Eucommia ulmoides medicinal material quality comprehensive evaluation method based on UHPLC (Ultra High Performance Liquid Chromatography) and application

Abstract

The invention discloses a method for comprehensively evaluating the quality of a eucommia ulmoides medicinal material based on a UHPLC fingerprint spectrum and quantitative analysis and application of the method. By optimizing the chromatographic conditions, sample analysis of the eucommia ulmoides medicinal material or formula granules or extracts can be completed within 17 minutes, and the detection and analysis time of the eucommia ulmoides medicinal material is shortened by more than 100% compared with that reported in literatures. The established fingerprint spectrum contains 20 common peaks, synchronous quantitative determination of 6 index components is realized, and a multi-index quality control standard is provided for eucommia ulmoides medicinal materials or formula granules or extracts. Further, efficient source tracing of eucommia ulmoides in three producing areas, namely angleru in Wild Shaanxi, Chengdu in Sichuan and Enshi in Hubei can be realized by combining a fingerprint spectrum with orthogonal partial least squares discriminant analysis (OPLS-DA), and recipient operation characteristic curve analysis (ROC) shows that the discriminant preparation rates of the three producing areas can reach 100% accuracy; and a component-based production place discrimination method can be provided for the source tracing of the production place of eucommia ulmoides.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis technology, specifically involving a comprehensive quality evaluation method for Eucommia ulmoides based on ultra-high performance liquid chromatography (UHPLC) fingerprint spectroscopy, and its application in the quality control of Eucommia ulmoides raw materials, formulation granules, extracts, and the identification of the origin of medicinal materials based on components. Background Technology

[0002] Eucommia ulmoides Oliv., a traditional and precious Chinese medicinal herb, possesses the effects of tonifying the liver and kidneys, strengthening tendons and bones, and calming the fetus. Eucommia ulmoides and its processed products are widely used in clinical and health care fields. With the advancement of the modernization of traditional Chinese medicine, the market demand for Eucommia ulmoides raw materials, granules, and extracts is increasing. However, due to its complex chemical composition and the influence of factors such as origin and processing technology, its quality varies greatly, necessitating the establishment of scientific and comprehensive quality control methods. The quality evaluation of Eucommia ulmoides largely relies on the content determination of single components (such as pinoresinol diglucoside) or traditional identification methods, which are insufficient to comprehensively reflect its overall chemical characteristics and cannot effectively distinguish between different origins. Furthermore, existing analytical methods (such as HPLC) suffer from long analysis times and low separation efficiency, making it difficult to meet the needs of large-scale standardized production.

[0003] CN109596763B describes a method for constructing and identifying characteristic chromatograms of Eucommia ulmoides and Eucommia ulmoides with salt. However, under the chromatographic separation conditions, only 5 chromatographic peaks can be identified (3 chromatographic peaks can be identified), and multi-component quantitative analysis of Eucommia ulmoides is not achieved, resulting in a limited number of quality markers. At the same time, the fingerprint chromatogram provided by this method offers very few variables, making it difficult to conduct multivariate analysis effectively and failing to provide support for the identification of the place of origin.

[0004] Therefore, UHPLC quality evaluation methods for Du courier products that take into account both fingerprint spectroscopy and multi-index quantification still need to be developed, especially UHPLC component analysis methods that can identify the origin of Du courier products. Summary of the Invention

[0005] The purpose of this invention is to provide a comprehensive quality evaluation method for Eucommia ulmoides based on UHPLC fingerprinting and multi-component quantitative analysis, in order to solve the problems of existing technologies, such as long detection and analysis time, incomplete overall quality control, lack of unified quality evaluation standards for formulation granules and deep-processed products such as extracts, reliance on subjective experience or a small number of markers for traditional origin determination methods, and lack of objective analysis methods based on multi-component fingerprinting.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A comprehensive quality evaluation method for Eucommia ulmoides based on UHPLC fingerprinting and quantitative analysis includes the following steps:

[0008] Step 1: Preparation of reference solutions: Take appropriate amounts of genipin, chlorogenic acid, caffeic acid, geniposide, pinoresinol diglucoside, and ferulic acid reference standards, respectively, accurately weigh them, and add methanol to prepare reference solution stock solutions.

