Establishment of Fingerprint Chromatogram of Chao Medicine Fang Liangyi Decoction and Its Fingerprint Chromatogram

Abstract

The application discloses a method for establishing a fingerprint spectrum of a Chao medicine recipe Liangyi decoction, and comprises the following steps: taking 5-HMF, protocatechuic acid, morroniside, loganin, oroxindin and verbascoside reference substances, dissolving the reference substances in chromatographic methanol, and fixing the volume to prepare a reference solution; taking rehmannia and cornus officinalis medicine, extracting the medicine with an aqueous solution to obtain a Liangyi decoction freeze-dried powder, dissolving the freeze-dried powder in chromatographic methanol, and fixing the volume to prepare a test solution; performing high-performance liquid separation detection, using a 0.15% formic acid solution as phase A and a 100% acetonitrile solution as phase B to form a mobile phase, performing gradient elution, and using relative retention time and relative peak area to calibrate fingerprint characteristics, so that the fingerprint spectrum is obtained. The application can provide a reliable basis for identification and quality control of the Chao medicine recipe Liangyi decoction.

Description

Technical Field

[0001] This invention relates to the field of quality control of traditional Chinese medicine prescriptions, specifically to a method for establishing the fingerprint spectrum of the Korean medicine prescription Liangyi Decoction and its fingerprint spectrum. Background Technology

[0002] The Liangyi Decoction is recorded in ancient Korean medical books such as the New Dongbo Yijian and the Chronicle of Korean Medicine, and is a traditional Korean medical prescription. It consists of Rehmannia glutinosa (processed) and Cornus officinalis in a 2:1 ratio. According to Korean medical theory, the combination of Rehmannia glutinosa and Cornus officinalis has the function of tonifying the kidney and replenishing essence, and is used to treat penile pain. The whole prescription reflects the principle of supporting the body's resistance and eliminating pathogenic factors, addressing both the root cause and the symptoms.

[0003] Fingerprint spectroscopy, as an effective means of controlling the quality of traditional Chinese medicine, can comprehensively reflect the intrinsic chemical characteristics of traditional Chinese medicine or its preparations. It has the characteristics of wholeness and fuzziness. Through chemical pattern recognition techniques such as cluster analysis, principal component analysis, and orthogonal partial least squares-discriminant analysis, it can identify the complex component information of classic prescriptions and their differences in drug quality, and can be used to evaluate the overall quality of prescriptions.

[0004] To date, there are few reports on the traditional Korean medicine formula Liangyi Decoction. This study established an HPLC fingerprint of Liangyi Decoction, evaluated its similarity, assigned common peaks and identified indicator components, determined the consistency of indicator components between batches, and evaluated the integrity, complexity and quality differences of the chemical components of Liangyi Decoction through chemical pattern recognition, providing a basis for the overall quality research of Liangyi Decoction. Summary of the Invention

[0005] This invention designs and develops a method for establishing the fingerprint spectrum of the Korean medicine formula Liangyi Decoction. The purpose of this invention is to provide a reliable basis for the identification and quality control of Liangyi Decoction through the establishment of the high-performance liquid chromatography fingerprint spectrum of Liangyi Decoction.

[0006] This invention designs and develops a method for establishing the fingerprint spectrum of the Korean medicine formula Liangyi Decoction. The purpose of this invention is to provide a reliable basis for the identification and quality control of Liangyi Decoction through the obtained high-performance liquid chromatography fingerprint spectrum.

[0007] The technical solution provided by this invention is as follows:

[0008] The method for establishing the fingerprint spectrum of the Korean medical formula Liangyi Decoction includes the following steps:

[0009] Take 5-HMF, protocatechuic acid, monoglucoside, loganin, scutellarin, and verbascoside as reference standards, dissolve them in chromatographic methanol, and dilute to volume to prepare the reference solution.

[0010] Prepared Rehmannia glutinosa and Cornus officinalis were extracted with aqueous solution to obtain the freeze-dried powder of Liangyi Decoction. The powder was then dissolved and diluted with chromatographic methanol to obtain the test solution.

[0011] The fingerprint spectrum was obtained by high performance liquid chromatography (HPLC) with a mobile phase consisting of 0.15% formic acid solution (phase A) and 100% acetonitrile solution (phase B). Gradient elution was used, and the fingerprint characteristics were calibrated by relative retention time and relative peak area.

[0012] Preferably, the high-performance liquid chromatography (HPLC) separation and detection conditions are as follows: using a chromatographic column with Promosil C20 ... 18 The stationary phase was used, the column temperature was 25℃, the detection wavelength was 260nm, the flow rate was 0.6mL / min, the analysis time was 80min, and the injection volume was 10μL into the high performance liquid chromatograph.

[0013] Preferably, the gradient elution procedure is as follows:

[0014] At 0 minutes, mobile phase A was a 95% formic acid solution and mobile phase B was a 5% acetonitrile solution;

[0015] At 20 minutes, mobile phase A was a 91% formic acid solution and mobile phase B was a 9% acetonitrile solution.

[0016] At 35 minutes, mobile phase A was an 85.5% formic acid solution, and mobile phase B was a 14.5% acetonitrile solution.

[0017] At 50 minutes, mobile phase A was an 82.5% formic acid solution, and mobile phase B was a 17.5% acetonitrile solution.

[0018] At 62 minutes, mobile phase A was a 77% formic acid solution and mobile phase B was a 23% acetonitrile solution;

[0019] At 67 minutes, mobile phase A was a 77% formic acid solution and mobile phase B was a 23% acetonitrile solution;

[0020] At 77 minutes, mobile phase A was a 71% methanol solution and mobile phase B was a 29% acetonitrile solution.

