A method for determining the quality of stems and leaves of black dragon bone.

Abstract

This invention discloses a method for determining the quality of *Hemiberlesia lingua* stems and leaves. The method includes fingerprint spectroscopy establishment and similarity analysis, content determination, cluster analysis, principal component analysis, and partial least squares discriminant analysis. The method of this invention involves UPLC fingerprint analysis and similarity analysis of 15 batches of *Hemiberlesia lingua* stems and leaves from different origins. A total of 28 common peaks were found, and 7 of these common peaks were identified using reference standards: peak 4 (neochlorogenic acid), peak 8 (chlorogenic acid), peak 9 (caffeic acid), peak 11 (cryptochlorogenic acid), peak 22 (isoquercetin), peak 24 (astragalin), and peak 28 (isochlorogenic acid C). This invention's method is highly sensitive, specific, stable, and feasible, providing a reference for the quality evaluation and control of *Hemiberlesia lingua* stems and leaves.

Description

Technical Field

[0001] This invention relates to methods for determining multiple components of medicinal materials, and in particular to a method for determining the quality of stems and leaves of black dragon bone. Background Technology

[0002] *Periploca forrestii* Schltr., commonly known as Southwest Periploca, is a plant belonging to the genus *Periploca* in the family Asclepiadaceae. Its vine, *Periploca forrestii*, is listed as a Miao medicine in the *Chinese Materia Medica: Miao Medicine Volume*. It possesses properties such as relaxing muscles and promoting blood circulation, dispelling wind and dampness, and clearing heat and detoxifying. For a long time, it has been used as a traditional Miao herbal medicine to treat rheumatism and other diseases, and it is one of the important Miao medicines currently being developed in Guizhou Province. There are few reports on the chemical composition and pharmacological effects of the non-medicinal parts of *Periploca forrestii*, including its stems and leaves. Furthermore, because it is not used clinically, it is largely discarded, resulting in significant resource waste and environmental pollution. Therefore, our research group conducted a preliminary study on the vine stems of *Periploca forrestii*, and the results showed that this part contains caffeoylquinic acid compounds, which have strong antibacterial and anti-inflammatory activities.

[0003] To better develop and utilize the stems and leaves of *Hemiberlesia lingua* and effectively ensure its quality, it is necessary to establish quality evaluation methods to better control its quality. Therefore, this study uses multi-index content determination combined with cluster analysis, principal component analysis, and partial least squares discriminant analysis to analyze the stems and leaves of *Hemiberlesia lingua* from different producing areas, clarifying the differential components among different producing areas, in order to provide a reference for the quality evaluation and quality control of *Hemiberlesia lingua* stems and leaves, and at the same time provide a reference for the effective utilization and development of its stem and leaf resources. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining the quality of stems and leaves of *Hemiberlesia lingua*, which can establish a fingerprint spectrum of stems and leaves and detect the content of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isoquercitrin, astragaloside, and isochlorogenic acid C in stems and leaves. This analytical method has high sensitivity, good specificity, stability, and feasibility. Furthermore, by combining cluster analysis, principal component analysis, and partial least squares discriminant analysis, it can distinguish quality differences from different production areas, providing a reference for the quality evaluation and control of stems and leaves of *Hemiberlesia lingua*.

[0005] The technical solution of this invention: A method for determining the quality of stems and leaves of *Hemiberlesia lingua*, comprising fingerprint spectrum establishment and similarity analysis, content determination, combined with cluster analysis, principal component analysis, and partial least squares discriminant analysis. The specific steps are as follows:

[0006] (1) Preparation of mixed reference solution: Accurately weigh 14.5-16.5 mg neochlorogenic acid, 11.5-13.5 mg chlorogenic acid, and 11.5-13.5 mg cryptochlorogenic acid and place them in a 10 ml volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ①. Separately, accurately weigh 2-4 mg caffeic acid, 4.5-6.5 mg isoquercitrin, 4-6 mg astragaloside, and 7.5-9.5 mg isochlorogenic acid C and place them in a 50 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ②. Take 1 ml from each of mixed reference solution ① and mixed reference solution ② and place them in the same 10 ml volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ③.

[0007] (2) Preparation of test solution: Accurately weigh 0.4-0.6g of black dragon bone stem and leaf sample, place it in a 20ml conical flask, add 10mL of 75% ethanol, accurately weigh, extract by ultrasonication for 20-40min, cool, make up the weight loss, shake well, filter through a 0.22μm microporous membrane to obtain the test solution.

[0008] (3) Chromatographic conditions: An Agilent Eclipse Plus C18 column with dimensions of 2.1 × 100 mm and 1.8 μm was used. The flow rate was 0.2 mL / min. Acetonitrile was used as mobile phase B, and 0.1% phosphoric acid water was used as mobile phase A. Gradient elution was performed. The injection volume was 0.8 μL, the column temperature was 30℃, and the detection wavelengths were 327 nm for 1-49 min; 254 nm for 49-70 min; and 327 nm for 70-85 min.

[0009] (4) Fingerprint chromatogram establishment and similarity analysis: The test solutions of black dragon bone stems and leaves from different origins were subjected to UPLC determination under chromatographic conditions. The sample chromatograms were converted to AIA format and the data were imported into the 2012 version of the Chinese herbal medicine chromatographic fingerprint chromatogram similarity evaluation system. The first batch of sample chromatograms were used as reference chromatograms. The time window width was set to 0.1. Multi-point correction was used. After marker peak matching, the fingerprint superimposed chromatogram of black dragon bone stems and leaves and the reference chromatogram were generated and the similarity was calculated to obtain the common peaks. Then, the mixed reference solution ③ was injected and determined under chromatographic conditions to obtain the reference chromatogram. By comparing the chromatographic peaks and ultraviolet absorption wavelengths of the reference, the characteristic peaks were identified and the fingerprint chromatogram was established.

[0010] (5) Content determination: Ultra-high performance liquid chromatography was used to detect the test solution and mixed reference solution ③ under chromatographic conditions to effectively separate neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, astragaloside and isochlorogenic acid C in the stems and leaves of black dragon bone. The peak area was recorded and the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, astragaloside and isochlorogenic acid C in the test sample were calculated.

[0011] (6) The stems and leaves of black dragon bone from different origins were analyzed by combining cluster analysis, principal component analysis and partial least squares discriminant analysis.