[0009] Step 2: Preparation of the test solution: Take Eucommia ulmoides medicinal materials from different origins, cut them into pieces, knead them into flocculent form, take 0.5g to 1.0g, weigh accurately, place them in a stoppered conical flask, accurately add 25mL of solvent, weigh, sonicate for 30min to 60min, take out, cool, weigh again, use the above solvent to make up the weight loss, shake well, filter, and take the filtrate to obtain the test solution;

[0010] Step 3: Ultra-high performance liquid chromatography (UHPLC) was used to detect the reference solution and the test solution respectively, and the fingerprint chromatograms of the reference solution and the test solution were recorded.

[0011] Step 4: Establish a control fingerprint spectrum: Import the fingerprint spectrum of the test solution in Step 3 into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) to generate a control fingerprint spectrum, and calculate the similarity between fingerprint spectra of Eucommia ulmoides from different origins and the similarity with the control fingerprint spectrum.

[0012] Step 5: Content determination of indicator components: Six marker components with a resolution Ri≥1.5 were selected, namely genipin, chlorogenic acid, caffeic acid, geniposide, pinoresinol diglucoside, and ferulic acid, and their contents were determined. Product quality control was carried out based on the content of each component.

[0013] Preferably, the solvent used in steps 1 and 2 is a 70% methanol solution, and the ultrasonic extraction in step 2 is performed with a power of 250W and a frequency of 40kHz.

[0014] Preferably, the concentrations of genipin, chlorogenic acid, caffeic acid, geniposide, pinoresinol diglucoside, and ferulic acid in the reference stock solution in step 1 are 196.2 μg / mL, 181.6 μg / mL, 128.6 μg / mL, 199.1 μg / mL, 575.2 μg / mL, and 149.1 μg / mL, respectively.

[0015] Preferably, the detection conditions for ultra-high performance liquid chromatography (UHPLC) in step 3 are as follows: the chromatographic column is a C18 small particle size column, the mobile phase is acetonitrile (A)-0.2% formic acid (B), and gradient elution is performed. The gradient elution program is: 0-4.5 min, 4% → 10.5% A; 4.5-6.5 min, 10.5% → 11% A; 6.5-14 min, 11% → 18% A; 14-17 min, 18% → 60% A; the flow rate of the mobile phase is 0.2-0.4 mL / min; the detection wavelength is 230 nm-280 nm; and the column temperature is 35-45 °C.

[0016] Preferably, in step 3, the fingerprint spectrum of the test solution was identified by chromatographic peak matching, resulting in 20 common peaks. Six chromatographic peaks were then identified by comparison with the reference standard: genipin (peak 3); chlorogenic acid (peak 11); caffeic acid (peak 12); geniposide (peak 15); pinoresinol diglucoside (peak 16); and ferulic acid (peak 18).

[0017] Preferably, the 20 common chromatographic peaks are used with the chromatographic peak of pinoresinol diglucoside (16) as the reference S peak. The relative retention times of other common chromatographic peaks are calculated, and the relative retention times of other characteristic peaks are specified to be within ±10% of a specified value. The specified values ​​for the relative retention times are: 0.24 (peak 1), 0.35 (peak 2), 0.36 (peak 3), 0.37 (peak 4), 0.40 (peak 5), 0.41 (peak 6), 0.44 (peak 7), 0.47 (peak 8), 0.49 (peak 9), 0.50 (peak 10), 0.56 (peak 11), 0.62 (peak 12), 0.70 (peak 13), 0.72 (peak 14), 0.78 (peak 15), 1.06 (peak 17), 1.10 (peak 18), 1.11 (peak 19), and 1.33 (peak 20). The similarity between the characteristic chromatogram of the sample to be tested and the reference fingerprint chromatogram is greater than 0.90.

[0018] The method for determining the content of the six indicator components in step 5 of this invention exhibits good linearity (R0). 2 The RSDs for precision, repeatability, and stability were all less than 2%, and the recovery rates were between 97.63% and 99.65%.

[0019] As another objective of this invention, it also provides the application of the aforementioned comprehensive evaluation method to the identification of the origin of Eucommia ulmoides medicinal materials. By combining fingerprint spectroscopy with orthogonal partial least squares discriminant analysis (OPLS-DA) and receiver operating characteristic (ROC) curve analysis, orthogonal partial least squares discriminant analysis (OPLS-DA) is performed on Eucommia ulmoides samples from different origins and 20 common peak components to identify the origin of Eucommia ulmoides. The model's discrimination accuracy can reach 100%.

