Quality detection method of beautiful millettia root medicinal material or extract thereof

Abstract

The invention relates to a quality detection method of a millettia specisoa medicinal material or an extract thereof, and belongs to the field of quality detection of traditional Chinese medicinal materials. The invention provides a quality detection method of a millettia specisoa medicinal material or an extract thereof, which is characterized in that erythrinine and flavonoid components in the millettia specisoa medicinal material or the extract thereof are detected by high performance liquid chromatography-mass spectrometry, and the flavonoid components comprise liquiritigenin, calycosin, formononin, isoliquiritigenin, formononetin and maackiain. According to the detection method, the contents of the seven components of the beautiful millettia root can be determined within 12 minutes, the liquiritigenin is used as an internal standard substance, a method for quantitative analysis of multiple components by one marker is established, the cost for purchasing other reference substances is saved, and the method is high in precision, good in repeatability and high in accuracy and can provide a method basis for comprehensive control of the quality of the beautiful millettia root.

Description

Technical Field

[0001] The invention relates to a quality detection method for a Herba Cynanchifoliae medicinal material or an extract thereof, and belongs to the field of quality detection of traditional Chinese medicinal materials. Background Art

[0002] Niu Dali Millettia speciose Champ. is the dried root of the beautiful Millettia genus of the Leguminosae family, also known as mountain lotus root, blood vine, sweet Niu Dali, Niu Gu Dali, Dalishu, etc. Niu Dali is used as medicine with its dried tubers, which is sweet and flat in nature. It has the effects of nourishing the lungs, strengthening tendons and activating collaterals. Clinically, it has certain effects on chronic diseases such as lumbar muscle strain, rheumatoid arthritis, tuberculosis, chronic bronchitis, etc. Niu Dali has a long history of use as a medicine. As early as in the "Essentials of Herbal Medicine", it was recorded that it "strengthens muscles and bones, relieves heat and toxins, regulates internal injuries, treats bruises, and nourishes the kidneys by soaking in wine". "Luchuan Materia Medica" also records that it "clears the lungs and stops coughing, cools and detoxifies. It treats coughing up blood, dysentery, warms the body, thirst, and dizziness".

[0003] There are many chemical components in Radix Codonopsis pilosulae, such as flavonoids, alkaloids, lignin, triterpenes, polysaccharides, etc. At present, scholars at home and abroad have done little research on the components of Radix Codonopsis pilosulae, and the research parts mainly include the chemical components of the roots and the chemical components of the vine leaves. The main flavonoid compounds in Radix Codonopsis pilosulae are formononetin, asterone, kaempferol, isoliquiritigenin, medipterocarpin, calycosin, homopterocarpin, pterocarpin, 3', 7-dihydroxy-2', 4'-dimethoxy isoflavone, etc.; the alkaloids isolated from Radix Codonopsis pilosulae include 6-methoxydihydrosanguinarine, N-methylcytisine, sanguinarine, erythrin, etc. The monosaccharide components that make up Radix Codonopsis pilosulae polysaccharides are mainly rhamnose, galactose, glucose, mannose and fructose.

[0004] Modern pharmacological studies have shown that Rhizoma Coptidis has a variety of pharmacological activities such as immunomodulation, anti-inflammatory, liver protection, anti-thrombosis, and anti-tumor. For example, the polysaccharide components in Rhizoma Coptidis can enhance the phagocytic function of phagocytes, increase the number of antibody-forming cells, promote lymphocyte transformation, and enhance the immune cells of mice. Rhizoma Coptidis also has a protective effect on chemical liver damage and alcoholic liver damage in mice. Rhizoma Coptidis can significantly increase the secretion of phenol red in the trachea of ​​mice, promote the movement of ink in the trachea of ​​pigeons, reduce the number of cough reactions induced by ammonia in mice and induced by tartrate in guinea pigs, prolong the cough latency, and counteract histamine-acetylcholine-induced bronchial asthma in guinea pigs. It has certain expectorant, antitussive and antiasthmatic effects. The water extract of Rhizoma Coptidis can significantly prolong the survival time of mice in weight-bearing swimming tests and hypoxia tolerance tests. The water extract of Rhizoma Coptidis also has obvious anti-inflammatory, analgesic, and antioxidant effects.