[0012] In step (1) above, the preparation of the mixed reference solution is as follows: accurately weigh 15-16 mg of neochlorogenic acid, 12-13 mg of chlorogenic acid, and 12-13 mg of cryptochlorogenic acid and place them in a 10 ml volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ①. Separately, accurately weigh 2.5-3.6 mg of caffeic acid, 5-6 mg of isoquercitrin, 4.5-5.5 mg of astragaloside, and 8-9 mg of isochlorogenic acid C and place them in a 50 mL volumetric flask. Add 75% ethanol to the mark and shake well to prepare mixed reference solution ②. Take 1 ml from each of mixed reference solution ① and mixed reference solution ② and place them in the same 10 ml volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ③.

[0013] Specifically, in step (1) above, the preparation of the mixed reference solution is as follows: accurately weigh 15.2 mg of neochlorogenic acid, 12.4 mg of chlorogenic acid, and 12.6 mg of cryptochlorogenic acid and place them in a 10 ml volumetric flask, add 75% ethanol solution to dilute to the mark, shake well, and prepare mixed reference solution ①; separately, accurately weigh 3.1 mg of caffeic acid, 5.6 mg of isoquercitrin, 4.9 mg of astragaloside, and 8.5 mg of isochlorogenic acid C and place them in a 50 mL volumetric flask, add 75% ethanol to the mark, shake well, and prepare mixed reference solution ②; take 1 ml from each of mixed reference solution ① and mixed reference solution ② and place them in the same 10 ml volumetric flask, add 75% ethanol solution to dilute to the mark, shake well, and prepare mixed reference solution ③.

[0014] In step (2) above, the preparation of the test solution is as follows: accurately weigh 0.45-0.55g of the stem and leaf sample of black dragon bone, place it in a 20ml conical flask, add 10mL of 75% ethanol, accurately weigh it, extract it by ultrasonication for 25-35min, ultrasonic power 200W, ultrasonic frequency 40KHZ, cool it, make up the weight loss, shake it well, and filter it through a 0.22μm microporous membrane to obtain the test solution.

[0015] Specifically, in step (2) above, the preparation of the test solution is as follows: accurately weigh 0.5g of the black dragon bone stem and leaf sample, place it in a 20ml conical flask, add 10mL of 75% ethanol, accurately weigh, ultrasonically extract for 30min, ultrasonic power 200W, ultrasonic frequency 40KHZ, cool, make up the weight loss, shake well, and filter through a 0.22μm microporous membrane to obtain the test solution.

[0016] In step (3) above, the elution gradient is 0-9 min, 7-7.5% B; 9-28 min, 7.5-9% B; 28-31 min, 9-13% B; 31-55 min, 13-13% B; 55-60 min, 13-14% B; 60-62 min, 14-16% B; 62-67 min, 16-18% B; 67-72 min, 18-18% B; 72-75 min, 18-21.5% B; 75-80 min, 21.5-65% B; 80-85 min, 65-95% B.

[0017] In the aforementioned step (4), the characteristic peaks are as follows: peak 4 is neochlorogenic acid, peak 8 is chlorogenic acid, peak 9 is caffeic acid, peak 11 is cryptochlorogenic acid, peak 22 is isoquercitrin, peak 24 is astragaloside, and peak 28 is isochlorogenic acid C.

[0018] In step (6) above, the combined cluster analysis involves importing the common peaks of the stems and leaves of black dragon bone from different origins into SPSS 26.0 software for cluster analysis, using the intergroup connection method and selecting Euclidean squared distance as the clustering formula.

[0019] In step (6) above, the principal component analysis is to use the area of ​​the common peak of the stems and leaves of black dragon bone from different producing areas as a variable to perform principal component analysis, extract principal components with eigenvalues ​​and variance contribution rates as standards, and make scree plots. Further principal component factor matrix analysis of the stems and leaves of black dragon bone is performed. Finally, the common peaks are imported into SIMCA14.0 software to obtain the principal component score map, and SPSS software is used to calculate the principal component score. The comprehensive score is calculated with the variance contribution rate corresponding to each principal component as the weight.

[0020] In step (6) above, the partial least squares discriminant analysis is performed based on principal component analysis to obtain the partial least squares discriminant analysis score map.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. This invention uses UPLC fingerprint analysis and similarity analysis on 15 batches of black dragon bone stem and leaf samples, and found a total of 28 common peaks. Seven of these common peaks were identified using reference standards: peak 4 (neochlorogenic acid), peak 8 (chlorogenic acid), peak 9 (caffeic acid), peak 11 (cryptochlorogenic acid), peak 22 (isoquercetin), peak 24 (astragalin), and peak 28 (isochlorogenic acid C). The similarity between the samples ranged from 0.810 to 0.999, indicating that there are certain differences among the samples.

[0023] 2. In the content detection, the maximum RSD of the precision test was 0.83%, the maximum RSD of the stability test was 0.59%, the maximum RSD of the repeatability test was 0.78%, and the maximum RSD of the spiking recovery test was 3.22%, with a maximum RSD of 96.01%-98.94%. This indicates that the method has good precision, good stability, good repeatability, and good spiking recovery.

[0024] 3. Through partial least squares discriminant analysis, peaks 9 (caffeic acid), 24, 8 (chlorogenic acid), 12, 4 (neochlorogenic acid), 11 (cryptochlorogenic acid), 17, 14, 23, 3, 13, 22 (isoquercetin), and peak 7 were identified as the main differential components. These components can be used to distinguish the quality differences between different production areas, providing a reference for the quality evaluation and control of black dragon bone stems and leaves. Attached Figure Description

[0025] Figure 1 : Overlay fingerprint spectrum of stems and leaves of Black Dragon Bone and control spectrum;

[0026] Figure 2 UPLC chromatograms of test solution, reference solution and blank solution overlay (4 is neochlorogenic acid; 8 is chlorogenic acid; 9 is caffeic acid; 11 is cryptochlorogenic acid; 22 is isoquercitrin; 24 is astragaloside; 28 isochlorogenic acid C; E and F are test solutions of sample S1; D and C are mixed reference solutions ③; A and B are negative solutions).