[0020] The above evaluation methods can also be applied to the quality control of Eucommia ulmoides formula granules and extracts.

[0021] This invention utilizes UHPLC to provide a comprehensive quality evaluation method for Eucommia ulmoides that can achieve characteristic chromatograms and determination of multiple component contents under a single chromatographic condition, while also shortening the detection cycle. Furthermore, this method can be used for quality control of fingerprint chromatograms of Eucommia ulmoides medicinal materials, formulation granules, or extracts from different origins; it can also be used for high-precision origin identification of Eucommia ulmoides medicinal materials based on fingerprint chromatograms. This is of great significance for improving the development, quality control, evaluation, and origin determination of Eucommia ulmoides medicinal materials and their extracts as described in this invention.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) Analytical efficiency is significantly improved

[0024] Existing HPLC methods typically require more than 40 minutes to analyze Eucommia ulmoides components, while the optimized UHPLC method of this invention can complete sample analysis in just 17 minutes, significantly increasing detection throughput and making it more suitable for large-scale production quality control.

[0025] (2) Simultaneous quantitative analysis of multiple indicators enables more comprehensive quality control.

[0026] Traditional methods often only determine a single component, pinoresinol diglucoside. However, this invention can simultaneously determine six components: genipinic acid, chlorogenic acid, caffeic acid, geniposide, pinoresinol diglucoside, and ferulic acid. Combined with a fingerprint spectrum of 20 common peaks, it achieves dual quality control of "overall + characteristic components". Compared with existing fingerprint spectrum technology, it can simultaneously perform quantitative analysis on more components, and can more scientifically and comprehensively reflect the quality differences of Eucommia ulmoides medicinal materials while maintaining detection efficiency.

[0027] (3) Fingerprint spectroscopy combined with chemometrics enables precise identification of place of origin.

[0028] Existing technologies struggle to objectively distinguish Eucommia ulmoides from different origins. However, this invention, through fingerprinting and orthogonal partial least squares discriminant analysis (OPLS-DA), achieves 100% accurate identification of the three major producing areas: Shaanxi (Ankang), Sichuan (Chengdu), and Hubei (Enshi). This provides a reliable basis for tracing the origin of Eucommia ulmoides and assists in the identification and market supervision of authentic medicinal materials.

[0029] (4) The method has wide applicability and covers the entire industry chain.

[0030] Existing methods are mostly limited to the detection of medicinal materials, while this invention is applicable to various forms of Eucommia ulmoides medicinal materials and their processed products, formula granules and extracts, etc., meeting the quality control requirements of the whole process from raw materials to finished products, and has a high degree of standardization.

[0031] (5) Good reproducibility and strong stability

[0032] By systematically optimizing chromatographic conditions (such as mobile phase gradient, column temperature, detection wavelength, etc.), the method of this invention has good peak shape, high resolution, and excellent reproducibility, which meets the technical requirements of fingerprinting of traditional Chinese medicine and is suitable for promotion and application in different laboratories.

[0033] (6) Provide technical support for the modernization of traditional Chinese medicine

[0034] The comprehensive strategy of "rapid analysis + multi-index quantification + chemical pattern recognition" established in this invention is not only applicable to Eucommia ulmoides, but can also provide a reference for the quality evaluation and origin identification of other Chinese medicinal materials, and promote the internationalization of Chinese medicine quality standards.

[0035] In summary, this invention is significantly superior to existing technologies in terms of analysis speed, detection comprehensiveness, accuracy of origin identification, and method universality, providing an efficient and reliable technical means for the quality control and standardized production of Eucommia ulmoides and its related products. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a chromatogram of Eucommia ulmoides medicinal material and different reference standards in Example 1 of this application;

[0038] Figure 2 These are the UHPLC fingerprint chromatograms of 18 batches of Eucommia ulmoides medicinal materials from different origins in Example 2 of this application, with S1-S18 being the fingerprint chromatograms of batches of Eucommia ulmoides medicinal materials from S1 to S18 respectively.

