solution;

[0009] (3) High performance liquid chromatography was used for detection to establish a fingerprint. In step (3), the conditions of the high performance liquid chromatography were as follows: a Waters ACQUITY UPLC HSS T3 column with specifications of 2.1 mm × 100 mm, 1.8 μm, and a flow rate of 0.2 mL / min; a column temperature of 35°C and an injection volume of 2 μL. Mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B) for gradient elution, and the gradient elution conditions are: 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 is used for measurement: 0-19 min: 192 nm; 19-24 min: 220 nm; 24-32 min: 256 nm; 32-40 min: 276 nm.

[0010] The method for establishing a traditional Chinese medicine fingerprint of the Tianma Uncaria decoction is described, wherein the concentrations of gastrodin, gardenia glycoside, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin, and wogonin in the mixed reference solution are 0.075 9, 0.920 0, 0.054 1, 0.139 6, 2.125 5, and 0.9555 mg / mL, respectively.

[0011] The method for establishing a traditional Chinese medicine fingerprint of a Tianma Uncaria drink decoction is as follows: weigh 9g each of Tianma, Gardenia, Scutellaria, Eucommia, Leonurus, Viscum album, Polygonum multiflorum, Poria, 12g each of Uncaria, Cyathula officinalis, and 18g of Semen Cassiae, add water and boil twice, wherein Semen Cassiae is first boiled for 40min, and Uncaria is added 15min before the end of the second decoction, the first decoction is soaked for 40min, the decoction time is 30min after boiling, and the amount of water added is 10 times, the amount of water added to the second decoction is 4 times, and the decoction time is 20min after boiling, the decoctions of the two decoctions are combined and fixed to equal volume.

[0012] A fingerprint spectrum of Gastrodia elata and Uncaria rhynchophylla decoction is constructed by detection using the above construction method.

[0013] The fingerprint of the Gastrodia elata and Uncaria rhynchophylla decoction has 6 common peak components, which are: No. 1 gastrodin; No. 6 gardenoside; No. 8 pinoresinol diglucoside; No. 9 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside; No. 11 baicalin; and No. 14 wogonin.

[0014] A method for detecting the quality of a decoction of Gastrodia elata and Uncaria rhynchophylla, comprising: detecting a sample of the decoction of Gastrodia elata and Uncaria rhynchophylla according to the construction method described above to obtain a high performance liquid chromatography spectrum of the sample of the decoction of Gastrodia elata and Uncaria rhynchophylla, constructing a fingerprint spectrum of the decoction of Gastrodia elata and Uncaria rhynchophylla according to the construction method described in claim 1, and comparing the high performance liquid chromatography spectrum of the sample of the decoction of Gastrodia elata and Uncaria rhynchophylla with the fingerprint spectrum of the decoction of Gastrodia elata and Uncaria rhynchophylla.

[0015] Beneficial effects:

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

[0017] (2) The method provided by the present invention can effectively indicate the variation of component amounts between samples by rationally selecting reference substance components and using a multi-wavelength method based on the maximum absorption wavelength of the components, thereby more accurately judging the quality of the samples;

[0018] (3) The method provided by the present invention screens and optimizes the configuration of reference substances, test solutions, etc. according to the efficacy of the Tianma Uncaria Decoction and the structural characteristics of the ingredients contained therein. The developed method has good repeatability and stability, and can more comprehensively monitor the quality of the Tianma Uncaria Decoction. This method provides a valuable reference for the quality evaluation and standardization of Chinese herbal decoctions.

[0019] (4) The Chinese medicine compound preparation involved in the present invention has many Chinese medicines and relatively complex effective ingredients. It is difficult to accurately evaluate the quality of the Chinese medicine preparation by only measuring the content of one or several effective ingredients. The present invention can well conduct qualitative analysis on the product quality of the Tianma Uncaria Decoction by constructing the HPLC fingerprint of the Tianma Uncaria Decoction, and provide a basis for comprehensively evaluating the quality standard of the Tianma Uncaria Decoction.