[0021] At 80 minutes, mobile phase A was a 5% formic acid solution and mobile phase B was a 95% acetonitrile solution.

[0022] Column temperature: 25℃; flow rate: 0.6 mL / min; injection volume: 10 μL.

[0023] Preferably, the preparation process of the test solution includes: accurately weighing 40.0g of Rehmannia glutinosa and 20.0g of Cornus officinalis, adding 8 times the amount of water, soaking for 30min, decocting twice, 1.5h each time, filtering while hot through a 200-mesh sieve, combining the filtrates, concentrating, and then freeze-drying to obtain the two-ingredient decoction lyophilized powder. Accurately weighing about 1.0g of the two-ingredient decoction lyophilized powder, making up to 20mL in a volumetric flask with chromatographic methanol, sonicating for 30min, allowing to stand, adding chromatographic methanol solution to the mark, mixing well, allowing to stand, and filtering through a 0.22μm microporous membrane to obtain the test sample.

[0024] Preferably, the preparation process of the reference solution includes: accurately weighing 2.1 mg, 0.5 mg, 4.6 mg, 2.7 mg, 1.9 mg, and 1.1 mg of 5-HMF, protocatechuic acid, monoglucoside, loganin, strychnoside, and verbascoside reference standards, respectively, placing them in a 10 mL volumetric flask, adding chromatographic methanol solution, shaking well to dissolve, and making up to volume to prepare mixed reference solutions with concentrations of 210 μg / mL, 50 μg / mL, 460 μg / mL, 270 μg / mL, 190 μg / mL, and 110 μg / mL, respectively;

[0025] Accurately transfer 2.5 mL of the prepared mixed reference solution of 5-HMF, protocatechuic acid, monoglucoside, loganin, strychnine, and verbascoside into a 10 mL volumetric flask, add chromatographic methanol solution, shake well to dissolve, and dilute to volume to prepare mixed reference solutions with concentrations of 52.5 μg / mL, 12.5 μg / mL, 115.0 μg / mL, 67.5 μg / mL, 47.5 μg / mL, and 27.5 μg / mL, respectively.

[0026] Preferably, the fingerprint spectrum contains 19 common fingerprint peaks, with the following relative retention times:

[0027] Peak 1: 0.368–0.370; Peak 2: 0.512–0.514; Peak 3: 0.553–0.556; Peak 4: 0.609–0.613; Peak 5: 0.657–0.662; Peak 6: 0.710–0.712; Peak 7: 0.759–0.760; Peak 8: 0.830–0.832; Peak 9: 0.854–0.857; Peak 10: 0.875–0. 0.879; Peak 11: 0.920~0.923; Peak 12 (reference peak): 1.000; Peak 13: 1.021~1.022; Peak 14: 1.107~1.108; Peak 15: 1.265~1.266; Peak 16: 1.469~1.471; Peak 17: 1.526~1.531; Peak 18: 1.570~1.573; Peak 19: 1.609~1.613.

[0028] Preferably, the fingerprint spectrum contains 19 common fingerprint peaks, with the following relative retention times:

[0029] Peak 1: 0.370; Peak 2: 0.513; Peak 3: 0.554; Peak 4: 0.611; Peak 5: 0.659; Peak 6: 0.712; Peak 7: 0.760; Peak 8: 0.832; Peak 9: 0.856; Peak 10: 0.877; Peak 11: 0.922; Peak 12 (reference peak): 1.000; Peak 13: 1.021; Peak 14: 1.107; Peak 15: 1.265; Peak 16: 1.470; Peak 17: 1.529; Peak 18: 1.571; Peak 19: 1.610.

[0030] The fingerprint spectrum of the Korean medicine formula Liangyi Decoction was obtained by extracting Rehmannia glutinosa and Cornus officinalis into a methanol solution, followed by high performance liquid chromatography (HPLC) separation and detection.

[0031] Preferably, the high-performance liquid chromatography (HPLC) separation and detection conditions are as follows: using a chromatographic column with Promosil C20 ... 18 The stationary phase was used, the column temperature was 25℃, the detection wavelength was 260nm, the flow rate was 0.6mL / min, the analysis time was 80min, and a 10μL injection volume was used in the high-performance liquid chromatograph.

[0032] The gradient elution procedure is as follows:

[0033] At 0 minutes, mobile phase A was a 95% formic acid solution and mobile phase B was a 5% acetonitrile solution;

[0034] At 20 minutes, mobile phase A was a 91% formic acid solution and mobile phase B was a 9% acetonitrile solution.

[0035] At 35 minutes, mobile phase A was an 85.5% acetic acid solution, and mobile phase B was a 14.5% acetonitrile solution.

[0036] At 50 minutes, mobile phase A was an 82.5% acetic acid solution, and mobile phase B was a 17.5% acetonitrile solution.

[0037] At 62 minutes, mobile phase A was a 77% acetic acid solution and mobile phase B was a 23% acetonitrile solution;

[0038] At 67 minutes, mobile phase A was a 77% formic acid solution and mobile phase B was a 23% acetonitrile solution;

[0039] At 77 minutes, mobile phase A was a 71% methanol solution and mobile phase B was a 29% acetonitrile solution.

[0040] At 80 minutes, mobile phase A was a 5% formic acid solution and mobile phase B was a 95% acetonitrile solution.

[0041] Column temperature: 25℃; flow rate: 0.6 mL / min; injection volume: 10 μL.