[0039] Figure 3 The reference fingerprint spectrum R in Example 2 of this application is shown, wherein peak 3 is genipin glycoside, peak 11 is chlorogenic acid, peak 12 is caffeic acid, peak 15 is geniposide, peak 16 is pinoresinol diglucoside, and peak 18 is ferulic acid.

[0040] Figure 4 This is an OPLS-DA score chart of samples from different origins in Example 6 of this application;

[0041] Figure 5 Replacement verification diagrams of samples from different origins in Example 6 of this application;

[0042] Figure 6 VIP images of samples from different origins in Example 6 of this application;

[0043] Figure 7 ROC plot of the OPLS-DA model in Embodiment 6 of this application;

[0044] Figure 8 This is a chromatogram of Eucommia ulmoides and salt-treated Eucommia ulmoides samples (medicinal materials, formula granules, extracts) in Example 7 of this application;

[0045] Figure 9 This is a comparative diagram of the present application and Example 1. Detailed Implementation

[0046] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0047] Example 1: UHPLC fingerprinting method for detecting Eucommia ulmoides medicinal materials

[0048] Preparation of the test sample: Take Eucommia ulmoides medicinal materials from different origins, cut them into pieces, knead them into flocculent form, take about 0.5g, weigh accurately, place them in a stoppered conical flask, accurately add 25ml of 70% methanol, seal tightly, weigh, sonicate (power 250W, frequency 40kHz) for 30min, take out, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test sample.

[0049] Preparation of reference solutions: Take appropriate amounts of geniposide, chlorogenic acid, caffeic acid, geniposide, pinoresinol diglucoside, and ferulic acid reference standards, respectively, accurately weigh them, and add methanol to prepare reference solutions containing 196.2 μg of geniposide, 181.6 μg of chlorogenic acid, 128.6 μg of caffeic acid, 199.1 μg of geniposide, 575.2 μg of pinoresinol diglucoside, and 149.1 μg of ferulic acid per 1 mL.

[0050] An octadecylsilane-bonded silica gel column was used as the packed column; gradient elution was performed using acetonitrile (A)-0.2% formic acid aqueous solution (B) as the mobile phase: 0–4.5 min, 4% → 10.5% A; 4.5–6.5 min, 10.5% → 11% A; 6.5–14 min, 11% → 18% A; 14–17 min, 18% → 60% A; the mobile phase flow rate was 0.3 mL / min; the detection wavelength was 254 nm; and the column temperature was 40 °C. The theoretical plate number, calculated based on the pinoresinol diglucoside peak, should be no less than 10,000.

[0051] Accurately pipette 1–2 μl each of the reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatograms. Figure 1 As shown.

[0052] Example 2: Methodological Investigation of Eucommia ulmoides Fingerprint Spectroscopy

[0053] Precision: Take 0.5g of the same batch of Eucommia ulmoides sample (S1), accurately weigh it, prepare it according to the preparation method of the test solution under Example 1 and conduct chromatographic experiments, and continuously inject and determine it 6 times.

[0054] Repeatability: Take 0.5g of the same batch of Eucommia ulmoides sample (S1), accurately weigh it, and prepare 6 test solutions in parallel according to the preparation method of the test solution in Example 1. Determine the test solution under the same chromatographic conditions as described above.

[0055] Stability: Take 0.5g of the same batch of Eucommia ulmoides sample (S1), accurately weigh it, and conduct experiments according to the preparation of the test solution and chromatographic conditions and methods in Example 1. The sample is injected and measured after being placed for 0, 4, 8, 12, 16 and 22 hours after preparation.

[0056] The precision, repeatability, and stability methodological tests described above all used 20 common chromatographic peaks as representatives, with the retention time and peak area of ​​peak 16 (pinoresinol diglucoside) serving as controls. The RSD values ​​of the relative retention times (RRTs) and relative peak areas (RPAs) of other chromatographic peaks were used to evaluate the feasibility of the methodological experiments. The results show that the RRTs and RSD values ​​of all chromatographic peaks in the precision, repeatability, and stability tests were all less than 2%. This demonstrates that the method is stable and feasible and can be used for fingerprint analysis in this technical solution.

