A method for detecting the contents of seven phenolic acids in ramie roots based on fingerprint analysis combined with a one-test-multiple-evaluation method

Abstract

This invention discloses a method for determining the content of seven phenolic acid components in ramie roots based on fingerprint spectroscopy combined with a multi-component assay, belonging to the technical field of detection methods. The method includes the preparation of a reference solution, the preparation of a test solution, the establishment of a fingerprint spectroscopy, the determination of multiple component contents, and the calculation of relative correction factors. The method of this invention establishes a fingerprint spectroscopy of ramie roots, identifies 16 common peaks, and identifies seven components: protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid, and p-coumaric acid. The similarity of 11 batches of ramie root samples was greater than 0.87, indicating that the quality of ramie roots from different origins is relatively uniform. The RSDs of the relative correction factors and relative retention times of each component are all less than 2.00%, and different flow rates, column temperatures, instruments, and chromatographic columns have little effect on them. There is no significant difference between the results determined by the multi-component assay and the external standard method. The fingerprint spectroscopy combined with the multi-component assay method established in this invention is feasible and can provide a theoretical basis for the quality control of ramie root medicinal materials.

Description

Technical Field

[0001] This invention relates to the field of detection methods, and in particular to a method for determining the content of seven phenolic acid components in ramie roots based on fingerprinting combined with a multi-analysis method. Background Technology

[0002] Ramie root is the dried rhizome and root of *Boehmeria nivea* (L.) Gaud., a plant in the Urticaceae family. It is mainly produced in Jiangsu, Shandong, and Shaanxi provinces. The best quality ramie root is grayish-brown in color and without hollow stems. It is bitter and cold in nature, and enters the heart, liver, kidney, and bladder meridians. Clinically, it is mainly used for colds with fever, measles with high fever, urinary tract infections, nephritis with edema, abdominal pain in pregnant women, sores and swelling, and hemorrhagic diseases. The *Compendium of Materia Medica Supplement* records that it "treats various poisons, invigorates blood, and stops bleeding. It also has the functions of dispersing, quenching thirst, calming the fetus, relieving abdominal distension, metrorrhagia, asthma, leukorrhea, spermatorrhea, toothache, sore throat, hernia, and traumatic injuries." Modern research shows that ramie root has the effects of cooling the blood and stopping bleeding, clearing heat and calming the fetus, promoting diuresis and detoxification, protecting the liver and anti-oxidation. Literature also reports that plants in the *Boehmeria* genus mainly contain triterpenoids, flavonoids, phenolic acids, and alkaloids. Studies by Zhang Hongqi et al. have found that phenolic acid components in ramie leaves not only have multiple effects such as anti-inflammatory, analgesic, and improvement of microcirculation and blood rheology, but also have a good repair effect on acute soft tissue injuries. Studies by Liu Xiaoyu et al. have found that chlorogenic acid in ramie roots can increase white blood cell levels and can be used for post-cancer nutritional support.

[0003] Traditional Chinese medicine (TCM) is characterized by its complex composition and multiple medicinal effects. Establishing fingerprint spectroscopy to simultaneously determine the content of multiple components can better reflect its quality. Modern clinical studies have found that ramie root, used as a single herb or in compound formulas, can treat various diseases. Traditional multi-component determination methods are costly, time-consuming, and complex to operate; the one-test-multiple-evaluation method can effectively overcome these shortcomings. Currently, the one-test-multiple-evaluation method is widely used for the determination of multiple component contents in TCM and TCM compound preparations, and is included in the Chinese Pharmacopoeia for many varieties. Simultaneously, fingerprint spectroscopy, as a technique for inspecting the overall quality control of medicinal materials, can effectively reflect the quality uniformity between different samples.

