Method for determining contents of seven phenolic acid components in ramie roots based on combination of fingerprint spectrum and quantitative analysis of multi-components by single marker

Abstract

The invention discloses a method for determining the contents of seven phenolic acid components in ramie roots based on a fingerprint spectrum in combination with a quantitative analysis of multi-components by single marker, and belongs to the technical field of detection methods, and the method comprises the following steps: preparing a reference substance solution, preparing a test solution, establishing the fingerprint spectrum, determining the contents of multiple components and calculating a relative correction factor. According to the method, the ramie root fingerprint spectrum is established, 16 common peaks are calibrated, seven components including protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid and p-coumaric acid are identified, the similarity of 11 batches of ramie root samples is larger than 0.87, and it is indicated that the quality of ramie roots of different producing areas is uniform. RSD (Relative Standard Deviation) of relative correction factors and relative retention time of each component is less than 2.00%, and different flow velocities, column temperatures, instruments and chromatographic columns have little influence. And the determination results of the quantitative analysis of multi-components by single marker and the external standard method have no obvious difference. The fingerprint spectrum established by the invention is feasible in combination with a quantitative analysis of multi-components by single marker, and a theoretical basis can be provided for quality control of the ramie root medicinal material.

Description

Technical Field

[0001] The invention relates to the technical field of detection methods, and in particular to a method for determining the contents of seven phenolic acid components in ramie roots based on a fingerprint spectrum combined with a one-measurement-multiple-evaluation method. Background Art

[0002] Ramie root is the dried rhizome and root of Boehmeria nivea (L.) Gaud. of Urticaceae. It is mainly produced in Jiangsu, Shandong, Shaanxi and other places. The medicinal material is gray-brown and not hollow. It tastes bitter and cold in nature. It enters the heart, liver, kidney and bladder meridians. It is mainly used for colds and fever, measles and high fever, urinary tract infection, nephritis edema, abdominal pain in pregnant women, sores and swelling, hemorrhagic diseases, etc. "Compendium of Materia Medica" records: "It can treat various poisons, promote blood circulation and stop bleeding. It has the functions of dispersing, quenching thirst, calming the fetus, relieving swelling, metrorrhagia, asthma, leucorrhea, spermatorrhea, toothache, throat closure, hernia, and traumatic injuries." Modern research shows that ramie root has the effects of cooling blood and stopping bleeding, clearing heat and calming the fetus, diuresis and detoxification, liver protection and anti-oxidation. At the same time, there are literature reports that Ramie plants mainly contain triterpenes, flavonoids, phenolic acids and alkaloids. Zhang Hongqi et al. found that the phenolic acid components of ramie leaves not only have multiple effects such as anti-inflammatory, analgesic, improving microcirculation and blood rheology, but also have a good repair effect on acute soft tissue injuries. Liu Xiaoyu et al. found that chlorogenic acid in ramie roots has the effect of increasing white blood cell levels and can be used for post-cancer nourishment.

[0003] The characteristics of traditional Chinese medicine are complex ingredients and multiple medicinal effects. By establishing a fingerprint spectrum and measuring the content of multiple ingredients at the same time, its quality can be better reflected. Modern clinical practice has found that ramie root can be used as a single medicine or a compound medicine to treat a variety of diseases. The traditional multi-component determination method has the disadvantages of high detection cost, long time, and complex operation. The one-test-multiple-evaluation method can make up for this shortcoming. At present, the one-test-multiple-evaluation method has been widely used in the determination of multi-component content of traditional Chinese medicine and traditional Chinese medicine compound preparations, and has been included in many varieties of the "Chinese Pharmacopoeia". At the same time, fingerprint spectrum, as a technology for testing the overall quality control of medicinal materials, can well reflect the quality uniformity between different samples.