[0057] Example 3: Fingerprint analysis and common peak identification of 18 batches of Eucommia ulmoides medicinal materials

[0058] Eighteen batches of Eucommia ulmoides medicinal materials were collected and analyzed by UHPLC according to the preparation method and chromatographic conditions described in Example 1. Fingerprint chromatograms of different batches of Eucommia ulmoides medicinal materials were obtained. The chromatograms of the 18 batches of Eucommia ulmoides were imported into the 2012 version of the "Similarity Evaluation System for Fingerprint Spectrum of Traditional Chinese Medicine" software. UHPLC fingerprint chromatograms were established using the median method, generating a control fingerprint chromatogram (R) consisting of 20 common peaks. Figure 2-3As shown. Of the 20 common peaks, 6 were identified by comparison with the reference standard: genipin (peak 3); chlorogenic acid (peak 11); caffeic acid (peak 12); geniposide (peak 15); pinoresinol diglucoside (peak 16); and ferulic acid (peak 18). Peak 16, pinoresinol diglucoside, was designated as the reference peak S. The relative retention times of the other common peaks were calculated. The relative retention times of other characteristic peaks were specified to be within ±10% of a given value. The specified values ​​for relative retention times were: 0.24 (peak 1), 0... The values ​​are: 0.35 (peak 2), 0.36 (peak 3), 0.37 (peak 4), 0.40 (peak 5), 0.41 (peak 6), 0.44 (peak 7), 0.47 (peak 8), 0.49 (peak 9), 0.50 (peak 10), 0.56 (peak 11), 0.62 (peak 12), 0.70 (peak 13), 0.72 (peak 14), 0.78 (peak 15), 1.06 (peak 17), 1.10 (peak 18), 1.11 (peak 19), and 1.33 (peak 20). The origin information for the 18 batches of medicinal materials is shown in Table 1 below, the relative retention time is shown in Table 2 below, and the similarity matching results are shown in Table 3 below.

[0059] Table 1. Origin Information of 18 Batches of Eucommia ulmoides Medicinal Materials

[0060] batch number Origin batch number Origin S1 Zhaofei Town, Xunyang County, Ankang City, Shaanxi Province S10 Baifusi Town, Laifeng County, Enshi City, Hubei Province S2 Bailiu Town, Xunyang County, Ankang City, Shaanxi Province S11 Xiangfeng Town, Laifeng County, Enshi City, Hubei Province S3 Tongmu Town, Xunyang County, Ankang City, Shaanxi Province S12 Longping Town, Jianshi County, Enshi City, Hubei Province S4 Tongmu Town, Xunyang County, Ankang City, Shaanxi Province S13 Qingchengshan Town, Dujiangyan City, Chengdu, Sichuan Province S5 Shimen Town, Xunyang County, Ankang City, Shaanxi Province S14 Qingchengshan Town, Dujiangyan City, Chengdu, Sichuan Province S6 Bailiu Town, Xunyang County, Ankang City, Shaanxi Province S15 Puyang Town, Dujiangyan City, Chengdu, Sichuan Province S7 Chunmuying Town, Xuan'en County, Enshi City, Hubei Province S16 Qingchengshan Town, Dujiangyan City, Chengdu, Sichuan Province S8 Banqiao Town, Enshi City, Hubei Province S17 Qingchengshan Town, Dujiangyan City, Chengdu, Sichuan Province S9 Dahe Town, Laifeng County, Enshi City, Hubei Province S18 Puyang Town, Dujiangyan City, Chengdu, Sichuan Province

[0061]

[0062]

[0063]

[0064]

[0065] Example 4: Methodological Investigation of Content Determination of Six Indicator Components

[0066] Linearity: The reference stock solution from Example 1 was used to obtain six reference solutions of different concentrations using the serial dilution method. 2 μl of each of the six concentrations was injected into a UHPLC ultra-high performance liquid chromatograph, and the test was conducted according to the preparation and chromatographic conditions described in Example 1. Linear regression analysis was performed with the injection concentration as the abscissa (X) and the peak area integral as the ordinate (Y). The results showed that all six components exhibited good linearity within the investigated range, with correlation coefficients all above 0.9998. Detailed results are shown in Table 4 below.

[0067] Precision: Take 0.5g of Eucommia ulmoides sample (S1) from the same batch of medicinal materials, weigh accurately, prepare the test solution according to the preparation method and chromatographic conditions in Example 1, and perform continuous injection and determination 6 times.