[0004] There are 120 species of ramie worldwide, mainly distributed in tropical and subtropical regions, with a few in temperate zones. 75 species are found in Asia, 30 in the Americas, and a few in Oceania and Africa. my country has abundant wild and cultivated ramie resources, accounting for over 90% of the world's ramie production. Ramie is not only an important fiber-rich economic crop, but its roots are also a traditional Chinese medicine with a long history of medicinal use in my country. However, the current Chinese Pharmacopoeia does not include ramie roots, and there are no reports of research on fingerprinting and multi-index component determination of ramie roots. Therefore, fingerprinting combined with multi-component and multi-index quality control methods is of great significance for the quality research of ramie roots. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for determining the content of seven phenolic acid components in ramie roots based on fingerprinting combined with a multi-analysis method. This method provides a theoretical basis for the quality control of ramie root medicinal materials. The contents of this invention are as follows:

[0006] A method for determining the content of seven phenolic acid components in ramie roots based on fingerprinting combined with a multi-analysis method, the technical points of which include the following steps:

[0007] Step 1, Preparation of reference solution: Dissolve protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid and p-coumaric acid in methanol in a volumetric flask to obtain the reference solution;

[0008] Step 2, preparation of the test solution: Take about 100 grams of ramie root medicinal material sample powder and place it in a stoppered conical flask. Add water, heat and reflux to extract, cool, filter, evaporate the filtrate to dryness, add methanol to dissolve and make up to volume, filter and take the filtrate to obtain the test solution.

[0009] Step 3, fingerprint chromatogram establishment: Take different test solutions, record the fingerprint chromatograms of each sample under liquid chromatography conditions, import the data into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", set the reference chromatogram for the fingerprint chromatogram, perform full peak matching, and generate the control fingerprint chromatogram using the average method.

[0010] Step 4, determination of multiple component contents;

[0011] Step 5, Calculation of relative correction factor: Take 1, 2, 4, 5, 8, and 10 μL of the test solution and determine the sample under the same liquid chromatography conditions as in Step 3. Use chlorogenic acid as an internal reference and calculate the relative correction factor according to the formula ƒsi=ƒs / ƒi=(As / Cs) / (Ai / Ci), where As is the peak area of ​​the chlorogenic acid internal reference, Cs is the concentration of the chlorogenic acid internal reference, Ai is the peak area of ​​the analyte, and Ci is the concentration of the analyte. Take the average value as ƒsi for quantification.

[0012] Step 6, Confirmation of relative retention time;

[0013] Step 7, Durability assessment;

[0014] Step 8, chromatographic peak localization;

[0015] Step 9, determination using the one-test-multiple-evaluation method.

[0016] This invention establishes a fingerprint chromatogram for determining the content of seven phenolic acid components in ramie roots based on fingerprinting combined with a single-analysis-multiple-evaluation method. Sixteen common peaks were identified, and seven components—protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid, and p-coumaric acid—were identified. The similarity of 11 batches of ramie root samples was greater than 0.87, indicating that the quality of ramie roots from different origins is relatively uniform. The RSDs of the relative correction factors and relative retention times for each component were all less than 2.00%, and different flow rates, column temperatures, instruments, and chromatographic columns had little impact on them. There was no significant difference between the results determined by the single-analysis-multiple-evaluation method and the external standard method. Conclusion: The fingerprinting method combined with the single-analysis-multiple-evaluation method established in this invention is feasible and can provide a theoretical basis for the quality control of ramie root medicinal materials. Attached Figure Description

[0017] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0018] Figure 1 Chromatograms of 11 batches of ramie root samples

[0019] In the figure: 5. Protocatechuic acid; 7. Neochlorogenic acid; 8. Protocatechuic aldehyde; 10. Chlorogenic acid; 11. Cryptochlorogenic acid; 12. Caffeic acid; 15. p-Coumaric acid;

[0020] Figure 2 This is a chromatogram comparing the mixed reference standard of the present invention with a ramie root sample.

[0021] In the figure: 5. Protocatechuic acid; 7. Neochlorogenic acid; 8. Protocatechuic aldehyde; 10. Chlorogenic acid; 11. Cryptochlorogenic acid; 12. Caffeic acid; 15. p-Coumaric acid;

[0022] Figure 3 Chromatograms of blank solvent (A), mixed reference standard (B), and ramie root sample (C).