[0042] Preferably, the fingerprint spectrum contains 19 common fingerprint peaks, with the following relative retention times:

[0043] Peak 1: 0.368–0.370; Peak 2: 0.512–0.514; Peak 3: 0.553–0.556; Peak 4: 0.609–0.613; Peak 5: 0.657–0.662; Peak 6: 0.710–0.712; Peak 7: 0.759–0.760; Peak 8: 0.830–0.832; Peak 9: 0.854–0.857; Peak 10: 0.875–0. 0.879; Peak 11: 0.920~0.923; Peak 12 (reference peak): 1.000; Peak 13: 1.021~1.022; Peak 14: 1.107~1.108; Peak 15: 1.265~1.266; Peak 16: 1.469~1.471; Peak 17: 1.526~1.531; Peak 18: 1.570~1.573; Peak 19: 1.609~1.613.

[0044] The beneficial effects of this invention compared to existing technologies are as follows: This invention established HPLC fingerprint chromatograms for 10 different batches of Liangyi Decoction, identified 19 common chromatographic peaks, and demonstrated that the peak groupings of the chromatograms from the 10 batches of Liangyi Decoction were basically consistent. Furthermore, chemical pattern recognition methods such as similarity evaluation, cluster analysis, principal component analysis, and orthogonal partial least squares-discriminant analysis were used to screen out the main marker components causing differences in the composition of different batches of Liangyi Decoction, providing a scientific basis for the quality control of Liangyi Decoction. This method is reliable, simple, and can serve as an effective method for the identification and quality control of Liangyi Decoction. Attached Figure Description

[0045] Figure 1 This is a full-wavelength scan 3D chromatogram of the test sample described in this invention.

[0046] Figure 2 The images show HPLC chromatograms of the Korean medicine formula Liangyi Decoction under different detection wavelengths as described in this invention.

[0047] Figure 3a This is the chromatogram of the test sample described in this invention.

[0048] Figure 3b This is the chromatogram of the mixed reference standard described in this invention.

[0049] Figure 4 This is a fingerprint map of the common pattern described in this invention.

[0050] Figure 5 These are the chromatograms of the 10 batches of test samples described in this invention. (S1~S10: HPLC fingerprint chromatograms of 10 batches of the Chaoyi Fang Liangyi Decoction; R: control HPLC fingerprint chromatogram)

[0051] Figure 6 This is the chromatographic peak assignment diagram of the present invention. (A is the Liangyi Decoction test sample; B is the Rehmannia glutinosa negative sample; C is the Cornus officinalis negative sample).

[0052] Figure 7 This is a schematic diagram of cluster analysis of 10 batches of the Korean medicine formula Liangyi Decoction described in this invention.

[0053] Figure 8 This is the principal component fragmentation diagram described in this invention.

[0054] Figure 9 This is the 3D-PCA score chart of the principal component analysis described in this invention.

[0055] Figure 10 This is an OPLS-DA score chart of 10 batches of the Korean medicine formula Liangyi Decoction described in this invention.

[0056] Figure 11 The VIP value is the orthogonal partial least squares discriminant analysis of the sample of the Korean medicine formula Liangyi Decoction described in this invention. Detailed Implementation

[0057] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0058] like Figures 1-11 As shown, the present invention provides a method for establishing the fingerprint spectrum of the Korean medicine formula Liangyi Decoction, which includes the following steps: taking 5-HMF, protocatechuic acid, monoglucoside, loganin, dangyao glycoside, and verbascoside reference standards, dissolving them in chromatographic methanol, and making up to volume to obtain the reference solution.

[0059] Prepared Rehmannia glutinosa and Cornus officinalis were extracted with aqueous solution to obtain the freeze-dried powder of Liangyi Decoction. The powder was then dissolved and diluted with chromatographic methanol to obtain the test solution.

[0060] The fingerprint spectrum was obtained by high performance liquid chromatography separation and detection, using a mobile phase consisting of 0.15% formic acid solution (phase A) and 100% acetonitrile solution (phase B) and gradient elution. The fingerprint characteristics were calibrated by relative retention time and relative peak area.

[0061] In another embodiment, the high-performance liquid chromatography (HPLC) injection and detection conditions are as follows: a chromatographic column is used with Promosil C20 ... 18The stationary phase was used, the column temperature was 25℃, the detection wavelength was 260nm, the flow rate was 0.6mL / min, the analysis time was 80min, and the injection volume was 10μL into the high performance liquid chromatograph.

[0062] In another embodiment, the gradient elution procedure is as follows:

[0063] At 0 minutes, mobile phase A was a 95% formic acid solution and mobile phase B was a 5% acetonitrile solution;

[0064] At 20 minutes, mobile phase A was a 91% formic acid solution and mobile phase B was a 9% acetonitrile solution.

[0065] At 35 minutes, mobile phase A was an 85.5% formic acid solution, and mobile phase B was a 14.5% acetonitrile solution.

[0066] At 50 minutes, mobile phase A was an 82.5% formic acid solution, and mobile phase B was a 17.5% acetonitrile solution.

[0067] At 62 minutes, mobile phase A was a 77% formic acid solution and mobile phase B was a 23% acetonitrile solution;

[0068] At 67 minutes, mobile phase A was a 77% formic acid solution and mobile phase B was a 23% acetonitrile solution;

[0069] At 77 minutes, mobile phase A was a 71% methanol solution and mobile phase B was a 29% acetonitrile solution.

[0070] At 80 minutes, mobile phase A was a 5% formic acid solution and mobile phase B was a 95% acetonitrile solution.

[0071] Column temperature: 25℃; flow rate: 0.6 mL / min; injection volume: 10 μL.