[0068] Repeatability: Take 0.5g of the same batch of Eucommia ulmoides sample (S1), accurately weigh it, and prepare 6 test solutions in parallel according to the preparation method of the test solution in Example 1. Determine the test solution under the same chromatographic conditions as described above.

[0069] Stability: Take 0.5g of the same batch of Eucommia ulmoides sample (S1), accurately weigh it, and conduct experiments according to the preparation of the test solution and chromatographic conditions and methods in Example 1. The sample is injected and measured after being placed for 0, 4, 8, 12, 16 and 22 hours after preparation.

[0070] The RSD values ​​of the peak areas of the six components were used to evaluate the methodological feasibility, precision, repeatability, and stability. Experimental results show that the method is stable and feasible, and can be used for the determination of the contents of the six components in the technical solution of this invention. Detailed experimental results are shown in Table 4 below.

[0071] Accuracy: Six portions of Eucommia ulmoides sample (S1) with known content were accurately weighed. The reference solutions of the six components were added at a ratio of 1:1. The samples were then analyzed under ultra-high performance liquid chromatography (UHPLC) conditions. The average recovery rate and RSD of each component were calculated. The results are shown in Table 4.

[0072] Table 4. Results of the methodological study for the determination of the content of six components.

[0073]

[0074]

[0075] Example 5: Determination of the content of 6 components in 18 batches of samples

[0076] According to the established method, the contents of 6 components in 18 batches of samples were determined by the preparation method and chromatographic conditions of the test solution in Example 1. The results are shown in Table 5 below.

[0077] Table 5. Results of content determination of six components in 18 batches of samples

[0078]

[0079]

[0080] Example 6: A component-based method for determining the origin of Eucommia ulmoides

[0081] Orthogonal partial little squares discriminant analysis (OPLS-DA) was performed on 18 batches of Eucommia ulmoides and 20 components. The results showed that OPLS-DA can effectively distinguish Eucommia ulmoides samples from three producing areas: Shaanxi, Sichuan, and Hubei. The model R²X, R²Y, and Q² values ​​were satisfactory. 2 All values ​​are greater than 0.5, and the 200 permutation validation results also indicate that the model has not overfitted (e.g. Figure 4 , 5(As shown). Differential components from the three origins were screened using "VIP > 1", and the results were as follows: Peak 19, genipin, Peak 1, Peak 13, geniposide, Peak 6, Peak 8, Peak 4, Peak 17, Peak 7, chlorogenic acid, Peak 14, and caffeic acid. Simultaneously, the model's classification ability was evaluated using ROC curve analysis. The AUC of this OPLS-DA model was 1 for each origin (as shown). Figure 7 As shown in the figure, this indicates that the model has excellent origin discrimination performance and an accuracy of 100%.

[0082] Example 7: Quality control of Eucommia ulmoides samples, granules, or salt-processed Eucommia ulmoides slices and granules

[0083] Salt-processed Eucommia ulmoides slices: Eucommia ulmoides raw materials are processed according to the processing method of salt-processed Eucommia ulmoides in the current edition of the Chinese Pharmacopoeia to obtain salt-processed Eucommia ulmoides slices.

[0084] Preparation of Eucommia ulmoides and salt-treated Eucommia ulmoides granules: Take 50-200g of Eucommia ulmoides and salt-treated Eucommia ulmoides slices, decoct 2-3 times, add 8-10 times the amount of water each time, decoct for 20-30 minutes, soak for 30 minutes the first time, filter, combine the filtrates, concentrate, add appropriate excipients, dry, add appropriate excipients again, granulate, and the product is obtained.

[0085] Preparation of Eucommia ulmoides and salt-treated Eucommia ulmoides extract: Take 50-100g of Eucommia ulmoides and salt-treated Eucommia ulmoides slices, add 3-6 times 30%-70% ethanol, reflux extract 1-3 times, 30-60 minutes each time, filter, combine the filtrates, concentrate and dry to obtain the extract.