[0023] In the diagram: 1. Protocatechuic acid; 2. Neochlorogenic acid; 3. Protocatechuic aldehyde; 4. Chlorogenic acid; 5. Cryptochlorogenic acid; 6. Caffeic acid; 7. p-Coumaric acid. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] The technical solutions provided by the various embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0026] This invention establishes a fingerprint spectroscopy combined with Quality Assay Multiple Selection (QAMS) to evaluate the quality of ramie roots. Chlorogenic acid, a common phenolic acid compound, is selected as an internal reference. The contents of six other phenolic acid components in ramie roots are calculated using relative correction factors. This invention is the first to establish a fingerprint spectroscopy for ramie roots and simultaneously determine seven phenolic acid components. It provides a simple, rapid, comprehensive, and accurate multi-index quality evaluation of ramie roots, helping to ensure controllable ramie root quality while saving testing costs and time. Furthermore, the established method has good reliability and can provide a theoretical basis for the quality control of ramie roots.

[0027] 1. Instruments and Materials

[0028] 1.1 Instruments

[0029] Ultra-high performance liquid chromatograph (UHPLC) (Waters PDA, Waters Corporation, USA); Ultra-high performance liquid chromatograph (Waters TUV, Waters Corporation, USA); Ultra-high performance liquid chromatograph (Agilent 1260, Agilent Technologies, USA); Agilent ZORBAX Eclipse XDB-C18 column (4.6mm × 250mm, 5μm); Phenomenex Luna C18 column (4.6mm × 250mm, 5μm); Thermo Acclaim™ 120C18 Chromatographic column (4.6mm×250mm, 5μm); 0.0001 g balance (Quintix-ICN, Sartorius GmbH, Germany); 0.1 million g balance (XP26, Mettler Toledo, Switzerland); 0.1 g balance (Quintix-ICN, Sartorius GmbH, Germany); Ultrasonic cleaner (SB-800DT, Ningbo Xinzhi Biotechnology Co., Ltd.); Ultrapure water system (UPT-II-20T, Sichuan Youpu Ultrapure Technology Co., Ltd.)

[0030] 1.2 Materials

[0031] Reagents: Methanol (Hunan Huihong Reagent Co., Ltd., analytical grade); phosphoric acid (Tianjin Kemeio Chemical Reagent Co., Ltd., chromatographic grade); acetonitrile (Merck AG, Germany, chromatographic grade); ultrapure water (laboratory-made).

[0032] Test reagents: Protocatechuic acid (batch number: 110809-202207, content 97.5%), protocatechuic aldehyde (batch number: 110810-202210, content 99.9%), chlorogenic acid (batch number: 110753-202119, content 96.3%), caffeic acid (batch number: 110885-201703, content 99.7%), p-coumaric acid (batch number: 112037-202102, content 99.7%) were all purchased from the National Institutes for Food and Drug Control, while neochlorogenic acid (batch number: 23103101, content 99.38%) and cryptochlorogenic acid (batch number: 3208, content 98.0%) were all purchased from Chengdu Glip Biotechnology Co., Ltd.

[0033] Eleven batches of ramie root medicinal materials were identified by Professor Sun Dongmei of Hunan Yifang Tianjiang Pharmaceutical Co., Ltd. as dried rhizomes and roots of Boehmeria nivea (L.) Gaud., a plant of the Urticaceae family. After testing, all of them met the requirements of the ramie root medicinal materials section of the 2010 edition of the "Hunan Province Standard for Processing Traditional Chinese Medicine Pieces". The source information of the medicinal materials is shown in Table 1.

[0034] Table 1. Source Information of 11 Batches of Ramie Roots

[0035]

[0036] 2. Methods and Results

[0037] 2.1 Chromatographic conditions

[0038] Chromatographic column: Agilent ZORBAX Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B); gradient elution (0-10 min, 5% → 7% A; 10-15 min, 7% → 10% A; 15-25 min, 10% → 12% A; 25-30 min, 12% → 15% A; 30-40 min, 15% A; 40-45 min, 15% → 18% A); flow rate: 0.8 mL / min; column temperature: 25℃; detection wavelength: 310 nm; injection volume: 10 μL.