[0027] Figure 3 Cluster analysis results of stems and leaves of black dragon bone;

[0028] Figure 4 Principal component analysis of black dragon bone stems and leaves: gravel plot;

[0029] Figure 5 Principal component analysis score chart of black dragon bone stems and leaves;

[0030] Figure 6 Black dragon bone stem and leaf OPLS-DA score chart;

[0031] Figure 7 Black Dragon Bone Stem and Leaf VIP Value. Detailed Implementation

[0032] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0033] Example 1: Establishment and Similarity Analysis of Stem and Leaf Fingerprints of Black Dragon Bone

[0034] (1) Preparation of mixed reference solution: Accurately weigh 15.2 mg neochlorogenic acid, 12.4 mg chlorogenic acid, and 12.6 mg cryptochlorogenic acid and place them in a 10 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ①. Separately, accurately weigh 3.1 mg caffeic acid, 5.6 mg isoquercitrin, 4.9 mg astragaloside, and 8.5 mg isochlorogenic acid C and place them in a 50 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ②. Take 1 mL from each of mixed reference solution ① and mixed reference solution ② and place them in the same 10 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ③.

[0035] (2) Preparation of test solution: Accurately weigh 0.5g of black dragon bone stem and leaf sample, place it in a 20ml conical flask, add 10mL of 75% ethanol, accurately weigh, ultrasonically extract for 30min, ultrasonic power 200W, ultrasonic frequency 40KHZ, cool, make up the weight loss, shake well, filter through a 0.22μm microporous membrane to obtain the test solution;

[0036] (3) Chromatographic conditions: An Agilent Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm) was used. The flow rate was 0.2 mL / min. Acetonitrile was used as mobile phase B, and 0.1% phosphoric acid water was used as mobile phase A. Gradient elution was performed with the following gradients: 0-9 min, 7-7.5% B; 9-28 min, 7.5-9% B; 28-31 min, 9-13% B; 31-55 min, 13-13% B; 55-60 min, 13-14% B; 60-62 min... Injection time: 14-16% B; 62-67 min: 16-18% B; 67-72 min: 18-18% B; 72-75 min: 18-21.5% B; 75-80 min: 21.5-65% B; 80-85 min: 65-95% B; Injection volume: 0.8 μL; Column temperature: 30℃; Detection wavelength: 1-49 min: 327 nm; 49-70 min: 254 nm; 70-85 min: 327 nm.

[0037] (4) Fingerprint chromatogram establishment and similarity analysis: The test solutions of black dragon bone stems and leaves from different origins were subjected to UPLC determination under chromatographic conditions. The sample chromatograms were converted to AIA format and the data were imported into the 2012 version of the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system. The first batch of sample chromatograms were used as reference chromatograms. The time window width was set to 0.1. Multi-point correction was used. After marker peak matching, the fingerprint superimposed chromatogram of black dragon bone stems and leaves and the reference chromatogram were generated and the similarity was calculated to obtain 28 common peaks. Then, the mixed reference solution ③ was injected and determined under chromatographic conditions to obtain the reference chromatogram. By comparing the reference chromatographic peaks and ultraviolet absorption wavelengths, 7 characteristic peaks were identified (peak 4 is neochlorogenic acid, peak 8 is chlorogenic acid, peak 9 is caffeic acid, peak 11 is cryptochlorogenic acid, peak 22 is isoquercitrin, peak 24 is astragaloside, and peak 28 is isochlorogenic acid C) to establish the fingerprint chromatogram.

[0038] Example 2: Determination of the content of black dragon bone stems and leaves

[0039] (2) Preparation of mixed reference solution: Accurately weigh 15.2 mg neochlorogenic acid, 12.4 mg chlorogenic acid, and 12.6 mg cryptochlorogenic acid and place them in a 10 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ①. Separately, accurately weigh 3.1 mg caffeic acid, 5.6 mg isoquercitrin, 4.9 mg astragaloside, and 8.5 mg isochlorogenic acid C and place them in a 50 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ②. Take 1 mL from each of mixed reference solution ① and mixed reference solution ② and place them in the same 10 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ③.

[0040] (2) Preparation of test solution: Accurately weigh 0.5g of black dragon bone stem and leaf sample, place it in a 20ml conical flask, add 10mL of 75% ethanol, accurately weigh, ultrasonically extract for 30min, ultrasonic power 200W, ultrasonic frequency 40KHZ, cool, make up the weight loss, shake well, filter through a 0.22μm microporous membrane to obtain the test solution;

[0041] (3) Chromatographic conditions: An Agilent Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm) was used. The flow rate was 0.2 mL / min. Acetonitrile was used as mobile phase B, and 0.1% phosphoric acid water was used as mobile phase A. Gradient elution was performed with the following gradients: 0-9 min, 7-7.5% B; 9-28 min, 7.5-9% B; 28-31 min, 9-13% B; 31-55 min, 13-13% B; 55-60 min, 13-14% B; 60-62 min... Injection time: 14-16% B; 62-67 min: 16-18% B; 67-72 min: 18-18% B; 72-75 min: 18-21.5% B; 75-80 min: 21.5-65% B; 80-85 min: 65-95% B; Injection volume: 0.8 μL; Column temperature: 30℃; Detection wavelength: 1-49 min: 327 nm; 49-70 min: 254 nm; 70-85 min: 327 nm.

[0042] (4) Content determination: Ultra-high performance liquid chromatography was used to detect the test solution and mixed reference solution ③ under chromatographic conditions to effectively separate neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, astragaloside and isochlorogenic acid C in the stems and leaves of black dragon bone. The peak areas were recorded and the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, astragaloside and isochlorogenic acid C in the test sample were calculated.

[0043] Example 3: Determination of the content of black dragon bone stems and leaves

[0044] (1) Preparation of mixed reference solution: Accurately weigh 16.5 mg neochlorogenic acid, 13.5 mg chlorogenic acid, and 13.5 mg cryptochlorogenic acid and place them in a 10 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ①. Separately, accurately weigh 4 mg caffeic acid, 6.5 mg isoquercitrin, 6 mg astragaloside, and 9.5 mg isochlorogenic acid C and place them in a 50 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ②. Take 1 mL from each of mixed reference solution ① and mixed reference solution ② and place them in the same 10 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ③.

[0045] (2) Preparation of test solution: Accurately weigh 0.6g of black dragon bone stem and leaf sample, place it in a 20ml conical flask, add 10mL of 75% ethanol, accurately weigh, extract by ultrasonication for 40min, cool, make up the weight loss, shake well, filter through a 0.22μm microporous membrane to obtain the test solution.