[0086] Take 0.5-1.0g of Eucommia ulmoides, salt-processed Eucommia ulmoides slices, formula granules, and extract, accurately weigh them, place them in a stoppered conical flask, accurately add 25mL of 70% methanol, seal tightly, weigh, sonicate (power 250W, frequency 40kHz) for 30min, remove, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0087] According to the chromatographic conditions and methods described in Example 1, fingerprint chromatograms of Eucommia ulmoides raw material, formulated granules, extract, and salt-processed Eucommia ulmoides slices, formulated granules, and extract were determined. The results showed that the relative retention times of the common peaks of Eucommia ulmoides, salt-processed Eucommia ulmoides formulated granules, and extract were consistent with those of Eucommia ulmoides raw material, with RSD ≤ 1%. The chromatographic peak resolution Ri was ≥ 1.5 for all peaks. The methodological validation of the fingerprint chromatograms and quantitative analysis met the analytical requirements. This indicates that the method has strong applicability in the quality control of raw materials and formulated granules (e.g., ...). Figure 8 (As shown).

[0088] Comparative Example 1

[0089] This application provides a comparative example of a fingerprint spectrum of Eucommia ulmoides and a method for establishing its content.

[0090] Accurately weigh 0.5g of Eucommia ulmoides material from Example 1, place it in a stoppered conical flask, accurately add 25mL of 10% methanol, seal tightly, weigh, and sonicate (250W, 40kHz) for 30min. Remove, cool, and weigh again. Replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the test sample. Detect according to the chromatographic conditions of Example 1. The obtained chromatogram is shown below. Figure 7 As shown.

[0091] The results showed that the peak signals of the chromatograms obtained under these chromatographic conditions were worse than those in Example 1, and the number of peaks was less than that in Example 1. Therefore, it was impossible to achieve the analysis and quality control of Eucommia ulmoides samples based on these chromatographic conditions.

[0092] Comparative Example 2

[0093] This application provides a comparative example of a method for establishing a fingerprint spectrum of Eucommia ulmoides medicinal material.

[0094] The Eucommia ulmoides samples prepared in Example 1 were subjected to UHPLC analysis. The wavelength of the UV detector was set to 280 nm, and a C18 high-pressure resistant small-particle-size column was selected. The column temperature during detection was 40 °C, and the flow rate was 0.4 mL / min. Acetonitrile (A) and 0.2% formic acid (B) were used as the mobile phase, and the elution was performed as follows: 0–4.5 min, 4% → 10.5% A; 4.5–6.5 min, 10.5% → 11% A; 6.5–14 min, 11% → 18% A; 14–17 min, 18% → 60% A. The obtained chromatograms are shown below. Figure 9 As shown.

[0095] The results showed that the intensity and abundance of the chromatographic peaks obtained under these chromatographic conditions were worse than those in Example 1, and the analysis and quality control of Eucommia ulmoides samples could not be achieved based on the chromatographic conditions.

[0096] The application of a comprehensive quality evaluation method for Eucommia ulmoides based on ultra-high performance liquid chromatography (UHPLC) fingerprinting and quantitative analysis can provide multi-index quality control standards for Eucommia ulmoides raw materials, formulation granules, or extracts. At the same time, by combining fingerprinting with orthogonal partial least squares discriminant analysis (OPLS-DA), the origin of Eucommia ulmoides from three producing areas can be determined with 100% accuracy, providing a component-based origin determination method for tracing the origin of Eucommia ulmoides.

[0097] The above description is merely a specific embodiment of this application and is not intended to limit this application. For those skilled in the art, this application can have various changes and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A comprehensive quality evaluation method for Eucommia ulmoides medicinal materials based on UHPLC fingerprinting and quantitative analysis, comprising the following steps: Step 1: Preparation of reference solutions: Take appropriate amounts of genipin, chlorogenic acid, caffeic acid, geniposide, pinoresinol diglucoside, and ferulic acid reference standards, respectively, accurately weigh them, and add methanol to prepare reference solution stock solutions. Step 2: Preparation of the test solution: Take Eucommia ulmoides medicinal materials from different origins, cut them into pieces, knead them into flocculent form, take 0.5g to 1.0g, weigh accurately, place them in a stoppered conical flask, accurately add 25mL of solvent, weigh, sonicate for 30min to 60min, take out, cool, weigh again, use the above solvent to make up the weight loss, shake well, filter, and take the filtrate to obtain the test solution; Step 3: Ultra-high performance liquid chromatography (UHPLC) was used to detect the reference solution and the test solution respectively, and the fingerprint chromatograms of the reference solution and the test solution were recorded. Step 4: Establish a control fingerprint spectrum: Import the fingerprint spectrum of the test solution in Step 3 into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) to generate a control fingerprint spectrum, and calculate the similarity between fingerprint spectra of Eucommia ulmoides from different origins and the similarity with the control fingerprint spectrum. Step 5: Content determination of indicator components: Six marker components with a resolution Ri≥1.5 were selected, namely genipin, chlorogenic acid, caffeic acid, geniposide, pinoresinol diglucoside, and ferulic acid, and their contents were determined. Product quality control was carried out based on the content of each component.