[0005] There are often confused products in the purchase of Niu Dali products. This counterfeit product is of the same family and genus as the sweet Niu Dali. It is the dried root of the green flowered Millettia plant of the genus Millettia in the Leguminosae family. Because of its bitter taste, the people are accustomed to calling it bitter Niu Dali. At present, the quality standards for Niu Dali are only included in the "Guangdong Chinese Medicinal Materials Standard" and the "Guangxi Chinese Medicinal Materials Standard". Its quality control indicators only include microscopic and thin layer identification methods. The standards are too simple and not perfect, and cannot meet the quality evaluation of the current Niu Dali medicinal materials. With the conversion of wild Niu Dali resources to domestic cultivation, Niu Dali planting bases have been established in many places across the country. In order to promote the further development and utilization of the Chinese medicinal material Niu Dali and reduce the abuse of medicinal materials, it is very necessary to establish a comprehensive and effective Niu Dali quality evaluation standard. Guo Peng, Research on the Quality Standard of Niu Dali Medicinal Materials, Master's thesis of Guangzhou University of Chinese Medicine, March 2019, disclosed that the index components such as erythrine, quercetin, sophora japonica and formononetin can be used as evaluation indicators of the quality of Niu Dali medicinal materials. The chromatographic conditions disclosed are to use acetonitrile and water as the mobile phase and complete the gradient elution within 40 minutes. The detection method reported in this literature has a small number of measured components and a long measurement time; Ma Bokai, et al., Determination of erythrine, formononetin and sophora japonica in the medicinal material Niu Dali by ultra-high performance liquid chromatography-tandem mass spectrometry, Journal of Analytical Science, April 2022, using ultra-high performance liquid chromatography-tandem mass spectrometry technology, established an analytical method for the simultaneous determination of erythrine, formononetin and sophora japonica in the Chinese medicinal material Niu Dali; using WatersACQUITY UPLC BEH C 18 Column (100mm×2.1mm, 1.7μm), with methanol-0.1% formic acid aqueous solution as mobile phase, gradient elution; mass spectrometry uses electrospray ionization, negative ion source, multiple reaction monitoring mode detection, and external standard method quantification. Yu Dandan, LC-MS technology is used for qualitative and quantitative study of chemical components of Radix Codonopsis pilosulae, Zhejiang University of Technology, June 2018, the sample recovery rate of some components reported in this document exceeds 120%, RSD exceeds 9%, and the accuracy of content determination is not high; the determined components gentisic acid and salicylic acid are not characteristic; Wu Yu, et al., UPLC determination of erythrine in different Radix Codonopsis pilosulae samples, Guangdong Chemical Industry, Issue 2, 2024, this document only detects a single component; Chen Mingquan, et al., HPLC simultaneous determination of erythrine and sophora japonica in Radix Codonopsis pilosulae, Modern Chinese Medicine, Volume 20, Issue 1, January 2018, establish a method for determining the content of erythrine and sophora japonica in Radix Codonopsis pilosulae. Ultrasonic method was used to extract medicinal materials, and the determination method was established by optimizing the extraction process and chromatographic conditions, and the contents of erythrine and kaempferol in Radix Codonopsis were determined at the same time. Peng Fuquan, et al., HPLC determination of erythrine, formononetin and kaempferol in different varieties of Radix Codonopsis, Journal of Guangzhou University of Chinese Medicine, Vol. 35, No. 3, May 2018. The determination components reported in the literature are relatively few and the detection time is long.

[0006] The main drawbacks of the currently reported detection methods are that they measure too few components, the analysis time is too long, and even the accuracy needs to be verified. Summary of the invention

[0007] The technical solution of the present invention is to provide a quality detection method for Herba Lycopodii or its extract.

[0008] The present invention provides a quality detection method for Herba Cynoglossi or its extract, which adopts high performance liquid chromatography-mass spectrometry to detect erythrine and flavonoid components in Herba Cynoglossi or its extract, wherein the flavonoid components include liquiritigenin, calycosin isoflavones, formononetin, isoliquiritigenin, formononetin and sophora japonica; The HPLC conditions were: The chromatographic column is Shimadzu HPLC Shim-pack GIST C 18 2.1×100mm, 2μm; mobile phase A is methanol A, mobile phase B is aqueous solution; gradient elution conditions are: 0-3min, 25%-60%A; 3-10min, 60%-90%A; 10-10.1min, 90%-25%A; 10.1-12min, 25%A; volume flow rate is 0.3 mL / min; column temperature is 30 ℃; injection volume is 5 μL; Mass spectrometry conditions: nebulizer gas flow rate 3 L / min, heating gas flow rate 10 L / min, interface temperature 300 °C, desolvation temperature 526 °C, DL temperature 275 °C, heating block temperature 400 °C, drying gas flow rate 10 L / min; ion source ESI; ionization mode: positive and negative ion scanning simultaneously; parent ion, daughter ion, fragmentation voltage CE, and ionization mode of the components to be measured are: Erythrine: m / z 244.8, m / z 141.8, 18 V, negative ions; Licorice root: m / z 255.0, m / z 118.9, 28 V, negative ions; Isoflavones: m / z 283.0, m / z 268.0, 14 V, negative ions; Formononetin: m / z 431.0, m / z 269.1, 16 V, positive ions; Isoliquiritigenin: m / z 255.0, m / z 118.9, 28 V, negative ions; Formononetin: m / z 266.9, m / z 252.0, 25 V, negative ion; Sophora flavescens: m / z 282.9, m / z 254.0, 25 V, negative ions.