[0020] (5) The present invention determines the mass fraction and dry extract rate of gastrodin, gardenia glycoside, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin and wogonin as evaluation indicators of the extraction process, and uses a multi-index comprehensive scoring method to comprehensively score the Box-Behnken design-response surface methodology test results to optimize the extraction process. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The fingerprints of 17 groups of test samples are shown in Figure 1. Peak 1 is gastrodin; Peak 6 is gardenoside; Peak 8 is pinoresinol diglucoside; Peak 9 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside; Peak 11 is baicalin; Peak 14 is wogonin;

[0022] Figure 2 It is a multivariate statistical analysis diagram, where A is the PCA score diagram; B is the OPLS-DA score diagram;

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

[0024] Figure 4 UPLC chromatograms of Tianma Gouteng Decoction, where A is the mixed reference sample; B is the test sample of Tianma Gouteng Decoction (S1);

[0025] Figure 5 The influence of each factor on the comprehensive score. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, but these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0027] Embodiment 1:

[0028] 1 Instruments and medicines

[0029] 1.1 Instruments

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

[0031] 1.2 Reagents and Reference Materials

[0032] Acetonitrile and methanol were of chromatographic grade purchased from MERCK, Germany. Formic acid was of chromatographic grade purchased from Tektronix (Guangzhou) Technology Service Co., Ltd. Reference substances: Gastrodin (batch number AFCB2851), Gardenia glycoside (batch number AFCB1356), Pinus resinol diglucoside (batch number AZCC1012), 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside (batch number AFCI2156), Baicalin (batch number AFCK2351) were all purchased from Chengdu Aibok Biotechnology Co., Ltd., and Wogonin (batch number 180811) was purchased from Beijing Century Aoke Biotechnology Co., Ltd. The purity of the above reference substances was ≥98%.

[0033] 1.3 Medicinal Materials

[0034] Gastrodia elata (origin Guizhou, batch number 20240301-03), Uncaria rhynchophylla (origin Hunan, batch number 20231202-01), Semen Cassiae (origin Fujian Quanzhou, batch number 23112414), Gardenia jasminoides (origin Jiangxi, batch number 240401), Scutellaria baicalensis (origin Shaanxi, batch number 240303), Cyathula capitata (origin Sichuan, batch number 20240301-03), Eucommia ulmoides (origin Guizhou, Batch number 20240302-01), Leonurus japonicus (originating from Nanyang, Henan, batch number 240101), Viscum album (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 of the above pieces were purchased from the Chinese medicine pharmacy of Jiangsu Provincial Hospital of Traditional Chinese Medicine and identified by Zhu Yufeng, chief Chinese medicine pharmacist 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 (specifications: 2.1 mm×100 mm, 1.8 μm) was used with a flow rate of 0.2 mL / min; column temperature: 35°C, and an injection volume of 2 μL. Mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B) for gradient elution, and the gradient elution conditions are: 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 is used for measurement: 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 solution

[0039] Accurately weigh an appropriate amount of each reference substance, add pure methanol solution to dissolve, and prepare mixed reference substance solutions with concentrations of 0.075 9, 0.920 0, 0.054 1, 0.139 6, 2.125 5, and 0.955 5 mg / mL for gastrodin, gardenia glycoside, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin, and wogonin, respectively.

[0040] 2.3 Preparation of Tianma Gouteng Drink Test Solution

[0041] Weigh 9g each of Gastrodia elata, Gardenia jasminoides, Scutellaria baicalensis, Eucommia ulmoides, Leonurus japonicus, Morus alba, Polygonum multiflorum, Poria, 12g each of Uncaria rhynchophylla, Cyathula officinalis, and 18g of Semen Cassiae, add water and decoct twice. Semen Cassiae is first decocted for 40min, and Uncaria rhynchophylla is added 15min before the end of the second decoction. The soaking time (A), decoction time (B), and water addition (C) of the first decoction are carried out according to the conditions of each test number in Table 1. The water addition amount of the second decoction is 4 times of water, and the decoction time is 20min after boiling. Combine the two decoctions and adjust the volume to equal volume.

[0042] Accurately measure 1 mL of Tianma Gouteng Decoction and place it in a 2 mL volumetric flask, add methanol to make up the volume, mix well and perform ultrasonic-assisted extraction for 1 min, then centrifuge at 14,000 r / min for 10 min, take the supernatant, and filter through a 0.22 μm microporous filter membrane to obtain the test solution.