[0072] In another embodiment, the preparation process of the test solution includes: accurately weighing 40.0g of Rehmannia glutinosa and 20.0g of Cornus officinalis, adding 8 times the amount of water, soaking for 30min, decocting twice, 1.5h each time, filtering while hot through a 200-mesh sieve, combining the filtrates, concentrating, and then freeze-drying to obtain the freeze-dried powder of Liangyi Decoction. Accurately weighing about 1.0g of the dried powder of Liangyi Decoction, making up to 20mL in a volumetric flask with chromatographic methanol, sonicating for 30min, allowing to stand, adding chromatographic methanol solution to the mark, mixing well, allowing to stand, and filtering through a 0.22μm microporous membrane to obtain the test sample.

[0073] In another embodiment, the preparation process of the reference solution includes: accurately weighing 2.1 mg, 0.5 mg, 4.6 mg, 2.7 mg, 1.9 mg, and 1.1 mg of 5-HMF, protocatechuic acid, monoglucoside, loganin, strychnoside, and verbascoside reference standards, respectively, placing them in a 10 mL volumetric flask, adding chromatographic methanol solution, shaking well to dissolve, and making up to volume to prepare mixed reference solutions with concentrations of 210 μg / mL, 50 μg / mL, 460 μg / mL, 270 μg / mL, 190 μg / mL, and 110 μg / mL, respectively;

[0074] Accurately transfer 2.5 mL of the prepared mixed reference solution of 5-HMF, protocatechuic acid, monoglucoside, loganin, strychnine, and verbascoside into a 10 mL volumetric flask, add chromatographic methanol solution, shake well to dissolve, and dilute to volume to prepare mixed reference solutions with concentrations of 52.5 μg / mL, 12.5 μg / mL, 115.0 μg / mL, 67.5 μg / mL, 47.5 μg / mL, and 27.5 μg / mL, respectively.

[0075] In another embodiment, the fingerprint spectrum contains 19 common fingerprint peaks, with the following relative retention times:

[0076] Peak 1: 0.368–0.370; Peak 2: 0.512–0.514; Peak 3: 0.553–0.556; Peak 4: 0.609–0.613; Peak 5: 0.657–0.662; Peak 6: 0.710–0.712; Peak 7: 0.759–0.760; Peak 8: 0.830–0.832; Peak 9: 0.854–0.857; Peak 10: 0.875–0. 0.879; Peak 11: 0.920~0.923; Peak 12 (reference peak): 1.000; Peak 13: 1.021~1.022; Peak 14: 1.107~1.108; Peak 15: 1.265~1.266; Peak 16: 1.469~1.471; Peak 17: 1.526~1.531; Peak 18: 1.570~1.573; Peak 19: 1.609~1.613.

[0077] In another embodiment, the fingerprint spectrum contains 19 common fingerprint peaks, with the following relative retention times:

[0078] Peak 1: 0.370; Peak 2: 0.513; Peak 3: 0.554; Peak 4: 0.611; Peak 5: 0.659; Peak 6: 0.712; Peak 7: 0.760; Peak 8: 0.832; Peak 9: 0.856; Peak 10: 0.877; Peak 11: 0.922; Peak 12 (reference peak): 1.000; Peak 13: 1.021; Peak 14: 1.107; Peak 15: 1.265; Peak 16: 1.470; Peak 17: 1.529; Peak 18: 1.571; Peak 19: 1.610.

[0079] This invention also provides a fingerprint spectrum of the Korean medicine formula Liangyi Decoction. After extracting Rehmannia glutinosa and Cornus officinalis into water, the solution is prepared into a methanol solution and then subjected to high performance liquid chromatography separation and detection to obtain the fingerprint spectrum of the Korean medicine formula Liangyi Decoction.

[0080] In another embodiment, the high-performance liquid chromatography (HPLC) separation and detection conditions are as follows: a chromatographic column is used with Promosil C20 ... 18 The stationary phase was used, the column temperature was 25℃, the detection wavelength was 260nm, the flow rate was 0.6mL / min, the analysis time was 80min, and the injection volume was 10μL into the high performance liquid chromatograph.

[0081] The gradient elution procedure is as follows:

[0082] At 0 minutes, mobile phase A was a 95% formic acid solution and mobile phase B was a 5% acetonitrile solution;

[0083] At 20 minutes, mobile phase A was a 91% formic acid solution and mobile phase B was a 9% acetonitrile solution.

[0084] At 35 minutes, mobile phase A was an 85.5% acetic acid solution, and mobile phase B was a 14.5% acetonitrile solution.

[0085] At 50 minutes, mobile phase A was an 82.5% acetic acid solution, and mobile phase B was a 17.5% acetonitrile solution.

[0086] At 62 minutes, mobile phase A was a 77% acetic acid solution and mobile phase B was a 23% acetonitrile solution;

[0087] At 67 minutes, mobile phase A was a 77% formic acid solution and mobile phase B was a 23% acetonitrile solution;

[0088] At 77 minutes, mobile phase A was a 71% methanol solution and mobile phase B was a 29% acetonitrile solution.

[0089] At 80 minutes, mobile phase A was a 5% formic acid solution and mobile phase B was a 95% acetonitrile solution.

[0090] Column temperature: 25℃; flow rate: 0.6 mL / min; injection volume: 10 μL.

[0091] Example

[0092] I. Instruments and Materials

[0093] 1. Instruments

[0094] The instruments used in the experiment are shown in Table 1.