[0046] (3) Chromatographic conditions: An Agilent Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm) was used. The flow rate was 0.2 mL / min. Acetonitrile was used as mobile phase B, and 0.1% phosphoric acid water was used as mobile phase A. Gradient elution was performed with the following gradients: 0-9 min, 7-7.5% B; 9-28 min, 7.5-9% B; 28-31 min, 9-13% B; 31-55 min, 13-13% B; 55-60 min, 13-14% B; 60-62 min... Injection time: 14-16% B; 62-67 min: 16-18% B; 67-72 min: 18-18% B; 72-75 min: 18-21.5% B; 75-80 min: 21.5-65% B; 80-85 min: 65-95% B; Injection volume: 0.8 μL; Column temperature: 30℃; Detection wavelength: 1-49 min: 327 nm; 49-70 min: 254 nm; 70-85 min: 327 nm.

[0047] (4) Content determination: Ultra-high performance liquid chromatography was used to detect the test solution and mixed reference solution ③ under chromatographic conditions to effectively separate neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, astragaloside and isochlorogenic acid C in the stems and leaves of black dragon bone. The peak area was recorded and the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, astragaloside and isochlorogenic acid C in the test sample were calculated.

[0048] Example 4: Determination of the content of black dragon bone stems and leaves

[0049] (1) Preparation of mixed reference solution: Accurately weigh 14.5 mg neochlorogenic acid, 11.5 mg chlorogenic acid, and 11.5 mg cryptochlorogenic acid and place them in a 10 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ①. Separately, accurately weigh 2 mg caffeic acid, 4.5 mg isoquercitrin, 4 mg astragaloside, and 7.5 mg isochlorogenic acid C and place them in a 50 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ②. Take 1 mL from each of mixed reference solution ① and mixed reference solution ② and place them in the same 10 mL volumetric flask. Add 75% ethanol solution to the mark and shake well to prepare mixed reference solution ③.

[0050] (2) Preparation of test solution: Accurately weigh 0.4g of black dragon bone stem and leaf sample, place it in a 20ml conical flask, add 10mL of 75% ethanol, accurately weigh, extract by ultrasonication for 20min, cool, make up the weight loss, shake well, filter through a 0.22μm microporous membrane to obtain the test solution.

[0051] (3) Chromatographic conditions: An Agilent Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm) was used. The flow rate was 0.2 mL / min. Acetonitrile was used as mobile phase B, and 0.1% phosphoric acid water was used as mobile phase A. Gradient elution was performed with the following gradients: 0-9 min, 7-7.5% B; 9-28 min, 7.5-9% B; 28-31 min, 9-13% B; 31-55 min, 13-13% B; 55-60 min, 13-14% B; 60-62 min... Injection time: 14-16% B; 62-67 min: 16-18% B; 67-72 min: 18-18% B; 72-75 min: 18-21.5% B; 75-80 min: 21.5-65% B; 80-85 min: 65-95% B; Injection volume: 0.8 μL; Column temperature: 30℃; Detection wavelength: 1-49 min: 327 nm; 49-70 min: 254 nm; 70-85 min: 327 nm.

[0052] (4) Content determination: Ultra-high performance liquid chromatography was used to detect the test solution and mixed reference solution ③ under chromatographic conditions to effectively separate neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, astragaloside and isochlorogenic acid C in the stems and leaves of black dragon bone. The peak area was recorded and the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, astragaloside and isochlorogenic acid C in the test sample were calculated.

[0053] Example 6: Cluster analysis, principal component analysis, and partial least squares discriminant analysis

[0054] Cluster analysis involves importing the common peaks of stems and leaves of black dragon bone from different origins into SPSS 26.0 software for cluster analysis, using the between-groups connection method and selecting Euclidean squared distance as the clustering formula;

[0055] Principal component analysis (PCA) uses the area of ​​the common peaks in the stems and leaves of black dragon bone from different origins as variables. Principal components are extracted using eigenvalues ​​and variance contribution rates as standards, and scree plots are created. Further PCA analysis of the stem and leaf principal component factor matrix is ​​performed. Finally, the 28 common peaks are imported into SIMCA14.0 software to obtain the principal component score plot. SPSS software is used to calculate the principal component scores, and the comprehensive score is calculated using the variance contribution rate corresponding to each principal component as the weight.

[0056] Partial least squares discriminant analysis was performed based on principal component analysis to obtain the partial least squares discriminant analysis score map.

[0057] The inventor conducted numerous experiments; the following are some of the experimental studies.

[0058] 1. Instruments and Materials

[0059] 1.1 Instruments

[0060] LC-2040C 3D-Plus Ultra-High Performance Liquid Chromatograph (Shimadzu, Japan); FA2204B 0.001% electronic balance (Shanghai Tianmei Instrument Co., Ltd.); MS205DU 0.001% electronic balance (Mettler Toledo AG, Switzerland); SB-4200DTD Ultrasonic Cleaner (Ningbo Xinyi Ultrasonic Equipment Co., Ltd.); SHZ-DⅢ Circulating Water Vacuum Pump (Shanghai Yuying Co., Ltd.); RHP-750A Multifunctional Grinder (Zhejiang Ronghao Industry & Trade Co., Ltd.)

[0061] 1.2 Reagents

[0062] Neochlorogenic acid (batch number: DZP10805), chlorogenic acid (batch number: DZP10526), ​​cryptochlorogenic acid (batch number: DZP10804), caffeic acid (batch number: DZP10444), isoquercitrin (batch number: DZP01483), and astragaloside (batch number: WP204023); isochlorogenic acid C (batch number: DZP10804) were all purchased from Tianjin Wanxiang Hengyuan Technology Co., Ltd.; acetonitrile and methanol (chromatographic grade), and all other reagents were analytical grade; purified water (Wahaha). Fifteen batches of samples were identified as stems and leaves of *Periploca forrestii* Schltr, a plant belonging to the genus *Periploca* of the family Asclepiadaceae (see Table 1).