[0004] There are 120 species of Ramie plants in the world, mainly distributed in tropical and subtropical areas, a few in temperate zones, 75 species distributed in Asia, 30 species distributed in America, and a few distributed in Oceania and Africa. my country is rich in wild and cultivated ramie resources, accounting for more than 90% of the world's ramie production. Ramie is not only an important fiber economic crop, but also ramie root is a traditional Chinese medicinal material with a long history of use in my country. The current "Chinese Pharmacopoeia" does not include ramie root, and there are no related research reports on ramie root fingerprint and multi-index component content determination. Therefore, the fingerprint combined with multi-component, multi-index quality control method is of great significance to the quality research of ramie root. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for determining the contents of 7 phenolic acid components in ramie root based on fingerprint spectrum combined with one-measurement-multiple-evaluation method. The method of the present invention can provide a theoretical basis for the quality control of ramie root medicinal materials. The content of the present invention is as follows: A method for determining the contents of seven phenolic acid components in ramie roots based on fingerprint spectrum combined with one-measurement-multiple-evaluation method, the technical point of which is that it comprises the following steps: Step 1, preparation of reference solution: add methanol to a volumetric flask to dissolve protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid and p-coumaric acid to obtain a reference solution; Step 2, preparation of the test solution: take the powder of the ramie root medicinal material sample and place it in a stoppered conical flask, add water, heat and reflux to extract, then cool, filter, evaporate the filtrate to dryness, add methanol to dissolve and make up the volume, filter and take the filtrate to obtain the test solution; Step 3, establishment of fingerprint: take different test sample solutions, record the fingerprint of each sample under liquid chromatography conditions, import the data into the "Chinese medicine chromatographic fingerprint similarity evaluation system", set the reference spectrum of the fingerprint spectrum, perform full peak matching, and generate a control fingerprint spectrum by the average method; Step 4, multi-component content determination; Step 5, calculation of relative correction factor: take 1, 2, 4, 5, 8, 10 μL of the test solution, and determine under the same liquid chromatography conditions as step 3, using chlorogenic acid as the internal reference, 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 component to be measured, and Ci is the concentration of the component to be measured. Calculate the relative correction factor and take the average value as ƒsi for quantification; Step 6, confirmation of relative retention time; Step 7, durability inspection; Step 8, chromatographic peak positioning; Step 9, determination of the one-test-multiple-evaluations method.

[0006] The method for determining the contents of 7 phenolic acid components in ramie root based on fingerprint combined with one-measurement-multiple-evaluation method of the present invention established the fingerprint of ramie root, 16 common peaks were calibrated, and 7 components including protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid and p-coumaric acid were identified. The similarities of 11 batches of ramie root samples were all greater than 0.87, indicating that the quality of ramie roots from different origins was relatively uniform. The RSD of the relative correction factor and relative retention time of each component was less than 2.00%, and different flow rates, column temperatures, instruments and chromatographic columns had little effect on them. There was no significant difference in the determination results between the one-measurement-multiple-evaluation method and the external standard method. Conclusion: The method of fingerprint combined with one-measurement-multiple-evaluation method established by the present invention is feasible and can provide a theoretical basis for the quality control of ramie root medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 Overlay of chromatograms for 11 batches of ramie root samples 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; Figure 2 The chromatogram of the mixed reference substance of the present invention and the ramie root sample comparison 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; Figure 3 Chromatograms of blank solvent (A), mixed reference substance (B) and ramie root sample (C) In the figure, 1. protocatechuic acid; 2. neochlorogenic acid; 3. protocatechuic aldehyde; 4. chlorogenic acid; 5. cryptochlorogenic acid; 6. caffeic acid; 7. p-coumaric acid. DETAILED DESCRIPTION

[0008] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention and the corresponding drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0009] The technical solutions provided by various embodiments of the present invention are described in detail below in conjunction with the accompanying drawings.