[0009] The detection method of the present invention comprises the following steps: a. Preparation of test sample: Take 0.5g of dry powder of Radix Codonopsis pilosulae, weigh accurately, place in a stoppered conical flask, add 50mL of 50% methanol accurately, weigh, ultrasonicate for 30min, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, accurately measure 1mL of the filtrate, transfer to a 10mL volumetric flask, add 50% methanol to the mark, shake well, and obtain; b. Preparation of reference solution: accurately weigh reference substances of erythrine, formononetin, licorice, calycosin, isoliquiritigenin, kaempferol, and formononetin, and prepare mixed reference solutions with concentrations of 6000-8000 ng / mL, 30-40 ng / mL, 120-140 ng / mL, 60-80 ng / mL, 70-90 ng / mL, 1400-1600 ng / mL, and 700-900 ng / mL, respectively, with methanol. Dilute the mixed reference solutions into 6 different concentrations, store at 4°C, and set aside. c. Detect according to the method described.

[0010] Among them, the amounts of erythrine, formononetin, liquiritigenin, verbascoside, isoliquiritigenin, sophora flavescens, and formononetin contained in the Herba Lycopodii are: 173.6-331.3 μg / g, 1.113-1.837 μg / g, 1.468-9.127 μg / g, 2.345-6.814 μg / g, 0.7250-2.544 μg / g, 36.43-157.7 μg / g, and 3.229-28.84 μg / g, respectively.

[0011] The present invention also provides a method for determining the contents of seven components in the Radix Codonopsis pilosulae medicinal material or its extract by a one-test multiple-evaluation method, which adopts the quality detection method for detection, takes licorice root as an internal reference, and calculates the relative correction factors fs / x of erythrine, formononetin, isoliquiritigenin, calycosin isoflavone, sophora japonica and formononetin according to the formula fs / x=fs / fx=AxCs / (AsCx); wherein Cs and Cx represent the mass concentrations of the internal reference and the component to be detected, respectively, and As and Ax represent the peak area values ​​of the internal reference and the component to be detected, respectively; the content of licorice root is usually determined, and the contents of erythrine, formononetin, isoliquiritigenin, calycosin isoflavone, sophora japonica and formononetin in the Radix Codonopsis pilosulae medicinal material or its extract are calculated according to the relative correction factors fs / x of erythrine, formononetin, isoliquiritigenin, calycosin isoflavone, sophora japonica and formononetin.

[0012] Among the seven components of the present invention, liquiritigenin, calycosin, formononetin, isoliquiritigenin, formononetin and kaempferol are flavonoid components, and erythrin is an alkaloid component. Among them, formononetin and erythrin have relatively poor ionization activities, have relatively low corresponding intensities in the mass spectrometry ESI ion source, and have relatively high quantitative limits.

[0013] The beneficial effects of the present invention are: The inventors previously screened out seven ingredients through network pharmacology and literature research as quality markers for Radix Codonopsis pilosulae for treating myasthenia gravis. The detection method of the present invention can determine the contents of seven ingredients of Radix Codonopsis pilosulae in 12 minutes, and uses glycyrrhizin as an internal standard to establish a one-test-multiple-evaluation method for multiple ingredients, saving the cost of purchasing other reference substances. The method has high precision, good repeatability and high accuracy, and can provide a methodological basis for comprehensively controlling the quality of Radix Codonopsis pilosulae. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the MRM ion flow chart of the determination of the content of Radix Codonopsis pilosula (wherein, A. Radix Codonopsis pilosula mixed reference solution; B. Radix Codonopsis pilosula test solution; C. blank solvent; 1-erythrine, 2-liquiritigenin, 3-calycosin isoflavone, 4-formononetin, 5-isoliquiritigenin, 6-formononetin, 7-kalocynone); Figure 2 The MRM ion chromatogram is for reference. DETAILED DESCRIPTION

[0015] Example 1 The detection method of Herba Lysimachiae of the present invention Preparation of reference solution: Take appropriate amount of erythrine, formononetin, liquiritigenin, calycosin, isoliquiritigenin, sophora japonica and formononetin reference substances, weigh them accurately, and prepare mixed reference solutions with mass concentrations of 7225, 35.25, 130.0, 72.50, 86.00, 1520 and 855.0 ng / ml, respectively, with methanol. Dilute the mixed reference solutions into 6 different mass concentrations, store at 4°C for later use.