[0095] Table 1 Experimental Instruments

[0096]

[0097] 2. Reagents and reagents

[0098] The drugs and reagents used in the experiment are shown in Tables 2 and 3. All medicinal materials used in the experiment were purchased from pharmacies in major hospitals in Yanbian Prefecture. Associate Professor Zheng Mingshan of Yanbian University identified the processed Rehmannia glutinosa root as the dried tuberous root of Rehmannia glutinosa Libosch. (family Scrophulariaceae). Cornus officinalis was identified as the dried, mature fruit pulp of Cornus officinalis Sieb. et Zucc. (family Cornaceae). All the above medicinal materials underwent quality testing according to the methods specified in the 2020 edition of the Chinese Pharmacopoeia and met the requirements.

[0099] Table 2 Experimental Reagents

[0100]

[0101]

[0102] Table 3. Origin and serial number of 10 batches of Liangyi Decoction Pieces

[0103]

[0104] II. Preparation of the test solution

[0105] Accurately weigh 40.0g of Rehmannia glutinosa (processed) and 20.0g of Cornus officinalis, add 8 times the amount of water, soak for 30 minutes, decoct twice, 1.5 hours each time, filter while hot through a 200-mesh sieve, combine the filtrates, concentrate, and then freeze-dry to obtain the freeze-dried powder of the two herbs. Accurately weigh about 1.0g of the freeze-dried powder of the two herbs, dilute to 20mL in a volumetric flask with chromatographic methanol, sonicate for 30 minutes (250W, 40kHz), let stand, add chromatographic methanol solution to the mark, mix well, let stand, and filter through a 0.22μm microporous membrane to obtain the final product.

[0106] III. Preparation of Negative Sample Solutions

[0107] Accurately weigh the prepared Rehmannia glutinosa slices and Cornus officinalis slices respectively, and prepare negative sample solutions of Rehmannia glutinosa and Cornus officinalis respectively according to the method under "Preparation of Test Solution".

[0108] IV. Preparation of Reference Solution

[0109] Accurately weigh 2.1 mg, 0.5 mg, 4.6 mg, 2.7 mg, 1.9 mg, and 1.1 mg of 5-HMF, protocatechuic acid, monoglucoside, loganin, strychnine, and verbascoside reference standards, respectively, and place them in a 10 mL volumetric flask. Add chromatographic methanol solution, shake well to dissolve, and dilute to volume to prepare mixed reference standard solutions with concentrations of 210 μg / mL, 50 μg / mL, 460 μg / mL, 270 μg / mL, 190 μg / mL, and 110 μg / mL, respectively.

[0110] Accurately transfer 2.5 mL of the prepared mixed reference solution of 5-HMF, protocatechuic acid, monoglucoside, loganin, strychnine, and verbascoside into a 10 mL volumetric flask, add chromatographic methanol solution, shake well to dissolve, and dilute to volume to prepare mixed reference solutions with concentrations of 52.5 μg / mL, 12.5 μg / mL, 115.0 μg / mL, 67.5 μg / mL, 47.5 μg / mL, and 27.5 μg / mL, respectively.

[0111] V. Selection and Determination of Experimental Conditions

[0112] 1. Selection of chromatographic column

[0113] This experiment examined C from three different manufacturers. 18 The chromatographic columns were Phenomenex Luna C10 and Phenomenex Luna C10. 18 (2)(4.6mm×250mm, 5μm), Agilent Promosil C 18 (4.6mm×250mm, 5μm), Waters SunFire C 18 (4.6mm × 250mm, 5μm), the test solution of the same sample was measured sequentially, and the results showed that using Promosil C (4.6mm × 250mm, 5μm) 18 The (4.6mm×250mm, 5μm) column yielded the most fingerprint information, with better peak shape, more stable baseline, and better resolution. Therefore, this column was selected.

[0114] 2. Selection of detection wavelength

[0115] like Figure 1 , Figure 2As shown, the sample was scanned across the entire wavelength using a diode array detector (DAD). The Liangyi Decoction test solution was taken, and the detection wavelengths of Rehmannia glutinosa and Cornus officinalis were referenced to determine the detection wavelengths to be investigated: 250 nm, 260 nm, and 270 nm. The Liangyi Decoction test solution showed the most peaks at a wavelength of 260 nm, with good separation between peaks and good peak shape. Therefore, 260 nm was selected as the detection wavelength.

[0116] 3. Selection of mobile phase

[0117] Gradient elution was performed using methanol-water and acetonitrile-water as mobile phases. The results showed that acetonitrile-water provided better peak separation. Therefore, acetonitrile-water was chosen as the mobile phase. To improve peak shape, an appropriate amount of acid was added to the mobile phase to adjust the pH. The results also showed that acetonitrile-0.15% formic acid aqueous solution provided a stable peak baseline and good separation. Therefore, gradient elution was performed using acetonitrile-0.15% formic acid aqueous solution as the mobile phase. The elution program is shown in Table 4.

[0118] Table 4 Mobile Phase Elution System

[0119]

[0120]

[0121] 4. Column temperature selection

[0122] The temperature of a chromatographic column can affect its efficiency to some extent. Changes in column temperature can lead to variations in retention time and thus affect separation. Therefore, a constant temperature environment was chosen in the experiment to minimize the impact of external temperature changes on the fingerprint chromatogram. The separation effects at column temperatures of 25℃, 30℃, and 35℃ were investigated, showing that the separation effect was best at a column temperature of 25℃.

[0123] 5. Selection of reconstitution solution

[0124] Three solvents with different concentrations were investigated: 50% methanol, 80% methanol, and methanol. The results showed that when methanol was used as the resolvent, the chromatographic peak area was the largest, the number of chromatographic peaks was greater, and the separation effect was better. Therefore, methanol was selected as the resolvent for the two-component decoction.