[0063] Table 1. Origin of 15 batches of Black Dragon Bone Stems and Leaves

[0064]

[0065]

[0066] 2. Methods and Results

[0067] 2.1 Preparation of test solution

[0068] Accurately weigh 0.5g of black dragon bone stem and leaf sample, place it in a 20ml conical flask, add 10mL of 75% ethanol, accurately weigh, and ultrasonically extract for 30min (power 200W, frequency 40KHZ). Cool to make up the weight loss, shake well, and filter through a 0.22μm microporous membrane to obtain the final product.

[0069] 2.2 Preparation of mixed reference solution

[0070] Accurately weigh 15.2 mg of neochlorogenic acid, 12.4 mg of chlorogenic acid, and 12.6 mg of cryptochlorogenic acid into a 10 mL volumetric flask, and add 75% ethanol solution to the mark to obtain mixed reference solution ①, with mass concentrations of 0.152 mg / mL, 0.124 mg / mL, and 0.126 mg / mL, respectively. Separately, accurately weigh 3.1 mg of caffeic acid, 5.6 mg of isoquercitrin, 4.9 mg of astragaloside, and 8.5 mg of isochlorogenic acid C into a 50 mL volumetric flask, and add 75% ethanol to the mark to obtain mixed reference solution ②, with mass concentrations of 0.062 mg / mL, 0.112 mg / mL, 0.098 mg / mL, and 0.17 mg / mL, respectively. Take 1 mL from each of mixed reference solution ① and mixed reference solution ② and place them into the same 10 mL volumetric flask, and make up to the mark to obtain mixed reference solution ③. The mass concentrations were 0.152 mg / mL neochlorogenic acid, 0.124 mg / mL chlorogenic acid, 0.126 mg / mL cryptochlorogenic acid, 0.0062 mg / mL caffeic acid, 0.0112 mg / mL isoquercitrin, 0.0098 mg / mL astragaloside, and 0.017 mg / mL isochlorogenic acid C.

[0071] 2.3 Chromatographic conditions

[0072] An Agilent Eclipse Plus C18 (2.1 × 100 mm, 1.8 μm) column was used with a flow rate of 0.2 mL / min. The mobile phase was acetonitrile (B)-0.1% phosphoric acid water (A). The elution gradient was as follows: 0–9 min, 7–7.5% (B); 9–28 min, 7.5–9% (B); 28–31 min, 9–13% (B); 31–55 min, 13–13% (B); 55–60 min, 13–14% (B); 60 min, 13–14% (B). -62 min, 14-16% (B); 62-67 min, 16-18% (B); 67-72 min, 18-18% (B); 72-75 min, 18-21.5% (B); 75-80 min, 21.5-65% (B); 80-85 min, 65-95% (B); injection volume 0.8 μL, column temperature 30℃, detection wavelength 327 nm for 1-49 min, 254 nm for 49-70 min, and 327 nm for 70-85 min.

[0073] 2.4 Establishment and Similarity Evaluation of Fingerprint Maps

[0074] 2.4.1 Precision Examination

[0075] Take sample S3, prepare the test solution according to the method in section 2.1, inject it 6 times consecutively under the chromatographic conditions in section 2.3, record the chromatogram, and use peak 4 (neochlorogenic acid) as a reference. The RSD of the relative retention time and relative peak area of ​​each common peak is less than 2.98%, which indicates that the instrument precision is good.

[0076] 2.4.2 Stability Assessment

[0077] Take sample S3 and prepare the test solution according to the method in section 2.1. Under the chromatographic conditions in section 2.3, inject the sample at 0h, 2h, 4h, 6h, 8h, 10h, 12h and 24h respectively, and record the chromatogram. Using peak 4 (neochlorogenic acid) as a reference, the RSD of the relative retention time and relative peak area of ​​each common peak is calculated to be <3.31%, which indicates good stability within 24h.

[0078] 2.4.3 Repeatability Test

[0079] Six S3 samples were taken, and test solutions were prepared according to the method in section 2.1. Chromatograms were recorded under the chromatographic conditions in section 2.3. With peak 4 (neochlorogenic acid) as a reference, the RSD of the relative retention time and relative peak area of ​​each common peak were calculated to be <2.92%, indicating that the method has good repeatability.

[0080] 2.5 Fingerprint pattern establishment and similarity evaluation

[0081] Fifteen batches of *Hemiberlesia lingua* stem and leaf samples (S1-S15) were collected. Test solutions were prepared according to the method described in section "2.1," and the samples were injected and analyzed under the chromatographic conditions described in section "2.3." Chromatograms of the 15 batches of samples were obtained. The chromatograms were converted to AIA format and imported into the Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) software. The chromatogram of sample S1 was used as the reference chromatogram. The time window width was set to 0.1. Using multi-point correction and marker peak matching, a superimposed fingerprint chromatogram of *Hemiberlesia lingua* stem and leaf samples and a control chromatogram were generated. Figure 1 The similarity was calculated, and a total of 28 common peaks were found in 15 batches of samples.

[0082] Preparation of negative solution: Accurately measure 10 mL of 75% ethanol solution and prepare the negative solution according to the preparation method of the stem and leaf sample solution of black dragon bone.

[0083] Take the mixed reference solution ③ and negative solution from section "2.3", and inject them according to the chromatographic conditions in section "2.3" to obtain the reference chromatogram. By comparing the reference standard with the ultraviolet absorption wavelength, seven components were identified: peak 4 (neochlorogenic acid), peak 8 (chlorogenic acid), peak 9 (caffeic acid), peak 11 (cryptochlorogenic acid), peak 22 (isoquercetin), peak 24 (astragalin), and peak 28 (isochlorogenic acid C) (see...). Figure 2The similarity of the samples ranged from 0.810 to 0.999, indicating that there were certain differences among the samples (see Table 2).

[0084] Table 2. Similarity calculation results of 15 batches of samples

[0085]

[0086] 2.6 Sample Content Determination

[0087] 2.6.1 Specificity Examination

[0088] To further examine the rationality of the experiment, the negative solution from section "2.5" and the mixed reference solution ③ prepared from section "2.2" were injected according to the chromatographic method described in section "2.3". The results showed that the blank solution had no chromatographic peaks at the corresponding retention times of the reference standard at detection wavelengths of 327 nm and 254 nm, indicating no interference and good specificity.