[0043] Table 1 Three factors and three levels of response surface optimization experiment

[0044]

[0045] 2.4 BBD-RSM Factor and Level Design

[0046] In the early stage of this project, several factors such as soaking time, decoction time, amount of water added, number of decoctions, length of time for first decoction of Semen Cassiae, and time of adding Uncaria rhynchophylla in the traditional decoction of Gastrodia elata and Uncaria rhynchophylla were preliminarily investigated. Under the premise that the number of decoctions was fixed at 2 times, the amount of water added in the second decoction was 4 times of water, the decoction time of the second decoction was 20 minutes after boiling, Semen Cassiae was first decocted for 40 minutes, and Uncaria rhynchophylla was added 15 minutes before the end of the second decoction, soaking time (A), decoction time (B), and amount of water added (C) were selected as the factors of BBD-RSM.

[0047] 2.5 Fingerprint and multivariate statistical analysis

[0048] 2.5.1 Precision

[0049] Take Gastrodia elata and Uncaria rhynchophylla decoction S1, prepare the test solution according to item 2.2, and make 6 consecutive injections under the chromatographic conditions of item 2.1. Take gardenia glycoside as the reference peak, and calculate the relative peak area RSD value of each chromatographic peak, which is less than 2.87%, and the relative retention time RSD value is less than 0.26%, indicating that the instrument precision is good.

[0050] 2.5.2 Stability

[0051] Take Gastrodia elata and Uncaria rhynchophylla decoction S1, prepare the test solution according to item 2.2, and inject 6 times at 0, 4, 8, 12, 16, and 24 h according to the chromatographic conditions of item 2.1, take gardenia glycoside as the reference peak, calculate the relative area RSD value of each chromatographic peak and the RSD value of the relative retention time is less than 2.77%, which indicates that the sample has good stability at room temperature.

[0052] 2.5.3 Repeatability

[0053] Take Gastrodia elata and Uncaria rhynchophylla decoction S1, prepare 6 test solutions repeatedly according to item 2.2, and inject them according to the chromatographic conditions of item 2.1. Take gardenia glycoside as the reference peak, and calculate the relative area RSD value of each chromatographic peak, which is less than 2.75%, and the relative retention time is less than 0.39%, indicating that the method has good repeatability.

[0054] 2.5.4 Fingerprint analysis

[0055] The data of 17 groups of test samples were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system (2012), and 14 peaks with good repeatability, good stability and obvious characteristics were selected as common peaks, and 6 common peak components were identified ( Figure 1). They are: No. 1 gastrodin; No. 6 gardenoside; No. 8 pinoresinol diglucoside; No. 9 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside; No. 11 baicalin; No. 14 wogonin, and the relative area of ​​each chromatographic peak was recorded (Table 2).

[0056] Table 2 Relative peak area of ​​each chromatographic peak in fingerprint

[0057]

[0058]

[0059] 2.5.5 Multivariate statistical analysis

[0060] The common peak data of the 17 groups of 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 analysis methods were basically consistent. The 17 groups of test samples were divided into two categories, with test samples 1, 3, 4, 5, 14, and 15 classified into one category and the remaining test samples classified into the other category. According to the variable importance in project (VIP) ranking, the peaks with VIP values ​​> 1 are peaks 11 (baicalin), 14 (wogonin), 4, 13, 12, 6 (gardenia glycoside), 2, 8 (pinoresinol diglucoside), which may be the differential components that divide the test samples into two categories. See the multivariate statistical analysis diagram for details. Figure 2 and Figure 3 A is the PCA score graph; B is the OPLS-DA score graph; C is the cluster analysis graph; D is the VIP value ranking graph in OPLS-DA.

[0061] The common peak data were then imported into SPSS27.0 for PCA analysis. There were 4 components with principal component eigenvalues ​​greater than 1, with a cumulative contribution rate of 84.282%, as shown in Table 3. The obtained eigenvector was multiplied by the standardized data to obtain the principal component expression, and the proportion of the eigenvalue corresponding to each principal component to the sum of the total eigenvalues ​​of the extracted principal components was used as the weight to obtain the principal component comprehensive model.