[0125] 6. Selection of extraction method

[0126] The chromatographic peak separation effect was investigated at extraction times of 20 min, 30 min, and 40 min. The results showed that when the extraction time was 30 min, there were more chromatographic peaks. Therefore, 30 min was selected as the extraction time for the test sample.

[0127] 7. Determination of experimental conditions

[0128] As shown in Table 5, the chromatographic column used was Promosil C10. 18 (4.6mm×250mm, 5μm); Mobile phase solvent system: A: 0.15% formic acid aqueous solution, B: acetonitrile; Column temperature: 25℃; Detection wavelength: 260nm; Flow rate: 0.6mL / min; Injection volume: 10μL.

[0129] Table 5 Chromatographic conditions for HPLC

[0130]

[0131]

[0132] VI. Methodological Examination

[0133] 1. Precision Experiment

[0134] As shown in Tables 6 and 7, the same sample solution was precisely pipetted and injected six times consecutively under the conditions specified in the "Selection and Determination of Experimental Conditions" section. The retention time and peak area of ​​the chromatographic peaks in the six chromatograms were recorded. The retention time of loganin in chromatographic peak 12 was stable and the resolution was good. Therefore, peak 12 was used as the reference peak (S). The relative retention time and relative peak area of ​​each common peak were calculated to have RSD values ​​of <5.00%, indicating that the instrument was stable and had good precision.

[0135] Table 6. Precision Relative Retention Time

[0136]

[0137] Table 7 Relative Peak Area for Precision

[0138]

[0139]

[0140] 2. Repeatability experiment

[0141] As shown in Tables 8 and 9, the same batch of samples was used to prepare 6 test sample (S1) solutions. The samples were injected separately according to the conditions under the "Selection and Determination of Experimental Conditions" section. The RSD values ​​of the relative retention time and relative peak area of ​​each common peak were all <5.00%, indicating that the method has good repeatability.

[0142] Table 8. Relative Retention Time for Repeatability

[0143]

[0144] Table 9 Relative Peak Area for Repeatability

[0145]

[0146]

[0147] 3. Stability test

[0148] As shown in Tables 10 and 11, the same sample solution was precisely pipetted, and appropriate amounts of the solution were injected into the liquid chromatograph at 0h, 2h, 4h, 6h, 12h, and 24h. The chromatograms at different times were recorded. The results showed that the RSD values ​​of the relative retention time and relative peak area of ​​the common peak were both <5.00%, indicating that the solution was stable within 24h.

[0149] Table 10 Relative retention time of stability

[0150]

[0151] Table 11 Relative Peak Area for Stability

[0152]

[0153]

[0154] VII. Establishment of Fingerprint Map

[0155] 1. Shared Mode

[0156] Based on the main indicator components of Rehmannia glutinosa and Cornus officinalis and previous research, this invention selects 5-HMF, protocatechuic acid, monoglucoside, loganin, strychnine, and verbascoside as reference standards.

[0157] like Figure 3a , 3b As shown, chromatographic analysis of the Liangyi Decoction test solution yielded 19 chromatographic peaks. Comparison with the retention times of the reference peaks revealed that peak 1 was 5-HMF, peak 2 was protocatechuic acid, peak 7 was monoglucoside, peak 12 was loganin, peak 13 was daunoside, and peak 16 was verbascoside. The remaining peaks require further identification.

[0158] like Figure 4 , Figure 5 As shown, chromatographic analysis was performed on 10 batches of Liangyi Decoction test solutions under the above chromatographic conditions. Chromatographic parameters were recorded. Using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A," the chromatographic data of the 10 batches of Liangyi Decoction test solutions were imported into the software. S1 was set as the reference chromatogram. Using the median method, after multi-point correction and data matching, a reference fingerprint chromatogram was generated. Based on the matching results, 19 common peaks were identified from the 10 batches of Liangyi Decoction, resulting in the fingerprint chromatograms for all 10 batches.

[0159] As shown in Tables 12 and 13, using chromatographic peak 12 (loganin) as a reference peak, the relative retention times and relative peak areas of the 19 common chromatographic peaks in 10 batches of samples were calculated. Relative retention time = retention time of each peak / retention time of the reference peak; relative peak area = peak area of ​​each peak / peak area of ​​the reference peak.

[0160] Table 12 Relative retention times of common peaks

[0161]

[0162]

[0163] Table 13 Relative peak areas of common peaks

[0164]

[0165]

[0166] As shown in the above charts, the RSD values ​​of the relative retention times of the 19 common chromatographic peaks in the HPLC fingerprint chromatograms of the 10 batches of Liangyi Decoction are relatively similar, with RSD values ​​less than 1.00%. The RSD values ​​of the relative peak areas of the common peaks range from 8.91 to 56.70, with significant differences in peak areas. This indicates that different batches of Liangyi Decoction contain the same chemical components, but there are significant differences in the content of components between different batches.

[0167] 2. Similarity Evaluation

[0168] As shown in Table 14, chromatographic analysis was performed on 10 batches of Liangyi Decoction samples under the above chromatographic conditions. Using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A," the chromatographic data of the 10 batches of Liangyi Decoction were imported into the system. The cosine similarity method was used to establish a common pattern diagram for different batches of Liangyi Decoction samples. The results showed that the similarity was all above 0.900, indicating that the overall chromatogram... Figure 1 They are quite similar.