[0016] Preparation method of test sample: Take 0.5g of Herba Lycopodiellae powder, weigh it accurately, put it in a stoppered conical flask, accurately add 50mL of 50% methanol, weigh it, ultrasonicate it for 30min, let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, accurately measure 1mL of the filtrate, transfer it to a 10mL volumetric flask, add 50% methanol to the scale, shake it well, and obtain it.

[0017] Chromatographic conditions: The chromatographic column is Shimadzu HPLC Shim-pack GIST C 18(2.1×100mm, 2μm); the mobile phase was methanol (A)-water solution (B); the gradient elution conditions were 0-3min, 25%-60%A; 3-10min, 60%-90%A; 10-10.1min, 90%-25%A; 10.1-12min, 25%A. The volume flow rate was 0.3 mL / min; the column temperature was 30 ℃; the injection volume was 5 μL.

[0018] Mass spectrometry conditions: nebulizer gas flow rate 3 L / min, heating gas flow rate 10 L / min, interface temperature 300 °C, desolvation temperature 526 °C, DL temperature 275 °C, heating block temperature 400 °C, drying gas flow rate 10 L / min. Ion source ESI; ionization mode: positive and negative ion scanning simultaneously. The mass spectrometry detection parameters in the MRM mode of the measured components are shown in Table 1, and the MRM ion flow diagram is shown in Figure 1 .

[0019] Table 1 Mass spectrometry parameters of the components to be tested Compound Precursor ion ( ) Daughter ion () Cracking voltage (V) Ionization mode Erythrinaine 244.8 141.8 18 Negative ions Licorice 255.0 118.9 28 Negative ions Isoflavone 283.0 268.0 14 Negative ions Formononetin 431.0 269.1 16 Positive ions Isoliquiritigenin 255.0 118.9 28 Negative ions Formononetin 266.9 252.0 25 Negative ions Sophora flavescens 282.9 254.0 25 Negative ions Example 2 Investigation of the linear relationship, precision, repeatability, stability and sample recovery of the detection method of the present invention Linear relationship investigation: 5 μL of each of the 6 mixed reference solutions with different mass concentrations were accurately aspirated and measured according to the chromatographic and mass spectrometric conditions, with the mass concentration of the reference solution as the horizontal axis ( X ), the peak area is the ordinate ( Y ), draw the standard curves of erythrine, formononetin, liquiritigenin, calycosin, isoliquiritigenin, kaempferol, and formononetin, perform linear regression, and obtain the regression equation and linear range of each component. The results are shown in Table 2. Among them, the correlation coefficient r All of them were greater than 0.9990, showing a good linear relationship.

[0020] Table 2 Linear relationships among the seven index components of Rhizoma Corydalis Compound Regression equation Linear range / (ng / mL) Erythrinaine = (379.592)+ (14093.6) 0.9996 144.5~7225 Formononetin = (4793.09)+ (1954.49) 0.9990 0.7050~35.25 Licorice = (34346.3)+ (-7364.56) 0.9997 0.3250~130.0 Isoflavone = (76128.4)+ (12376.4) 0.9998 1.450~72.50 Isoliquiritigenin = (77094.3)+ (-4841.26) 0.9998 0.2150~86.00 Sophora flavescens = (3324.08)+ (-14534.6) 0.9991 19.00~1520 Formononetin = (113511)+ (21459.8) 0.9998 17.10~855.0 Precision test: The mixed reference solution was sampled continuously for 6 times, and the peak areas of erythrine, formononetin, liquiritigenin, calycosin, isoliquiritigenin, sophora flavanoids, and formononetin were determined. The RSD values ​​were 2.64%, 2.29%, 1.40%, 2.12%, 0.68%, 1.61%, and 0.89%, respectively, all less than 3%, indicating that the instrument has high precision.

[0021] Repeatability test: About 0.5 g of Radix Erythrinae sample was accurately weighed, and 6 test solutions were prepared in parallel. The peak areas of erythrine, formononetin, liquiritigenin, calycosin, isoliquiritigenin, sophora flavanoids and formononetin were determined, and their RSD values ​​were 3.57%, 3.06%, 2.01%, 0.93%, 3.34%, 3.86% and 3.65%, respectively, all less than 4%, indicating that the method had good repeatability.