[0010] The present invention establishes a fingerprint combined with a one-measurement-multiple-evaluations method (QAMS) to evaluate the quality of ramie roots, selects chlorogenic acid, a common phenolic acid compound, as an internal reference, and uses relative correction factors to calculate the contents of another 6 phenolic acid components in the ramie roots. The ramie root fingerprint is established for the first time and the 7 phenolic acid components in the ramie roots are simultaneously determined. At the same time, the ramie roots can be simply, quickly, comprehensively and accurately evaluated for multiple indicators of quality, which helps to ensure that the quality of the ramie roots is controllable and saves detection costs and time. The established method has good credibility and can provide a theoretical basis for quality control of ramie roots.

[0011] 1. Instruments and Materials 1.1 Instrument Ultra-high performance liquid chromatograph (Waters PDA, Waters Corporation, USA); Ultra-high performance liquid chromatograph (WatersTUV, Waters Corporation, USA); Ultra-high performance liquid chromatograph (Agilent 1260, Agilent Technologies, USA); Agilent ZORBAX Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm); Phenomenex Luna C18 column (4.6 mm × 250 mm, 5 μm); Thermo AcclaimTM 120C18 Chromatographic column (4.6mm×250mm, 5μm); one-tenth balance (Quintix-ICN, Sartorius, Germany); one-millionth balance (XP26, Mettler-Toledo, Switzerland); one-thousandth balance (Quintix-ICN, Sartorius, Germany); ultrasonic cleaning machine (SB-800DT, Ningbo Xinzhi Biotechnology Co., Ltd.); ultrapure water machine (UPT-II-20T, Sichuan Youpu Ultrapure Technology Co., Ltd.).

[0012] 1.2 Materials Reagents: methanol (Hunan Huihong Reagent Co., Ltd., analytical grade); phosphoric acid (Tianjin Komiou Chemical Reagent Co., Ltd., chromatographic grade); acetonitrile (Merck AG, Germany, chromatographic grade); water was ultrapure water (self-made in the laboratory).

[0013] Test drugs: 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 China Food and Drug Inspection Institutes, neochlorogenic acid (batch number: 23103101, content 99.38%); cryptochlorogenic acid (batch number: 3208, content 98.0%) were all purchased from Chengdu Glip Biotechnology Co., Ltd.

[0014] The 11 batches of ramie root medicinal materials were identified by Professor Sun Dongmei of Hunan Yifang Tianjiang Pharmaceutical Co., Ltd. as the dried rhizomes and roots of Boehmeria nivea (L.) Gaud. of the Urticaceae family. After inspection, they all met the requirements of the ramie root medicinal materials in the 2010 edition of the "Hunan Province Traditional Chinese Medicine Preparation Specifications". The source information of the medicinal materials is shown in Table 1.

[0015] Table 1 Source information of 11 batches of ramie roots

[0016] 2 Methods and Results 2.1 Chromatographic conditions Chromatographic column: Agilent ZORBAX Eclipse XDB-C18 column (4.6mm×250mm, 5μm); mobile phase is acetonitrile (A)-0.1% phosphoric acid aqueous solution (B); gradient elution (0-10min, 5%→7%A; 10-15min, 7%→10%A; 15-25min, 10%→12%A; 25-30min, 12%→15%A; 30-40min, 15%A; 40-45min, 15%→18%A); flow rate is 0.8mL per minute; column temperature is 25℃; detection wavelength is 310nm; injection volume is 10μL.

[0017] 2.2 Solution preparation 2.2.1 Preparation of reference solution Accurately weigh appropriate amounts of protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid, and p-coumaric acid reference substances, place in a volumetric flask, add 50% methanol to dissolve, 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.

[0018] 2.2.2 Preparation of test solution Take about 2.0g of ramie root medicinal material sample powder, accurately weigh it, put it in a stoppered conical flask, add 100mL of water, heat and reflux to extract for 45min, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 50% methanol, make up to volume in a 10mL volumetric flask, shake well, filter, and take the filtrate to obtain.