[0089] 2.6.2 Examination of Linear Relationships

[0090] Inject 0.1 μL, 0.2 μL, 0.4 μL, 0.6 μL, and 0.8 μL of mixed reference solution ① under section “2.2”, 0.2 μL, 0.4 μL, 0.6 μL, 0.8 μL, and 1.2 μL of mixed reference solution ②, and 0.1 μL, 0.2 μL, 0.4 μL, 0.8 μL, 1.2 μL, and 2 μL of mixed reference solution ③. Perform chromatographic analysis according to the chromatographic conditions in section “2.3”. Linear regression analysis was performed with the concentration of the reference solution as the abscissa (X) and the peak area as the ordinate (Y). The results showed that each component had a good linear relationship within the corresponding concentration range (Table 3).

[0091] Table 3 Results of linear relationship investigation of 7 components

[0092]

[0093] 2.6.3 Precision Test

[0094] Accurately pipette the mixed reference solution ③ under section “2.2”, inject it according to the chromatographic conditions in section 2.3, record the peak area, and calculate the RSD of the peak areas of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, and astragaloside. The RSDs are 0.69%, 0.65%, 0.66%, 0.63%, 0.43%, 0.83%, and 0.73%, respectively, indicating that the instrument has good precision.

[0095] 2.6.4 Stability Test

[0096] Take sample S3 and prepare the test solution according to the method in section "2.1". Under the chromatographic conditions in section "2.3", inject the sample at 0h, 2h, 4h, 6h, 8h, 10h, 12h and 24h respectively, record the peak area, and calculate the RSD of the peak areas of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin and astragaloside. The RSDs are 0.31%, 0.39%, 0.33%, 0.55%, 0.55%, 0.52%, 0.59% and 0.54% respectively, indicating that the method has good stability within 24h.

[0097] 2.6.5 Repeatability Test

[0098] Take 6 S3 samples and prepare test solutions according to the method in section "2.1". Record the peak areas according to the chromatographic conditions in section "2.3". Calculate the RSDs of the peak areas of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, and astragaloside, which are 0.35%, 0.12%, 0.46%, 0.58%, 0.78%, 0.74%, and 0.78%, respectively.

[0099] 2.6.6 Spiking Recovery Test

[0100] Accurately weigh 11.963 mg of neochlorogenic acid, 9.560 mg of chlorogenic acid, 6.80 mg of caffeic acid, 9.67 mg of cryptochlorogenic acid, 5.36 mg of isoquercitrin, 1.69 mg of astragaloside, and 3.40 mg of isochlorogenic acid C into volumetric flasks of 10 mL, 10 mL, 200 mL, 10 mL, 10 mL, 10 mL, and 10 mL respectively. Dilute to the mark with 75% and shake well to obtain 1.1963 mg / mL, 0.9560 mg / mL, 0.034 mg / mL, 0.967 mg / mL, 0.536 mg / mL, 0.169 mg / mL, and 0.34 mg / mL respectively.

[0101] Accurately weigh 0.25 g of *Hemiberlesia lingua* stem and leaves (S3) with known component content, place them in a 20 mL Erlenmeyer flask, and divide into six parallel flasks. Add 1 mL of each of the seven single reference solutions mentioned above, then add 3 mL of 75% ethanol, weigh the samples, and prepare them according to the method in section "2.1". Inject the samples according to the chromatographic conditions in section "2.3" and record the peak areas. The average recoveries of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoquercitrin, and astragaloside were 96.01%, 98.93%, 98.52%, 98.94%, 98.06%, 96.22%, and 98.20%, respectively, with RSDs of 2.29%, 3.13%, 3.22%, 2.71%, 1.92%, 2.86%, and 2.38%, respectively. The results indicate that the method has good recovery (see Table 4).

[0102] Table 4 Results of the recovery test

[0103]

[0104]

[0105] 2.6.7 Sample Determination

[0106] Fifteen batches of samples were collected, and test solutions were prepared according to the method in section "2.1". The contents were determined by injection under the chromatographic conditions in section "2.3", and the contents of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, isoquercitrin, astragaloside, and isochlorogenic acid C were calculated. The results showed that the contents of the seven chemical components in the stems and leaves of *Hemiberlesia lingua* varied depending on the origin, with neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid having relatively high contents (see Table 5).

[0107] Table 5. Results of content determination of 7 components

[0108]

[0109]

[0110] 2.7 Chemical Pattern Recognition Analysis

[0111] 2.7.1 Cluster Analysis

[0112] The common peaks of stems and leaves from 15 batches of black dragon bone from different origins were imported into SPSS 26.0 software for cluster analysis. The between-groups linkage method was used, and Euclidean squared distance was selected as the clustering formula. The clustering results showed that when the distance was less than 15, the samples could be clustered into three classes: S3, S11, S1, S9, S2, and S5 belonged to class I; S8, S10, S4, S12, S6, and S7 belonged to class II; and S14, S15, and S13 belonged to class III (see...). Figure 3 ).

[0113] 2.7.2 Principal Component Analysis

[0114] Principal component analysis was performed on the peak areas of 28 common peaks from 15 batches of black dragon bone stems and leaves from different origins. Using eigenvalues ​​>1 and variance contribution rates >80% as criteria, seven principal components were extracted, with a cumulative contribution rate of 93.155%, which can reflect 93.155% of the information in the fingerprint spectrum of black dragon bone stems and leaves (see Table 6). The scree plot further demonstrates that the extracted seven principal components can characterize most of the information in the black dragon bone stems and leaves (see Table 6). Figure 4 ).

[0115] Table 6. Principal component analysis eigenvalues ​​and variance contribution rates of Heilongjiang stem and leaf samples.

[0116]

[0117] The factor loading matrix reflects the correlation coefficients between the principal components and the original variables. Table 7 shows that principal component 1 reflects the information of peak 4 (neochlorogenic acid), peak 22 (isoquercetin), peak 23, peak 11 (cryptochlorogenic acid), peak 17, and peak 8 (chlorogenic acid); principal component 2 mainly reflects the information of peak 20, peak 21, peak 6, and peak 7; principal component 3 mainly reflects the information of peak 3 and peak 16; principal component 4 mainly reflects the information of peak 26 and peak 28 (isochlorogenic acid C); principal component 5 reflects the information of peak 10 and peak 20; principal component 6 mainly reflects the information of peak 25; and principal component 7 mainly reflects the information of peak 5.