[0039] 2.2 Solution Preparation

[0040] 2.2.1 Preparation of reference solution

[0041] Accurately weigh appropriate amounts of protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid, and p-coumaric acid reference standards, place them in a volumetric flask, dissolve them in 50% methanol, and prepare a mixed reference solution containing 8.4084 μg of protocatechuic acid, 20.2536 μg of neochlorogenic acid, 3.4462 μg of protocatechuic aldehyde, 19.4526 μg of chlorogenic acid, 19.3256 μg of cryptochlorogenic acid, 1.9653 μg of caffeic acid, and 5.7986 μg of p-coumaric acid per 1 mL.

[0042] 2.2.2 Preparation of the test solution

[0043] Accurately weigh approximately 2.0g of ramie root powder and place it in a stoppered conical flask. Add 100mL of water, heat under reflux for 45min, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 50% methanol, dilute to 10mL in a volumetric flask, shake well, filter, and collect the filtrate to obtain the final product.

[0044] 2.3 Establishment of fingerprint patterns

[0045] 2.3.1 Precision Test

[0046] Ramie root powder (sample number S2) was used to prepare a test solution according to the method described in section "2.2.2". The solution was then injected six times consecutively under the chromatographic conditions described in section "2.1". Using the chlorogenic acid peak as a reference peak, the RSD values ​​of the relative retention times and relative peak areas of the remaining common peaks were calculated. The results showed that the RSDs of the relative retention times and relative peak areas of all common peaks were less than 2.0%, indicating good instrument precision.

[0047] 2.3.2 Repeatability Test

[0048] Six test solutions were prepared from the same batch of ramie root powder (number S2) according to the method described in section "2.2.2". These solutions were then injected and analyzed under the chromatographic conditions described in section "2.1". Using the chlorogenic acid peak as a reference peak, the RSD values ​​of the relative retention times and relative peak areas of the remaining common peaks were calculated. The results showed that the RSD of the relative retention time of each common peak was less than 1.0%, and the RSD of the relative peak area was less than 3.0%, indicating good repeatability of the method.

[0049] 2.3.3 Stability Test

[0050] Ramie root powder (No. S2) was used to prepare a test solution according to the method described in section "2.2.2". The solution was then injected and analyzed at 0, 2, 4, 8, 12, and 24 hours under the chromatographic conditions described in section "2.1". Using the chlorogenic acid peak as a reference peak, the RSD values ​​of the relative retention time and relative peak area of ​​the remaining common peaks were calculated. The results showed that the RSD of the relative retention time of each common peak was less than 2.0%, and the RSD of the relative peak area was less than 3.0%, indicating that the test sample was stable within 24 hours.

[0051] 2.3.4 Fingerprint pattern establishment and similarity evaluation

[0052] Eleven batches of ramie root samples were collected, and test solutions were prepared according to the method described in section "2.2.2". The samples were then injected and analyzed under the chromatographic conditions described in section "2.1", and the fingerprint chromatograms of each sample were recorded. The data were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine". Using the fingerprint chromatogram of sample S1 as a reference chromatogram, full peak matching was performed, and a control fingerprint chromatogram was generated using the mean method. A total of 16 common peaks were identified. The overlay plot is shown below. Figure 1 Seven chromatographic peaks were identified by comparison with the reference standard; the comparison chromatogram is shown below. Figure 2 Peak 5 is protocatechuic acid, peak 7 is neochlorogenic acid, peak 8 is protocatechuic aldehyde, peak 10 is chlorogenic acid (reference peak), peak 11 is cryptochlorogenic acid, peak 12 is caffeic acid, and peak 15 is p-coumaric acid. The similarity between the fingerprint chromatograms and those of 11 batches of ramie root samples was evaluated, and the results are shown in Table 2. The results show that the similarity of the fingerprint chromatograms of all 11 batches of ramie root samples is greater than 0.87, indicating that the fingerprint chromatograms of common peaks of ramie roots from different origins have high similarity, and the quality of the medicinal materials is relatively uniform.