[0019] 2.3 Establishment of fingerprint 2.3.1 Precision test Take the powder of ramie root medicinal material (No. S2), prepare the test solution according to the method under "2.2.2", and continuously inject 6 needles for determination under the chromatographic conditions under "2.1". Take the chlorogenic acid peak as the reference peak, and calculate the RSD values ​​of the relative retention time and relative peak area of ​​the remaining common peaks. The results show that the RSD of the relative retention time and relative peak area of ​​each common peak is less than 2.0%, indicating that the instrument precision is good.

[0020] 2.3.2 Repeatability test Take the same batch of ramie root powder (No. S2), prepare 6 test solutions according to the method under "2.2.2", and inject and measure under the chromatographic conditions under "2.1". Take the chlorogenic acid peak as the reference peak, and calculate the RSD values ​​of the relative retention time and relative peak area of ​​the remaining common peaks. The results show that the RSD of the relative retention time of each common peak is less than 1.0%, and the RSD of the relative peak area is less than 3.0%, indicating that the method has good repeatability.

[0021] 2.3.3 Stability test Take the powder of ramie root medicinal material (No. S2), prepare the test solution according to the method under "2.2.2", and inject and measure at 0, 2, 4, 8, 12, and 24 hours under the chromatographic conditions under "2.1", and calculate the RSD values ​​of the relative retention time and relative peak area of ​​the remaining common peaks with the chlorogenic acid peak as the reference peak. The results show that the relative retention time RSD of each common peak is less than 2.0%, and the relative peak area RSD is less than 3.0%, indicating that the test sample is stable within 24 hours.

[0022] 2.3.4 Fingerprint establishment and similarity evaluation Take 11 batches of ramie root medicinal material samples, prepare the test solution according to the method under "2.2.2", inject and measure under the chromatographic conditions under "2.1", and record the fingerprint of each sample. Import the data into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", use the fingerprint of S1 sample as the reference spectrum, perform full peak matching, generate the reference fingerprint by the average method, and calibrate a total of 16 common peaks. The overlay diagram is shown in Figure 1 By comparing with the reference substance, 7 chromatographic peaks were identified, and the comparison diagram is shown in Figure 2, where 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 fingerprint was compared with the fingerprints of 11 batches of ramie root samples for similarity evaluation, and the results are shown in Table 2. The results showed that the similarity of the 11 batches of ramie root medicinal samples was greater than 0.87, indicating that the common peak fingerprints of ramie roots from different origins have high similarity and the quality of the medicinal materials is relatively uniform.

[0023] Table 2 Similarity table of 11 batches of ramie roots

[0024] 2.4 Determination of multi-component content 2.4.1 Specificity test Take the powder of ramie root medicinal material (No. S2), prepare the test solution according to the method in "2.2.2", and inject the blank solvent, mixed standard and test solution under the chromatographic conditions in "2.1". The results are shown in Figure 3 The separation of each chromatographic peak was greater than 1.5, and there was no interference from the blank solvent, indicating that the method had good specificity.

[0025] 2.4.2 Investigation of linear relationship Take the mixed reference solution under "2.2.1" and inject 2μL, 5μL, 10μL, 20μL, 50μL and 100μL respectively according to the chromatographic conditions under "2.1" for determination. The target peak area is used as the ordinate (Y) and the concentration is used as the abscissa (X). The results are shown in Table 3. It shows that the linear relationship of each component is good within its respective range.

[0026] Table 3 Linear relationship of each component

[0027] 2.4.3 Precision test Take the mixed reference solution under "2.2.1" and inject it 6 times continuously according to the chromatographic conditions under "2.1" to measure the protocatechuic acid, The RSDs of the peak areas 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 had good precision.

[0028] 2.4.4 Repeatability test Take the same batch of ramie root powder (No. S3), prepare 6 test solutions according to the method under "2.2.2", and inject and measure under the chromatographic conditions under "2.1". The RSDs of protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid and p-coumaric acid are 0.62%, 0.50%, 0.63%, 2.25%, 0.17%, 0.70% and 0.54%, respectively, indicating that the method has good repeatability.