[0118] Table 7 Principal Component Factor Matrix of Black Dragon Bone Stems and Leaves

[0119]

[0120] Twenty-eight common peaks from the stems and leaves of 15 batches of black dragon bone from different origins were imported into SIMCA14.0 software to obtain principal component score plots. Figure 5 The principal component scores were calculated using SPSS software, and the comprehensive score was calculated using the variance contribution rate corresponding to each principal component as the weight. Zcomprehensive = 0.315Z1 + 0.183Z2 + 0.174Z3 + 0.123Z4 + 0.088Z5 + 0.069Z6 + 0.045Z7 (Table 8). The results in Table 8 show that S14 (No. 2, Yongle Township, Nanming District, Guiyang City, Guizhou Province) had the highest comprehensive score, followed by S12 (Liuzhi, Liupanshui City, Guizhou Province) and S6 (Xixiu District, Anshun City, Guizhou Province). This further illustrates that among the 15 batches of production areas, the stem and leaf quality of Black Dragon Bone from No. 2, Yongle Township, Nanming District, Guiyang City, Guizhou Province is the best, followed by Liuzhi, Liupanshui City, Guizhou Province, and then Xixiu District, Anshun City, Guizhou Province.

[0121] Table 8. Principal component scores and overall scores of Black Dragon Bone Stem and Leaves

[0122]

[0123] 2.7.3 Partial Least Squares Discriminant Analysis

[0124] To further analyze the differences among the stem and leaf samples of *Hemiberlesia lingua*, partial least squares discriminant analysis was performed based on principal component analysis, resulting in a partial least squares discriminant analysis score map. Figure 6 ). RY 2 =0.947, Q 2＝ The values ​​were all greater than 0.500, indicating a good model fit. Variable projection importance (VIP) is an important indicator for screening differences between groups. Using a VIP value greater than 1 as the screening criterion, 13 differentially expressed components were selected. Figure 7The peaks were 9 (caffeic acid), 24, 8 (chlorogenic acid), 12, 4 (neochlorogenic acid), 11 (cryptochlorogenic acid), 17, 14, 23, 3, 13, 22 (isoquercetin), and peak 7, indicating that these components were the main reasons for the quality differences among the 15 batches.

[0125] 3 Discussion

[0126] This study investigated the effects of mobile phases (methanol-0.1% formic acid solution, methanol-0.1% acetic acid solution, methanol-0.1% phosphoric acid solution, acetonitrile-0.1% phosphoric acid solution, and acetonitrile-0.1% formic acid solution) on baseline stability, peak shape, and resolution. The effects of column temperature (25℃, 30℃, 35℃), flow rate (0.1 mL / min, 0.2 mL / min, 0.3 mL / min), and injection volume (0.5 vL, 0.8 vL, 1 vL, 2 vL) on these parameters were determined. Ultimately, the optimal chromatographic conditions were selected: mobile phase of acetonitrile-0.1% phosphoric acid solution, column temperature of 30℃, flow rate of 0.2 mL / min, and injection volume of 0.8 vL.

[0127] This study used a DAD detector to perform full-wavelength scanning of single reference standards for seven components. The results showed that neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, and isochlorogenic acid C had the maximum UV absorption wavelength at 327 nm. Isoquercetin and astragaloside both showed UV absorption at 202 nm and 254 nm. The baseline was more stable and the separation was higher at 254 nm, so the detection wavelength was selected as 254 nm.

[0128] Cluster analysis showed that the 15 batches of black dragon bone samples could be clustered into three groups: S1-S3, S9, S5, and S11 clustered in one group; S6-S8, S10, S4, and S12 clustered in another group; and S13-S15 clustered in yet another group. The OPLS-DA analysis results were consistent with the HCA results. In the PCA analysis, based on the principal component eigenvalues ​​and variance contribution rates, seven principal components were extracted, with a cumulative contribution rate of 93.155%, reflecting most of the information from the stems and leaves of the 15 batches of black dragon bone. The comprehensive principal component scores indicated that sample S14 (No. 2, Yongle Township, Nanming District, Guiyang City, Guizhou Province) had superior quality, consistent with the component content determination results. OPLS-DA, using VIP>1, identified 9 (caffeic acid), 24, 8 (chlorogenic acid), 12, 4 (neochlorogenic acid), 11 (cryptochlorogenic acid), 17, 14, 23, 3, 13, 22 (isoquercetin), and peak 7 as the main differentiating components. These components can be used to distinguish the quality differences between different production areas, providing a reference for the quality evaluation and control of black dragon bone stems and leaves.

Claims

1. A method for measuring the quality of rhizoma drynariae, characterized by: The quality determination method of the stem and leaf of Smilax china includes the establishment of fingerprint, similarity analysis, content determination, cluster analysis, principal component analysis and partial least squares discriminant analysis. The specific steps are as follows: (1) Preparation of mixed reference solution: precisely take 14.5-16.5 mg of neochlorogenic acid, 11.5-13.5 mg of chlorogenic acid and 11.5-13.5 mg of cryptochlorogenic acid into a 10 ml volumetric flask, add 75% ethanol solution to the mark, shake well, and prepare mixed reference solution 1; precisely take 2-4 mg of caffeic acid, 4.5-6.5 mg of isoquercitrin, 4-6 mg of astragalin and 7.5-9.5 mg of isochlorogenic acid C into a 50 mL volumetric flask, add 75% ethanol to the mark, shake well, and prepare mixed reference solution 2; take 1 ml from mixed reference solution 1 and mixed reference solution 2 into the same 10 ml volumetric flask, add 75% ethanol solution to the mark, shake well, and prepare mixed reference solution 3; (2) Preparation of test solution: precisely take 0.4-0.6 g of Smilax china stem and leaf sample into a 20 ml conical flask, add 75% ethanol 10 mL, precisely weigh, ultrasonic extraction for 20-40 min, cool, make up the weight loss, shake well, and pass through a 0.22 μm microporous filter to obtain the test solution; (3) Chromatographic conditions: Agilent Eclipse Plus C18 column, column specification 2.1x100mm, 1.8 μm, volume flow rate 0.2 mL / min, acetonitrile as mobile phase B, 0.1% phosphoric acid water as mobile phase A, gradient elution, injection volume 0.8 μL, column temperature 30℃, detection wavelength 1-49 min, 327 nm; 49-70 min, 254 nm; 70-85 min, 327 nm; Elution gradient: 0-9 min, 7-7.5% B; 9-28 min, 7.5-9% B; 28-31 min, 9-13% B; 31-55 min, 13-13% B; 55-60 min, 13-14% B; 60-62 min, 14-16% B; 62-67 min, 16-18% B; 67-72 min, 18-18% B; 72-75 min, 18-21.5% B; 75-80 min, 21.5-65% B; 80-85 min, 65-95% B; (4) Fingerprint establishment and similarity analysis: The test solution of Radix et Caulis Aristolochiae from different producing areas was determined by UPLC under chromatographic conditions, the sample chromatogram was converted into AIA format, the data was imported into the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint 2012 edition, the chromatogram of the first batch of samples was taken as the reference chromatogram, the time window width was set to 0.1, the multi-point correction was used, the Radix et Caulis Aristolochiae fingerprint superimposed chromatogram and the control chromatogram were generated through marker peak matching, and the similarity calculation was performed to obtain common peaks; the mixed control solution ③ was determined by injection under chromatographic conditions to obtain the control chromatogram, the characteristic peaks were identified through comparison of the control chromatogram peaks and the ultraviolet absorption wavelength, and the fingerprint was established; (5) Content determination: the test solution and the mixed control solution ③ were detected by UPLC under chromatographic conditions, neogreen acid, green acid, cryptogreen acid, caffeic acid, isoquercitrin, astragalin and isogreen acid C in Radix et Caulis Aristolochiae were effectively separated, the peak area was recorded, and the contents of neogreen acid, green acid, cryptogreen acid, caffeic acid, isoquercitrin, astragalin and isogreen acid C in the test solution were calculated; (6) Different producing areas of Radix et Caulis Aristolochiae were analyzed by combining cluster analysis, principal component analysis and partial least squares discriminant analysis.