[0053] Table 2. Similarity Table of Ramie Roots in 11 Batches

[0054]

[0055] 2.4 Determination of the content of multiple components

[0056] 2.4.1 Specificity Test

[0057] Take ramie root powder (No. S2), prepare the test solution according to the method in section "2.2.2", and inject the blank solvent, mixed standard, and test solution under the chromatographic conditions in section "2.1" for determination. The results are shown below. Figure 3 The resolution of each chromatographic peak was greater than 1.5, and there was no interference from the blank solvent. This indicates that the method has good specificity.

[0058] 2.4.2 Examination of Linear Relationships

[0059] Take the mixed reference solution from section "2.2.1", and inject 2 μL, 5 μL, 10 μL, 20 μL, 50 μL, and 100 μL respectively under the chromatographic conditions described in section "2.1" for determination. Plot the peak area of ​​the target peak as the ordinate (Y) and the concentration as the abscissa (X), and perform regression analysis. The results are shown in Table 3. This indicates that each component has a good linear relationship within its respective range.

[0060] Table 3 Linear Relationships of Each Component

[0061]

[0062] 2.4.3 Precision Test

[0063] Take the mixed reference solution from section "2.2.1", and inject it six times consecutively under the chromatographic conditions described in section "2.1" to determine protocatechuic acid and neocatechuic acid.

[0064] The peak area RSDs of chlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid, and p-coumaric acid were 0.65%, 0.37%, 0.62%, 0.27%, 0.22%, 0.43%, and 0.29%, respectively, indicating that the instrument has good precision.

[0065] 2.4.4 Repeatability Test

[0066] Six test solutions were prepared from the same batch of ramie root powder (No. S3) according to the method in section "2.2.2". The solutions were injected and determined under the chromatographic conditions in section "2.1". The RSDs of protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid, and p-coumaric acid were 0.62%, 0.50%, 0.63%, 2.25%, 0.17%, 0.70%, and 0.54%, respectively, indicating that the method has good repeatability.

[0067] 2.4.5 Stability Test

[0068] Take the same test solution under section "2.1.3" and inject it at 0, 2, 4, 6, 8, 12 and 24 hours according to the chromatographic conditions under section "2.1". The RSDs of protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid and p-coumaric acid were 0.87%, 1.22%, 1.67%, 1.86%, 0.65%, 1.14% and 0.69%, respectively, indicating that the test solution has good stability within 24 hours.

[0069] 2.4.6 Recovery Test

[0070] Accurately weigh 1.0 g of ramie root sample (S3) with determined content, in six parallel applications. Separately, accurately weigh 3.144 mg of protocatechuic acid reference standard, 4.987 mg of neochlorogenic acid reference standard, 5.811 mg of chlorogenic acid reference standard, 4.813 mg of cryptochlorogenic acid reference standard, and 2.307 mg of p-coumaric acid reference standard, placing each in a separate 5 mL volumetric flask. Separately, accurately weigh 3.326 mg of protocatechuic aldehyde reference standard and 2.129 mg of caffeic acid reference standard, placing each in a separate 20 mL volumetric flask. Add an appropriate amount of 50% methanol, sonicate to dissolve, and dilute to the mark. Shake well to obtain single reference standard solutions. Accurately measure 1 mL of each of the above reference standard solutions and place them in the same 10 mL volumetric flask. Add 50% methanol to the mark and shake well to obtain the mixed reference standard stock solution. 1 mL of the mixed reference standard stock solution was precisely added to each of the above 6 samples. The test solution was prepared according to the method in section "2.2.2". The chromatographic conditions in section "2.1" were used for determination. The peak areas of each component were recorded and the recovery rate was calculated. The results are shown in Table 4.