[0169] Table 14 Similarity of 10 batches of Liangyi Decoction

[0170]

[0171] VIII. Attribution of Common Peaks in the Fingerprint Spectrum of the Two Elements Decoction

[0172] The assignment of common peaks in the fingerprint chromatogram of Liangyi Decoction refers to comparing the 19 common peaks identified in the fingerprint chromatogram of Liangyi Decoction with the peaks in the chromatogram of the negative sample solution to determine which of the 19 common peaks belong to specific medicinal pieces in Liangyi Decoction. The Liangyi Decoction test solution and the negative sample solution were injected and analyzed according to the fingerprint chromatogram chromatographic conditions under the "Selection and Determination of Experimental Conditions" section to assign the common peaks in the fingerprint chromatogram of Liangyi Decoction. The results are as follows: Figure 6As shown in the figure, by comparing retention times, the origins of the 19 common peaks in Liangyi Decoction were traced. Peak 1 is a common component of Rehmannia glutinosa and Cornus officinalis; peaks 3, 4, 11, 14, 15, 16, and 19 are from Rehmannia glutinosa; and peaks 2, 5, 6, 7, 8, 9, 10, 12, 13, 17, and 18 are from Cornus officinalis. This indicates that the fingerprint spectrum results of Liangyi Decoction can reflect the information of each decoction piece in the Liangyi Decoction prescription.

[0173] IX. Chemical Pattern Recognition

[0174] 1. Cluster Analysis (CA)

[0175] like Figure 7 As shown, the data from 10 batches of samples were imported into SPSS 22.0 software. Using peak area as the variable, a cluster analysis chart was generated using the between-group mean linking method. The horizontal axis represents the critical value, and the vertical axis represents the sample numbers of the 10 different batches of Liangyi Decoction. The smaller the difference in the distance between groups, the smaller the difference between the samples. When the classification distance was 15, S1, S4, S5, S8, S9, and S10 clustered into one group, and S2, S3, S6, and S7 clustered into another group. When the classification distance was 10, S1, S4, S5, S8, S9, and S10 clustered into one group, S2, S6, and S7 clustered into another group, and S3 clustered into another group. The results indicate that S2, S6, S7, and S3 differ from other batches. This classification may be related to the origin of the prescription decoction pieces and the processing technology of the medicinal materials.

[0176] 2. Principal Component Analysis (PCA)

[0177] To more directly evaluate the resolving power of the 19 common peaks for the samples, principal component analysis was performed on 10 batches of Liangyi Decoction samples using SPSS 22.0 software, with the peak area of ​​the common peaks as the variable. The results are shown in Table 15. The cumulative variance contribution rate of the first four principal components was 95.482%, which can represent most of the information of the common peaks in the fingerprint spectrum of Liangyi Decoction. Specifically, the eigenvalue of the first principal component (PC1) was 11.874, with a variance contribution rate of 62.496%; the eigenvalue of the second principal component (PC2) was 3.064, with a variance contribution rate of 16.128%; the eigenvalue of the third principal component (PC3) was 1.703, with a variance contribution rate of 8.926%; and the eigenvalue of the fourth principal component (PC4) was 1.500, with a variance contribution rate of 7.896%. Figure 8As shown, the slope of the first four principal components in the PCA chromatogram is relatively steep, gradually becoming gentler in the later stages, indicating that the first four principal components are likely the most important chromatographic peaks for identifying Liangyi Decoction. The principal component factor loading matrix results are shown in Table 16. The information for the first principal component mainly comes from peaks 1–18; the information for the second principal component mainly comes from peaks 8–11, 13–17, and 19; the information for the third principal component mainly comes from peaks 2–3, 5–6, 8–11, and 13–15; and the information for the fourth principal component mainly comes from peaks 1–6, 9, 11, 14–15, 17, and 19. This indicates that the quality of Liangyi Decoction is not affected by a single component, but rather by the combined effect of multiple components. Figure 9 As shown, principal component analysis was performed on 10 batches of Liangyi Decoction samples using SIMCA 14.1 software. The results showed that the 10 batches of samples could be divided into two categories: samples S1, S4, S5, S8, S9, and S10 belonged to the first category, and samples S2, S3, S6, and S7 belonged to the second category. The results were consistent with the cluster analysis results.

[0178] Table 15 Eigenvalues ​​and Variance Contribution Rate

[0179]

[0180] Table 16 Initial Factor Loading Matrix

[0181]

[0182]

[0183] 3. Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA)

[0184] Using the relative peak areas of 19 common peaks in the fingerprint spectra of 10 batches of Liangyi Decoction as variables, a 10×19 original data matrix was constructed. Orthogonal partial least squares-discriminant analysis (OPLS-DA) was performed on the 10 batches of Liangyi Decoction using SIMCA 14.1 software. The score graph is shown below. Figure 10 As shown. By Figure 10 It can be seen that the 10 batches of Liangyi Decoction samples were divided into 2 categories. This result is basically consistent with the results of cluster analysis and similarity evaluation analysis. The reason is due to the difference in the content of chemical components in the 10 batches of Liangyi Decoction. Combining the variable importance projection value (VIP), the main marker components that can cause differences in the composition of different batches of samples were screened out. Variables with a VIP value greater than 1 were selected, indicating that the larger the VIP value, the greater the contribution to classification. Figure 11As shown, the vertical axis represents the VIP value, and the horizontal axis represents the contributing components in the medicinal material. The VIP value represents the contribution rate of the substance to the classification. Using a VIP value greater than 1 as the standard, three variables with significant influence were selected: chromatographic peaks 1, 7, and 12. Chromatographic peak 1 is considered to be 5-HMF, chromatographic peak 7 is considered to be monoglobulin, and chromatographic peak 12 is considered to be loganin.