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

[0062] Table 3 Principal component factor eigenvalues ​​and variance contribution rates

[0063]

[0064] Table 4 Comprehensive scores and rankings of principal components

[0065]

[0066] 2.6 Comprehensive evaluation of multi-index component content and dry cream rate

[0067] 2.6.1 System adaptability The mixed reference solution and the Tianma Gouteng Decoction test solution were injected according to the chromatographic conditions in 2.1. The obtained chromatographic peak separation, theoretical plate number and tailing factor were all good. Figure 3 , A is the mixed reference product picture, and B is the Tianma Gouteng Decoction test product picture (S1).

[0068] 2.6.2 Linear relationship investigation

[0069] Take the standard solution prepared in 2.2, add pure methanol solution, dilute it step by step by 2, 4, 8, 16, and 32 times, and inject it according to the chromatographic conditions of 2.1, record the peak area respectively, and perform linear regression with the peak area as the ordinate (Y) and the concentration as the abscissa (X). Calculate the regression equation of each index component and the correlation coefficient (R 2 ) and the linear range are shown in Table 5.

[0070] Table 5 Regression equations and R of each index component 2 , Linear range

[0071]

[0072] 2.6.3 Precision

[0073] Take the standard solution prepared in 2.2, and inject it 6 times continuously according to the chromatographic conditions of 2.1, record the area of ​​each chromatographic peak, and calculate the RSD value. The RSD values ​​of the peak areas of gastrodin, gardenia glycoside, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin and wogonin are 0.72%, 0.80%, 0.74%, 0.87%, 0.81%, and 0.82%, respectively, indicating that the instrument has good precision.

[0074] 2.6.4 Stability

[0075] Take the test solution of Tianma Gouteng Decoction S1 and inject it at 0, 4, 8, 12, 16 and 24 h according to the chromatographic conditions of Item 2.1, and record the area of ​​each chromatographic peak. The RSD values ​​of the peak areas of gastrodin, gardenia glycoside, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin and wogonin are

[0076] 1.30%, 0.78%, 1.79%, 1.97%, 0.98%, and 0.73%, indicating that the test solution was stable within 24 h at room temperature.

[0077] 2.6.5 Repeatability

[0078] Take Tianma Gou Teng Decoction S1, prepare 6 test sample solutions according to 2.3, inject under the chromatographic conditions of 2.1, and record the peak area. The RSD values ​​of the mass concentrations of gastrodin, gardenia glycoside, 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.

[0079] 2.6.7 Sample recovery rate

[0080] Take 6 portions of Tianma Gouteng decoction solution with known contents of each index component, add gastrodin, gardenia glycoside, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin and wogonin at 100% level, prepare the test solution according to 2.3, and inject under the chromatographic conditions of 2.1 for content determination. The average values ​​of the sample recovery rates of the index components were calculated to be 106.46%, 107.19%, 101.26%, 102.29%, 107.91%, and 106.72%, respectively, and the RSD values ​​were 0.60%, 0.50%, 0.85%, 0.63%, 0.21%, and 0.35%, respectively, indicating that the sample recovery rate of this method meets the requirements.

[0081] 2.6.8 Dry paste rate

[0082] Accurately measure 20 mL of each of the 17 groups of Tianma Gouteng decoction solutions and add them to a constant weight evaporating dish. After evaporating to dryness in a water bath at 100°C, transfer to a 105°C oven and dry for 3 hours until constant weight is reached. Take out and cool in a desiccator and weigh quickly.

[11] , calculate its dry paste rate.

[0083] Dry paste rate = total weight of dry paste / weight of medicinal materials × 100%

[0084] 2.6.9 AHP weighted method to calculate comprehensive scores

[0085] The AHP method (Table 6) was used to calculate each weight, and the total weight coefficient was set to 1. The weight coefficients were 0.1344 for gastrodin, 0.1344 for jasminoidin, 0.1344 for pinoresinol diglucoside, 0.1344 for 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, 0.1344 for baicalin, 0.0700 for wogonin, and 0.2580 for dry cream rate. X1 to X7 represent the above comprehensive scoring indicators, and the comprehensive score = (X1 / X 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 is test number 17 (soaking for 40 minutes, decocting for 30 minutes after boiling, and adding 10 times the amount of water), which is consistent with the comprehensive score of the main component.