[0071] Table 4. Results of recovery tests for each component (n=6)

[0072]

[0073] 2.5 Calculation of Relative Correction Factor

[0074] Accurately pipette 1, 2, 4, 5, 8, and 10 μL of the mixed reference solution under section "2.2.1" and determine the chromatographic conditions under section "2.1". Use chlorogenic acid as an internal reference and calculate the relative correction factor according to the formula ƒsi = ƒs / ƒi = (As / Cs) / (Ai / Ci), where As is the peak area of ​​the chlorogenic acid internal reference, Cs is the concentration of the chlorogenic acid internal reference, Ai is the peak area of ​​the analyte, and Ci is the concentration of the analyte. Take the average value as ƒsi for quantification. The results are shown in Table 5.

[0075] Table 5 Relative Correction Factors for Each Component

[0076]

[0077] The results showed that the relative correction factors for protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, cryptochlorogenic acid, caffeic acid, and p-coumaric acid were 4.588, 0.985, 0.690, 1.063, 0.513, and 0.362, respectively, with RSDs of 1.38%, 0.22%, 0.39%, 0.26%, 1.52%, and 0.61%, respectively.

[0078] 2.6 Confirmation of relative retention time

[0079] Accurately pipette 1, 2, 4, 5, 8, and 10 μL of the mixed reference solution under section "2.2.1" and determine the relative retention times of the other six components under the chromatographic conditions under section "2.1", using chlorogenic acid as an internal reference. The results are shown in Table 6.

[0080] Table 6 Relative retention times of each component

[0081]

[0082] The results showed that the relative retention times of protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, cryptochlorogenic acid, caffeic acid, and p-coumaric acid were 0.601, 0.717, 0.845, 1.096, 1.170, and 1.593, respectively, with RSDs of 0.23%, 0.12%, 0.10%, 0.00%, 0.03%, and 0.07%, respectively.

[0083] 2.7 Durability Test

[0084] 2.7.1 Flow rate

[0085] Take the mixed reference solution from section "2.2.1" and determine it according to the chromatographic conditions in section "2.1". Investigate the effect of three different flow rates (0.7, 0.8, and 0.9 mL / min) on the relative correction factor. The results are shown in Table 7.

[0086] Table 7 Relative correction factors for different flow rates

[0087]

[0088] The results showed that different flow rates had no significant effect on the relative correction factor. The relative correction factors RSD for protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, cryptochlorogenic acid, caffeic acid, and p-coumaric acid were 0.51%, 0.31%, 0.28%, 0.50%, 0.35%, and 0.15%, respectively.

[0089] 2.7.2 Column Temperature

[0090] Take the mixed reference solution from section "2.2.1" and determine it according to the chromatographic conditions in section "2.1". Investigate the effect of three different column temperatures (20, 25, and 30℃) on the relative correction factor. The results are shown in Table 8.

[0091] Table 8. Relative correction factors at different column temperatures

[0092]

[0093] The results showed that different column temperatures had no significant effect on the relative correction factor. The relative correction factors RSD for protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, cryptochlorogenic acid, caffeic acid, and p-coumaric acid were 0.82%, 0.21%, 0.37%, 0.32%, 0.58%, and 0.60%, respectively.

[0094] 2.7.3 Instruments and Chromatographic Columns

[0095] Take the mixed reference solution from section "2.2.1", and select Waters PDA, Waters TUV, and Agilent 1260 high-performance liquid chromatographs, as well as Agilent ZORBAX Eclipse XDB-C18 (4.6mm × 250mm, 5μm), Phenomenex Luna C18 (4.6mm × 250mm, 5μm), and Thermo Acclaim™ 120 C18 (4.6mm × 250mm, 5μm) columns, and determine the chromatographic conditions from section "2.1" to investigate the effect of different instruments and different columns on the relative correction factor. The results are shown in Table 9.