[0029] 2.4.5 Stability test The same test solution under "2.1.3" was taken and sampled at 0, 2, 4, 6, 8, 12 and 24 hours according to the chromatographic conditions under "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 had good stability within 24 hours.

[0030] 2.4.6 Sample recovery test Weigh 1.0 g of the ramie root medicinal material sample (S3) with the content determined, and make 6 parallel portions. Weigh 3.144 mg of protocatechuic acid reference substance, 4.987 mg of neochlorogenic acid reference substance, 5.811 mg of chlorogenic acid reference substance, 4.813 mg of cryptochlorogenic acid reference substance, and 2.307 mg of p-coumaric acid reference substance, and place them in different 5 mL volumetric bottles; Weigh 3.326 mg of protocatechuic aldehyde reference substance and 2.129 mg of caffeic acid reference substance, and place them in 20 mL volumetric bottles, add appropriate amount of 50% methanol to dissolve and dilute to the scale, shake well, and obtain a single reference solution. Weigh 1 mL of the above reference solution accurately, place it in the same 10 mL volumetric bottle, add 50% methanol to make up to volume, shake well, and obtain a mixed reference stock solution. Accurately add 1 mL of the mixed reference stock solution to each of the above 6 samples, prepare the test solution according to the method in "2.2.2", and measure under the chromatographic conditions in "2.1", record the peak area of ​​each component and calculate the recovery rate. The results are shown in Table 4.

[0031] Table 4 Results of sample recovery test for each component (n=6)

[0032] 2.5 Calculation of relative correction factor Accurately pipette 1, 2, 4, 5, 8, 10 μL of the mixed reference solution under "2.2.1", and determine according to the chromatographic conditions under "2.1", using chlorogenic acid as the internal reference, and 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 component to be measured, and Ci is the concentration of the component to be measured, calculate the relative correction factor, and take the average value as the quantitative ƒsi. The results are shown in Table 5.

[0033] Table 5 Relative correction factors of each component

[0034] The results showed that the relative correction factors of 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, and the RSDs were 1.38%, 0.22%, 0.39%, 0.26%, 1.52% and 0.61%, respectively.

[0035] 2.6 Confirmation of relative retention time Accurately pipette 1, 2, 4, 5, 8, 10 μL of the mixed reference solution under item "2.2.1", and determine according to the chromatographic conditions under item "2.1", using chlorogenic acid as the internal reference, and calculate the relative retention times of the remaining 6 components. The results are shown in Table 6.

[0036] Table 6 Relative retention time of each component

[0037] 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, and the RSDs were 0.23%, 0.12%, 0.10%, 0.00%, 0.03% and 0.07%, respectively.

[0038] 2.7 Durability test 2.7.1 Flow rate Take the mixed reference solution under "2.2.1" and measure it according to the chromatographic conditions under "2.1" to investigate the effect of three different flow rates of 0.7, 0.8, and 0.9 mL / min on the relative correction factor. The results are shown in Table 7.

[0039] Table 7 Relative correction factors for different flow rates

[0040] The results showed that different flow rates had no obvious effect on the relative correction factor. The RSDs of the relative correction factors of 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.

[0041] 2.7.2 Column temperature Take the mixed reference solution under "2.2.1" and measure it according to the chromatographic conditions under "2.1" to investigate the effect of three different column temperatures of 20, 25, and 30℃ on the relative correction factor. The results are shown in Table 8.

[0042] Table 8 Relative correction factors for different column temperatures

[0043] The results showed that different column temperatures had no obvious effect on the relative correction factor. The RSDs of the relative correction factors of 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.

[0044] 2.7.3 Instruments and chromatographic columns Take the mixed reference solution under "2.2.1", select Waters PDA, Waters TUV and Agilent 1260 high performance liquid chromatographs, and Agilent ZORBAX Eclipse XDB-C18 (4.6mm × 250mm, 5μm), Phenomenex Luna C18 (4.6mm × 250mm, 5μm), Thermo Acclaim TM 120 C18 (4.6mm × 250mm, 5μm) chromatographic columns, and measure according to the chromatographic conditions under "2.1" to investigate the effects of different instruments and different chromatographic columns on the relative correction factor. The results are shown in Table 9.