[0086] Table 6 AHP method weight calculation

[0087]

[0088] Table 7 Weighted comprehensive scores of 17 groups of samples

[0089]

[0090]

[0091] 2.7 Establishment of BBD-RSM regression model and variance analysis

[0092] Design-Expert 13 software was used to establish a model through response surface methodology to optimize the decoction process. Linear and nonlinear regression were performed on each factor, and the quadratic regression model equation of the comprehensive score (Y) against the three factors (A-immersion time; B-decoction time; C-water addition) was established. 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 P value of the established model is 0.0072<0.05, which is significant. The P value of the lack of fit term is 0.8499>0.05. The correlation coefficient R 2 =0.9063, Correction coefficient Adjusted R 2=0.7858, indicating that the model has a good fit and a small experimental error. This model can be used to analyze and predict the comprehensive score. The contour shape of the 3D response surface diagram reflects the strength of the interaction between the two factors. The ellipse indicates a stronger interaction, and the circle indicates a weaker interaction. For details, see Figure 4 The decoction process fitted by the model is soaking time 47.372min, decoction time 31.556min after boiling, and water addition 10.388 times. Combining the fingerprint spectrum and multi-index content analysis as well as the actual operation situation, the optimal process is finally determined to be soaking time 50min, decoction time 30min after boiling, and water addition 10 times.

[0093] 2.8 Process Validation

[0094] The above-mentioned optimal process was selected for three batches of verification tests, and 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 was relatively stable and feasible (Table 8).

[0095] Table 8 Verification test results

[0096]

[0097]

[0098] 3 Discussions

[0099] Box-Behnken design-response surface method describes the relationship between independent variables and dependent variables through mathematical modeling, and obtains the optimal combination of independent variables through model testing and optimization. Compared with the point analysis of orthogonal test, the analysis is more comprehensive and the prediction is more accurate. In this study, the samples of Tianma Gou Teng decoction in the design were qualitatively and quantitatively evaluated from two perspectives: "Chinese medicine fingerprint-multivariate statistical analysis" and "multi-index component content-comprehensive evaluation of dry paste rate", which is more scientific than single content determination.

[0100] In selecting the index ingredients, the 2020 edition of the "Chinese Pharmacopoeia" stipulates two content detection items for gastrodin and baicalin for Tianma Gouteng Granules. Combined with the content detection items of single medicinal materials in the pharmacopoeia and the preliminary experimental results, the six detection indicators of gastrodin, gardenia glycoside, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin and wogonin were finally determined. Gastrodin is one of the main active ingredients of Gastrodia elata, which has the effects of increasing cardiovascular and cerebrovascular flow, protecting the nervous system, enhancing immunity, and calming and inducing sleep. Gardenia glycoside is the main pharmacological substance of Gardenia, which has the effects of protecting the liver and promoting bile secretion, lowering blood sugar, resisting atherosclerosis, improving neurodegenerative diseases and anti-depression. Pinoresinol diglucoside is the main active ingredient of Eucommia ulmoides, and it mainly has anti-inflammatory, antioxidant, and neuroprotective functions. 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside is the main component of Polygonum multiflorum, which has anti-inflammatory, antioxidant and protective effects on neurons, cardiomyocytes and endothelial cells, and is helpful in treating cerebral ischemia, Parkinson's disease, Alzheimer's disease, atherosclerosis and other diseases. Baicalin is a flavonoid glycoside and the main active ingredient in Scutellaria baicalensis. Baicalin has multiple effects such as anti-inflammatory, antiviral, anti-tumor, anti-thrombotic, immunomodulatory, and neuroprotective, and has therapeutic effects on cardiovascular, respiratory, digestive and other multi-system diseases. Wogonin is also one of the flavonoid components in Scutellaria baicalensis, which mainly plays anti-inflammatory, anti-tumor, antioxidant, neuroprotective and cardiovascular protective effects.