[0096] Table 9. Relative correction factors for different instruments and chromatographic columns

[0097]

[0098] The results showed that the influence of different instruments and chromatographic columns on the relative correction factors was within an acceptable range. The relative correction factor RSDs for protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, cryptochlorogenic acid, caffeic acid, and p-coumaric acid were 3.41%, 0.58%, 1.43%, 0.39%, 1.84%, and 1.92%, respectively.

[0099] 2.8 Chromatographic Peak Localization

[0100] The relative retention time was used to identify the analyte. Using chlorogenic acid as a reference, the mixed reference solution from section "2.2.1" was taken and determined under the chromatographic conditions described in section "2.1". The reproducibility of the relative retention time under different flow rates, column temperatures, instruments, and columns was investigated. The results are shown in Table 10.

[0101] Table 10 Relative retention times for different flow rates, column temperatures, instruments, and columns.

[0102]

[0103] The results showed that the relative retention times of each analyte varied little, and chlorogenic acid could be used as a reference for chromatographic peak localization.

[0104] 2.9 Comparison of results between the single-measurement-multiple-evaluation method and the external standard method

[0105] Eleven batches of ramie root powder samples were collected, and test solutions were prepared according to the method described in section "2.2.2". The samples were injected and analyzed under the chromatographic conditions described in section "2.1". The contents of protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, cryptochlorogenic acid, caffeic acid, and p-coumaric acid in the samples were determined using the external standard method (ESM). The established quality-assay-multiple-analyses (QAMS) method was then used to calculate the results. To examine the accuracy of the QAMS method, the results were compared with those obtained using the external standard method (ESM). The results are shown in Table 11.

[0106] Table 11 Results of content determination using external standard method and one-test-multiple-evaluation method

[0107]

[0108] The results showed that there was no significant difference in the content of each component measured by the one-test-multiple-evaluation method and the external standard method, indicating that the established method has good reliability.

[0109] 3 Discussion

[0110] 3.1 Selection of internal reference materials

[0111] The current Chinese Pharmacopoeia does not include ramie root, and there are no research reports on fingerprint spectroscopy and multi-index component content determination of ramie root. Using fingerprint spectroscopy combined with multi-component and multi-index quantification can more accurately reflect the quality of ramie root. Chlorogenic acid, an indicator component in ramie root, has a high content, moderate retention time, and is relatively inexpensive. Therefore, this invention uses chlorogenic acid as an internal reference. The RSDs of the relative correction factors and relative retention times of each component are all less than 2.00%, indicating that the established one-test-multiple-evaluation method has good accuracy and high reliability.

[0112] 3.2 Sample pretreatment and selection of mobile phase

[0113] This invention compares ultrasonic extraction, reflux extraction, and water decoction extraction for 30, 45, and 60 min respectively, and compares extraction solvents such as water, methanol, ethanol, 50% methanol, and 50% ethanol. Ultimately, water decoction for 45 min was selected, followed by evaporation to dryness.

[0114] Dissolve in 50% methanol and bring to a final volume of 10 mL. Ramie roots contain a variety of phenolic acids with similar structures. If isocratic elution is used, compounds with similar structures are difficult to separate. Therefore, mobile phase systems of methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, and acetonitrile-0.2% phosphoric acid were compared and investigated. The results showed that gradient elution with acetonitrile-0.1% phosphoric acid aqueous solution provided good resolution of the chromatographic peaks of each indicative component.

[0115] 3.3 Sample content determination

[0116] The quality of Chinese medicinal materials is affected by both natural and human factors, which may lead to differences in quality between materials from different producing areas. This invention establishes a fingerprint spectrum for ramie roots and uses a multi-evaluation method to determine the content of seven phenolic acid components in ramie roots. Existing literature has not reported on fingerprint spectrum analysis and quantitative studies of multiple components in ramie roots. The qualitative and quantitative methods established in this invention can more comprehensively control and evaluate the quality of ramie roots. A total of 16 common peaks were identified in 11 batches of ramie root materials, and seven components were identified using reference standards. Except for S5, the similarity of all batches of samples was greater than 0.9, indicating that the quality of ramie root materials from different producing areas is relatively uniform. The lower similarity of sample S5 may be related to harvesting, sampling, and storage, reflecting the importance of these stages in the harvesting, processing, storage, and transportation of medicinal materials. This invention uses chlorogenic acid as an internal reference and calculates the content of six other phenolic acid components through relative correction factors. It is the first time that seven phenolic acid components in ramie roots have been simultaneously determined. Comparison with values ​​obtained using the external standard method revealed no significant difference between the two methods. The established fingerprint spectrum, combined with the one-test-multiple-evaluation method, is scientific, simple, specific, reproducible, accurate, and reliable, providing a theoretical basis for the quality control of ramie roots.