[0045] Table 9 Relative correction factors for different instruments and columns

[0046] The results showed that the influence of different instruments and chromatographic columns on the relative correction factor was within an acceptable range. The RSDs of the relative correction factors of 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.

[0047] 2.8 Chromatographic peak positioning The compounds to be tested were identified by relative retention time. Taking chlorogenic acid as a reference, the mixed reference solution under "2.2.1" was taken and measured according to the chromatographic conditions under "2.1". The reproducibility of relative retention time in different flow rates, column temperatures, instruments and chromatographic columns was examined. The results are shown in Table 10.

[0048] Table 10 Relative retention times at different flow rates, column temperatures, instruments and columns

[0049] The results showed that the relative retention time of each component to be tested changed little, and chlorogenic acid could be used as a reference to locate the chromatographic peak.

[0050] 2.9 Comparison of the results between the one-measurement-multiple-evaluation method and the external standard method Take 11 batches of ramie root medicinal material sample powder, prepare the test solution according to the method under "2.2.2", inject and measure under the chromatographic conditions under "2.1", use external standard method (ESM) to measure the content of protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, cryptochlorogenic acid, caffeic acid, and p-coumaric acid in the sample, and then calculate them using the established one measurement multiple evaluation method (QAMS). In order to investigate the accuracy of one measurement multiple evaluation method (QAMS), the determination results of one measurement multiple evaluation method (QAMS) were compared with those of external standard method (ESM). The results are shown in Table 11.

[0051] Table 11 Content determination results of external standard method and one-measurement-multiple-evaluation method

[0052] The results showed that there was no significant difference in the contents of each component measured by the one-measurement-multiple-evaluations method and the external standard method, indicating that the established method has good credibility.

[0053] 3 Discussions 3.1 Selection of internal reference The current "Chinese Pharmacopoeia" does not include ramie root, and there are no related research reports on ramie root fingerprint and multi-index component content determination. Selecting fingerprints combined with multi-component and multi-index quantification can more accurately reflect the quality of ramie root to a certain extent. The index component chlorogenic acid in ramie root has a high content, a moderate retention time, and a relatively cheap price. Therefore, the present invention uses chlorogenic acid as an internal reference. The RSD of the relative correction factor and relative retention time of each component is less than 2.00%, indicating that the established one-measurement-multiple-evaluation method has good accuracy and high credibility.

[0054] 3.2 Sample pretreatment and selection of mobile phase The present invention compares ultrasonic extraction, heating reflux extraction and water decoction extraction for 30, 45 and 60 min respectively, and compares water, methanol, ethanol, 50% methanol, 50% ethanol and other extraction solvents. Finally, water decoction for 45 min, evaporation and use Dissolve in 50% methanol and dilute to 10mL. There are many types of phenolic acid components in ramie roots with similar structures. If isocratic elution is used, compounds with similar structures are not easy to separate. Therefore, the 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 the chromatographic peak separation of each index component was good when eluted with acetonitrile-0.1% phosphoric acid aqueous solution.

[0055] 3.3 Sample content determination The quality of Chinese medicinal materials is affected by natural and human factors, which may lead to different qualities of medicinal materials from different origins. While establishing the fingerprint of ramie root, the present invention uses a one-measurement-multiple-evaluation method to determine the content of 7 phenolic acid components in ramie root. No reports on ramie root fingerprint and multi-component quantitative research have been found in the existing literature. The qualitative and quantitative methods established by the present invention can more comprehensively control and evaluate the quality of ramie root. A total of 16 common peaks were calibrated in 11 batches of ramie root medicinal materials, and 7 components were identified through reference substances. The similarity of each batch of samples except S5 was greater than 0.9, indicating that the quality of ramie root medicinal materials from different origins is relatively uniform. The similarity of S5 samples is low, which may be related to harvesting, sampling, storage, etc., which indirectly reflects the importance of medicinal materials in the harvesting, processing, storage, transportation and other links. The present invention uses chlorogenic acid as an internal reference, calculates the contents of the other six phenolic acid components through relative correction factors, and realizes the simultaneous determination of seven phenolic acid components in ramie roots for the first time. At the same time, the measured values ​​are compared with the measured values ​​of the external standard method, and it is found that there is no significant difference in the content values ​​obtained by the two methods, which realizes the simultaneous determination of seven phenolic acid components in ramie roots for the first time. The established fingerprint spectrum combined with the one-measurement-multiple-evaluation method is scientific, simple, has good specificity, strong repeatability, good accuracy, and high credibility, and can provide a theoretical basis for ramie root quality control.