2. The method for measuring the quality of Asparagus racemosus according to claim 1, wherein: In step (1), preparation of the mixed control solution: 15-16 mg of neogreen acid, 12-13 mg of green acid and 12-13 mg of cryptogreen acid were precisely weighed and placed in a 10 ml volumetric flask, 75% ethanol solution was added to constant volume, shaken and mixed to prepare the mixed control solution ①; in addition, 2.5-3.6 mg of caffeic acid, 5-6 mg of isoquercitrin, 4.5-5.5 mg of astragalin and 8-9 mg of isogreen acid C were precisely weighed and placed in a 50 mL volumetric flask, 75% ethanol was added to constant volume, shaken and mixed to prepare the mixed control solution ②; 1 ml of the mixed control solution ① and 1 ml of the mixed control solution ② were taken and placed in the same 10 ml volumetric flask, 75% ethanol solution was added to constant volume, shaken and mixed to prepare the mixed control solution ③.

3. The method of measuring the quality of Asparagus racemosus stem and leaf as claimed in claim 2, wherein: In step (1), preparation of the mixed control solution: 15-16 mg of neogreen acid, 12-13 mg of green acid and 12-13 mg of cryptogreen acid were precisely weighed and placed in a 10 ml volumetric flask, 75% ethanol solution was added to constant volume, shaken and mixed to prepare the mixed control solution ①; in addition, 2.5-3.6 mg of caffeic acid, 5-6 mg of isoquercitrin, 4.5-5.5 mg of astragalin and 8-9 mg of isogreen acid C were precisely weighed and placed in a 50 mL volumetric flask, 75% ethanol was added to constant volume, shaken and mixed to prepare the mixed control solution ②; 1 ml of the mixed control solution ① and 1 ml of the mixed control solution ② were taken and placed in the same 10 ml volumetric flask, 75% ethanol solution was added to constant volume, shaken and mixed to prepare the mixed control solution ③.

4. The method of claim 1, wherein the mass of the rhizome is determined by the method of claim 1. In step (2), the preparation of the test solution: accurately weigh 0.45-0.55g of the stem and leaf sample of Adiantum capillus-veneris, put it in a 20ml conical flask, add 75% ethanol 10mL, accurately weigh, ultrasonic extraction for 25-35min, ultrasonic power 200W, ultrasonic frequency 40KHZ, cool, make up the weight loss, shake well, pass through a 0.22μm microporous filter membrane, and the test solution is obtained.

5. The method of claim 4, wherein the mass of the rhizome is determined by the method of claim 1. In step (2), the preparation of the test solution: accurately weigh 0.45-0.55g of the stem and leaf sample of Adiantum capillus-veneris, put it in a 20ml conical flask, add 75% ethanol 10mL, accurately weigh, ultrasonic extraction for 25-35min, ultrasonic power 200W, ultrasonic frequency 40KHZ, cool, make up the weight loss, shake well, pass through a 0.22μm microporous filter membrane, and the test solution is obtained.

6. The method of claim 1, wherein the mass of the rhizome is determined by the method of claim 1. In step (4), the characteristic peaks are: peak 4 is neochlorogenic acid, peak 8 is chlorogenic acid, peak 9 is caffeic acid, peak 11 is cryptochlorogenic acid, peak 22 is isoquercitrin, peak 24 is astragalin, and peak 28 is isochlorogenic acid C.

7. The method of claim 1, wherein the mass of the rhizome is determined by the method of claim 1. In step (6), the cluster analysis is to import the common peaks of the stem and leaf samples of Adiantum capillus-veneris from different producing areas into SPSS 26.0 software for cluster analysis, to use the intergroup connection method, and to select the Euclidean square distance as the cluster formula.

8. The method of claim 1, wherein the mass of the rhizome is determined by the method of claim 1. In step (6), the principal component analysis is to use the peak area of the common peaks of the stem and leaf samples of Adiantum capillus-veneris from different producing areas as variables for principal component analysis, to extract principal components according to the characteristic value and variance contribution rate, and to make a scatter plot for further analysis of the principal component factor matrix of the stem and leaf samples of Adiantum capillus-veneris. Finally, the common peaks are imported into SIMCA 14.0 software to obtain the principal component score plot, and the principal component score is calculated by using SPSS software, and the comprehensive score is calculated by using the variance contribution rate corresponding to each principal component as the weight.

9. The method of claim 1, wherein the mass of the rhizome is determined by the method of claim 1. In step (6), the partial least squares discriminant analysis is based on the principal component analysis to obtain the partial least squares discriminant analysis score plot.

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