[0117] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for detecting the content of seven phenolic acid components in ramie roots based on fingerprinting combined with a single-test-multiple-evaluation method, characterized in that, Includes the following steps: Step 1, Preparation of reference solution: Dissolve protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid and p-coumaric acid in a volumetric flask with methanol-water solution to obtain the reference solution; Step 2, preparation of the test solution: Take the ramie root sample powder and place it in a stoppered conical flask. Add water, heat and reflux to extract, cool, filter, evaporate the filtrate to dryness, add methanol aqueous solution to dissolve and make up to volume, filter and take the filtrate to obtain the test solution. Step 3, fingerprint chromatogram establishment: Take different test solutions, record the fingerprint chromatograms of each sample under liquid chromatography conditions, import the data into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", set the reference chromatogram for the fingerprint chromatogram, perform full peak matching, and generate the control fingerprint chromatogram using the mean method. Step 4, Multi-component content determination: Calculation of relative correction factor: Take 1, 2, 4, 5, 8, and 10 μL of the test solution and determine the content under the same liquid chromatography conditions as in Step 3. Use chlorogenic acid as the internal reference and calculate the relative correction factor according to the formula ƒsi=ƒs / ƒi=(As / Cs) / (Ai / Ci), where As is the peak area of ​​the chlorogenic acid internal reference, Cs is the concentration of the chlorogenic acid internal reference, Ai is the peak area of ​​the analyte, and Ci is the concentration of the analyte. Take the average value as ƒsi for quantification. The chromatographic conditions in step 3 include: Chromatographic column: Agilent ZORBAX Eclipse XDB-C18 column; The mobile phases are as follows: mobile phase A is acetonitrile, and mobile phase B is a 0.1% aqueous solution of phosphoric acid. The gradient elution program was as follows: 0-10 min, 5% → 7% acetonitrile; 10-15 min, 7% → 10% acetonitrile; 15-25 min, 10% → 12% acetonitrile; 25-30 min, 12% → 15% acetonitrile; 30-40 min, 15% acetonitrile; 40-45 min, 15% → 18% acetonitrile. The flow rate was 0.7-0.9 mL per minute; the column temperature was 20-30℃; the detection wavelength was 310 nm; and the injection volume was 10 μL.

2. The method for detecting the content of seven phenolic acid components in ramie roots based on fingerprinting combined with a multi-analysis method, as described in claim 1, is characterized in that... Each 1 mL of the reference solution in step 1 contains a mixed reference solution of 8.4084 μg protocatechuic acid, 20.2536 μg neochlorogenic acid, 3.4462 μg protocatechuic aldehyde, 19.4526 μg chlorogenic acid, 19.3256 μg cryptochlorogenic acid, 1.9653 μg caffeic acid, and 5.7986 μg p-coumaric acid.

3. The method for detecting the content of seven phenolic acid components in ramie roots based on fingerprinting combined with a multi-analysis method according to claim 1, characterized in that, The preparation of the test solution in step 2 is as follows: 2.0 g of ramie root sample powder is accurately weighed, placed in a stoppered conical flask, 100 mL of water is added, and the mixture is heated under reflux for 45 min. After cooling, the sample is filtered, the filtrate is evaporated to dryness, the residue is dissolved in 50% methanol, and the solution is diluted to 10 mL in a volumetric flask. The solution is shaken well, filtered, and the filtrate is collected.

Images