[0101] In this experiment, the data of the fingerprint of the samples of Tianma Gou Teng decoction under different decoction processes were first analyzed and scored by CA, OPLS-DA and PCA, and the process of the 17th group of samples was the best process with the highest score (soaking for 40 minutes, decoction time for 30 minutes after boiling, and 10 times the amount of water added). Secondly, the samples were scored by multi-index component content determination and dry paste rate, and the conclusions were consistent with the fingerprint data. The experimental process was model fitted by BBD-RSM design, and the recommended process was obtained as 47.372 minutes of soaking time, 31.556 minutes of decoction time, and 10.388 times of water addition. Considering that the stone cassia of this prescription is a shell medicine, Tianma, Fuling, Chuanxiong, Shouwu Teng, etc. are all rhizome medicines with a relatively hard texture, and the soaking time can be appropriately extended. Therefore, the best optimized decoction process was selected: add 10 times the water to the whole prescription and soak for 50 minutes, decoct Semen Cassiae for 40 minutes first, decoct the whole prescription 30 minutes after boiling, and add Uncaria rhynchophylla 15 minutes before the decoction is completed. Experiments have verified that the process is stable and feasible.

[0102] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for establishing a traditional Chinese medicine fingerprint of Tianma Uncaria Decoction, characterized in that: The following steps are involved: (1) Preparation of reference solution: accurately weigh gastrodin, gardenia glycoside, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin and wogonin reference substances, and add methanol to prepare a mixed reference solution; (2) Preparation of test solution: accurately measure 1 mL of Tianma Gouteng decoction and place it in a 2 mL volumetric flask, add methanol to make up to volume, mix well and perform ultrasonic-assisted extraction for 1 min, then centrifuge at 14,000 r / min for 10 min, take the supernatant, and filter through a 0.22 μm microporous membrane to obtain the test solution; (3) High performance liquid chromatography was used for detection to establish a fingerprint. In step (3), the conditions of the high performance liquid chromatography were as follows: a Waters ACQUITY UPLC HSS T3 column with specifications of 2.1 mm × 100 mm, 1.8 μm, and a flow rate of 0.2 mL / min; a column temperature of 35°C and an injection volume of 2 μL. Mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B) for gradient elution, and the gradient elution conditions are: 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 is used for measurement: 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 of the Tianma Uncaria Decoction according to claim 1, characterized in that: The concentrations of gastrodin, gardenia glycoside, pinoresinol diglucoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, baicalin, and wogonin in the mixed reference solution were 0.075 9, 0.920 0, 0.0541, 0.139 6, 2.125 5, and 0.955 5 mg / mL, respectively.

3. The method for establishing the traditional Chinese medicine fingerprint of the Tianma Uncaria Decoction according to claim 1, characterized in that: The preparation method of Tianma Uncaria Decoction is as follows: weigh 9g each of Tianma, Gardenia, Scutellaria, Eucommia, Leonurus, Viscum album, Polygonum multiflorum, Poria, 12g each of Uncaria and Cyathula, and 18g of Semen Cassiae, add water and boil twice, wherein Semen Cassiae is boiled for 40min first, and Uncaria is added 15min before the end of the second decoction. The first decoction is soaked for 40min, the decoction time is 30min after boiling, and the amount of water added is 10 times. The amount of water added to the second decoction is 4 times, and the decoction time is 20min after boiling. The decoctions of the two decoctions are combined and fixed to equal volume.

4. A fingerprint spectrum of Tianma Uncaria Decoction, characterized in that: It is constructed by detection according to the construction method of claim 1.

5. The fingerprint of Tianma Uncaria Decoction according to claim 4 is characterized in that: The six common peak components in the fingerprint are: No. 1 gastrodin; No. 6 gardenoside; No. 8 pinoresinol diglucoside; No. 9 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside; No. 11 baicalin; and No. 14 wogonin.

6. A method for testing the quality of Gastrodia elata and Uncaria rhynchophylla decoction, characterized in that: The sample to be tested of the Gastrodia elata and Uncaria rhynchophylla decoction is tested according to the construction method described in claim 1 to obtain a high performance liquid chromatography spectrum of the test sample of the Gastrodia elata and Uncaria rhynchophylla decoction, a fingerprint spectrum of the Gastrodia elata and Uncaria rhynchophylla decoction is constructed according to the construction method described in claim 1, and the high performance liquid chromatography spectrum of the sample to be tested of the Gastrodia elata and Uncaria rhynchophylla decoction is compared with the fingerprint spectrum of the Gastrodia elata and Uncaria rhynchophylla decoction.

Images