2. The method for comprehensive quality evaluation of Eucommia ulmoides based on UHPLC according to claim 1, characterized in that: The solvent used in steps 1 and 2 is a 70% methanol solution, and the ultrasonic extraction in step 2 is performed at a power of 250W and a frequency of 40kHz.

3. The method for comprehensive quality evaluation of Eucommia ulmoides based on UHPLC according to claim 1, characterized in that: The concentrations of genipin, chlorogenic acid, caffeic acid, geniposide, pinoresinol diglucoside, and ferulic acid in the reference stock solution described in step 1 were 196.2, 181.6, 128.6, 199.1, 575.2, and 149.1 μg / mL, respectively.

4. The method for comprehensive quality evaluation of Eucommia ulmoides based on UHPLC fingerprint spectroscopy according to claim 1, characterized in that: The detection conditions for ultra-high performance liquid chromatography (UHPLC) in step 3 are as follows: the chromatographic column is a C18 small particle size column; the mobile phase is acetonitrile (A)-0.2% formic acid (B); gradient elution is performed with the following gradient elution program: 0–4.5 min, 4% → 10.5% A; 4.5–6.5 min, 10.5% → 11% A; 6.5–14 min, 11% → 18% A; 14–17 min, 18% → 60% A; the flow rate of the mobile phase is 0.2–0.4 mL / min; the detection wavelength is 230 nm–280 nm; and the column temperature is 35–45 °C.

5. The comprehensive quality evaluation method for Eucommia ulmoides based on UHPLC according to claim 1, characterized in that: In step 3, the fingerprint spectrum of the test solution was identified by chromatographic peak matching, and 6 chromatographic peaks were identified by comparison with the reference standard, namely: genipin (peak 3); chlorogenic acid (peak 11); caffeic acid (peak 12); geniposide (peak 15); pinoresinol diglucoside (peak 16); ferulic acid (peak 18).

6. The comprehensive quality evaluation method for Eucommia ulmoides based on UHPLC according to claim 1, characterized in that: The 20 common chromatographic peaks were used with peak 16 (pinoresinol diglucoside) as a reference S peak. The relative retention times of other common chromatographic peaks were calculated, and the relative retention times of other characteristic peaks were specified to be within ±10% of a specified value. The specified value for relative retention time is as follows: The values ​​were 0.24 (peak 1), 0.35 (peak 2), 0.36 (peak 3), 0.37 (peak 4), 0.40 (peak 5), 0.41 (peak 6), 0.44 (peak 7), 0.47 (peak 8), 0.49 (peak 9), 0.50 (peak 10), 0.56 (peak 11), 0.62 (peak 12), 0.70 (peak 13), 0.72 (peak 14), 0.78 (peak 15), 1.06 (peak 17), 1.10 (peak 18), 1.11 (peak 19), and 1.33 (peak 20), and the similarity between the characteristic spectrum of the sample to be tested and the control fingerprint spectrum was greater than 0.

90.

7. The method for comprehensive quality evaluation of Eucommia ulmoides based on UHPLC fingerprint spectroscopy according to claim 1, characterized in that: The method for determining the content of the six index components in step 5 showed good linearity (R≥0.9998), with RSDs of precision, repeatability, and stability all less than 2%, and recovery rates between 97.63% and 99.65%.

8. The method according to any one of claims 1-7 is used for the identification of the origin of Eucommia ulmoides medicinal materials, characterized in that: By combining fingerprint spectroscopy with orthogonal partial least squares discriminant analysis and receiver operating characteristic (ROC) analysis, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed on Eucommia ulmoides samples from different origins and 20 common peak components to identify the origin of Eucommia ulmoides.

Images