[0185] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for establishing the fingerprint spectrum of the Korean medical formula Liangyi Decoction, characterized in that, Includes the following steps: Take 5-HMF, protocatechuic acid, monoglucoside, loganin, scutellarin, and verbascoside as reference standards, dissolve them in chromatographic methanol, and dilute to volume to prepare the reference solution. Prepared Rehmannia glutinosa and Cornus officinalis were extracted with aqueous solution to obtain the freeze-dried powder of Liangyi Decoction. The powder was then dissolved and diluted with chromatographic methanol to obtain the test solution. The fingerprint spectrum was obtained by high performance liquid chromatography (HPLC) with a mobile phase consisting of 0.15% formic acid solution (phase A) and 100% acetonitrile solution (phase B). Gradient elution was used, and the fingerprint features were calibrated by relative retention time and relative peak area. The high-performance liquid chromatography (HPLC) separation and detection conditions are as follows: a chromatographic column is used with Promosil C60 ... 18 The stationary phase was used, the column temperature was 25℃, the detection wavelength was 260nm, the flow rate was 0.6mL / min, the analysis time was 80min, and the injection volume was 10μL into the high performance liquid chromatograph. The gradient elution procedure is as follows: At 0 minutes, mobile phase A is 95% and mobile phase B is 5%. At 20 minutes, mobile phase A was 91% and mobile phase B was 9%. At 35 minutes, mobile phase A was 85.5% and mobile phase B was 14.5%. At 50 minutes, mobile phase A was 82.5% and mobile phase B was 17.5%. At 62 minutes, mobile phase A was 77% and mobile phase B was 23%. At 67 minutes, mobile phase A was 77% and mobile phase B was 23%. At 77 minutes, mobile phase A was 71% and mobile phase B was 29%. At 80 minutes, mobile phase A was 5% and mobile phase B was 95%. The preparation process of the test solution includes: accurately weighing 40.0g of Rehmannia glutinosa and 20.0g of Cornus officinalis, adding 8 times the amount of water, soaking for 30min, decocting twice, 1.5h each time, filtering while hot through a 200-mesh sieve, combining the filtrates, concentrating, and then freeze-drying to obtain the two-in-one decoction freeze-dried powder. Accurately weighing about 1.0g of the two-in-one decoction freeze-dried powder, making up to 20mL in a volumetric flask with chromatographic methanol, sonicating for 30min, letting stand, adding chromatographic methanol solution to the mark, mixing well, letting stand, and filtering through a 0.22μm microporous membrane to obtain the test sample.

2. The method for establishing the fingerprint spectrum of the Korean medicine formula Liangyi Decoction as described in claim 1, characterized in that, The preparation process of the reference solution includes: accurately weighing 5-HMF, protocatechuic acid, monoglucoside, loganin, strychnine, and verbascoside reference standards at concentrations of 2.1 mg, 0.5 mg, 4.6 mg, 2.7 mg, 1.9 mg, and 1.1 mg, respectively, placing them in a 10 mL volumetric flask, adding chromatographic methanol solution, shaking well to dissolve, and making up to volume to prepare mixed reference solutions with concentrations of 210 μg / mL, 50 μg / mL, 460 μg / mL, 270 μg / mL, 190 μg / mL, and 110 μg / mL, respectively; Accurately transfer 2.5 mL of the prepared mixed reference solution of 5-HMF, protocatechuic acid, monoglucoside, loganin, strychnine, and verbascoside into a 10 mL volumetric flask, add chromatographic methanol solution, shake well to dissolve, and dilute to volume to prepare mixed reference solutions with concentrations of 52.5 μg / mL, 12.5 μg / mL, 115.0 μg / mL, 67.5 μg / mL, 47.5 μg / mL, and 27.5 μg / mL, respectively.

3. The method for establishing the fingerprint spectrum of the Korean medicine formula Liangyi Decoction as described in claim 2, characterized in that, The fingerprint spectrum contains 19 common fingerprint peaks, with the following relative retention times: Peak 1: 0.368–0.370; Peak 2: 0.512–0.514; Peak 3: 0.553–0.556; Peak 4: 0.609–0.613; Peak 5: 0.657–0.662; Peak 6: 0.710–0.712; Peak 7: 0.759–0.760; Peak 8: 0.830–0.832; Peak 9: 0.854–0.857; Peak 10: 0.875–0. 0.879; Peak 11: 0.920~0.923; Peak 12 (reference peak): 1.000; Peak 13: 1.021~1.022; Peak 14: 1.107~1.108; Peak 15: 1.265~1.266; Peak 16: 1.469~1.471; Peak 17: 1.526~1.531; Peak 18: 1.570~1.573; Peak 19: 1.609~1.

613.

4. The method for establishing the fingerprint spectrum of the Korean medicine formula Liangyi Decoction as described in claim 2, characterized in that, The fingerprint spectrum contains 19 common fingerprint peaks, with the following relative retention times: Peak 1: 0.370; Peak 2: 0.513; Peak 3: 0.554; Peak 4: 0.611; Peak 5: 0.659; Peak 6: 0.712; Peak 7: 0.760; Peak 8: 0.832; Peak 9: 0.856; Peak 10: 0.877; Peak 11: 0.922; Peak 12 (reference peak): 1.000; Peak 13: 1.021; Peak 14: 1.107; Peak 15: 1.265; Peak 16: 1.470; Peak 17: 1.529; Peak 18: 1.571; Peak 19: 1.610.

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