[0056] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for determining the contents of seven phenolic acid components in ramie roots based on fingerprint and one-measurement-multiple-evaluation method, characterized in that: The following steps are involved: Step 1, preparation of reference solution: add methanol to a volumetric flask to dissolve protocatechuic acid, neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, caffeic acid and p-coumaric acid to obtain a reference solution; Step 2, preparation of the test solution: take the powder of the ramie root medicinal material sample and place it in a stoppered conical flask, add water, heat and reflux to extract, then cool, filter, evaporate the filtrate to dryness, add methanol to dissolve and make up the volume, filter and take the filtrate to obtain the test solution; Step 3, establishment of fingerprint: take different test sample solutions, record the fingerprint of each sample under liquid chromatography conditions, import the data into the "Chinese medicine chromatographic fingerprint similarity evaluation system", set the reference spectrum of the fingerprint spectrum, perform full peak matching, and generate a control fingerprint spectrum by the average method; Step 4, multi-component content determination; Step 5, calculation of relative correction factor: take 1, 2, 4, 5, 8, 10 μL of the test solution, and determine under the same liquid chromatography conditions as step 3, using chlorogenic acid as the internal reference, 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 component to be measured, and Ci is the concentration of the component to be measured. Calculate the relative correction factor and take the average value as ƒsi for quantification; Step 6, confirmation of relative retention time; Step 7, durability inspection; Step 8, chromatographic peak positioning; Step 9, determination of the one-test-multiple-evaluations method.

2. The method for determining the contents of seven phenolic acid components in ramie roots based on fingerprint and one-measurement-multiple-evaluation method according to claim 1, characterized in that: Each 1 mL of the reference solution in step 1 contains a mixed reference solution of 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.

3. The method for determining the contents of 7 phenolic acid components in ramie roots based on fingerprint and one-measurement-multiple-evaluation method according to claim 1, characterized in that: The preparation of the test solution in step 2 is as follows: about 2.0 g of ramie root medicinal material sample powder is taken, accurately weighed, placed in a stoppered conical flask, 100 mL of water is added, heated to reflux extraction for 45 min, cooled, filtered, the filtrate is evaporated to dryness, 50% methanol is added to dissolve the residue, the volume is fixed in a 10 mL volumetric flask, shaken well, filtered, and the filtrate is obtained.

4. The method for determining the contents of seven phenolic acid components in ramie roots based on fingerprint and one-measurement-multiple-evaluation method according to claim 1, characterized in that: In the liquid chromatography conditions in step 3, mobile phase A is acetonitrile and mobile phase B is 0.1% phosphoric acid aqueous solution.

5. The method for determining the contents of seven phenolic acid components in ramie roots based on fingerprint spectrum combined with one-measurement-multiple-evaluation method according to claim 4, characterized in that: The gradient elution program in the liquid chromatography conditions in step 3 is: 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.

6. The method for determining the contents of seven phenolic acid components in ramie roots based on fingerprint and one-measurement-multiple-evaluation method according to claim 5, characterized in that: The liquid chromatography conditions in step 3 are as follows: flow rate of 0.7-0.9 mL per minute; column temperature of 20-30° C.; detection wavelength of 310 nm; injection volume of 10 μL.

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