[0022] Stability test: Accurately weigh about 0.5 g of Radix Codonopsis pilosulae sample to prepare the test solution, and inject the sample at 0, 2, 4, 6, 12, and 24 h after preparation, and determine the peak areas of erythrine, formononetin, liquiritigenin, calycosin, isoliquiritigenin, sophora flavanoids, and formononetin. The RSD values ​​were 2.26%, 3.16%, 2.00%, 1.52%, 1.11%, 2.60%, and 1.70%, respectively, all less than 4%, indicating that the test solution had good stability within 24 h.

[0023] Sample recovery test: accurately weigh about 0.25g of the sample of Radix Codonopsis pilosulae with the amount of each index component determined, prepare 6 copies in parallel, add a certain amount of reference solution in the ratio of each component amount to reference solution (1:1), prepare the test solution according to the method, determine the peak area of ​​7 active ingredients, and calculate the average sample recovery and RSD of each component. Results The average sample recovery of erythrine, formononetin, liquiritigenin, calycosin, isoliquiritigenin, kaempferol, and formononetin were 100.66%, 97.69%, 99.46%, 93.03%, 95.01%, 95.87%, and 99.67%, respectively; the RSD values ​​were 1.54%, 3.13%, 1.26%, 2.73%, 2.82%, 1.64%, and 2.74%, respectively, all less than 4%. It shows that the recovery rate of this method is good.

[0024] Sample determination: 5 μl of 10 batches of test sample solution were accurately aspirated and injected into the liquid chromatography-tandem triple quadrupole mass spectrometer for determination. The results are shown in Table 3.

[0025] Table 3 Determination results of the content of Niu Dali

[0026] Example 3 Establishment of the One Test Multiple Evaluation (QAMS) Method of the Present Invention Calculation of relative correction factor (fs / x): The content of each component increases or decreases in direct proportion to the peak area within the linear range, that is, f = W / A, where f represents the correction factor, W represents the content of the component, and A represents the peak area value. Liquoricerin (s) was used as an internal reference to control the quality of the six active ingredients in Radix Codonopsis pilosulae. The fs / x between the internal standard liquoricerin (s) and erythrine (B), formononetin (C), isoliquiritigenin (D), calycosin (E), koreanolide (F) and formononetin (G) was calculated according to the formula.

[0027] fs / x=fs / fx=AxCs / (AsCx) In the formula, Cs and Cx represent the mass concentration of the internal reference and the component to be measured, respectively, and As and Ax represent the mass concentration of the internal reference and the component to be measured, respectively. Represents the peak area value of the internal reference and the component to be tested) Take 6 mixed reference solutions of different mass concentrations, inject 5 μL respectively, determine the peak areas of erythrine, liquiritigenin, calycosin, formononetin, isoliquiritigenin, sophora japonica, and formononetin, use liquiritigenin as internal standard, calculate the fs / x between the 6 tested components and liquiritigenin respectively, and the results are shown in Table 4. The fs / x of erythrine, formononetin, calycosin, isoliquiritigenin, sophora japonica, formononetin and liquiritigenin are determined to be 0.01153, 0.1529, 2.268, 2.288, 0.09510, and 3.316, respectively.

[0028] The amount (mass or concentration) of the component within a certain range (linear range) is proportional to the detector response, that is, W=fA. In multi-index quality evaluation, a typical component in the medicinal material (with a reference material supplier) is used as the internal standard to establish a relative correction factor between the component and other components, and the content of other components is calculated by the correction factor. This method of measuring one component and achieving quantitative analysis of multiple components is named one-measurement-multiple-evaluation method.

[0029] Table 4 fs / x of 6 index components and liquiritigenin serial number <![CDATA[f S / B ]]> <![CDATA[f S / C ]]> <![CDATA[f S / D ]]> <![CDATA[f S / E ]]> <![CDATA[f S / F ]]> <![CDATA[f S / G ]]> 1 0.01179 0.1592 2.310 2.373 0.09585 3.302 2 0.01129 0.1560 2.174 2.248 0.09070 3.155 3 0.01148 0.1539 2.254 2.280 0.09439 3.301 4 0.01155 0.1503 2.315 2.330 0.09754 3.371 5 0.01162 0.1531 2.258 2.236 0.09710 3.349 6 0.01144 0.1447 2.296 2.261 0.09501 3.418 average value 0.01153 0.1529 2.268 2.288 0.09510 3.316 RSD / % 1.46 3.26 2.32 2.31 2.59 2.73 Comparison of the results of determination by QAMS method and external standard method (ESM): 10 batches of Herba Lycopodii samples were used to prepare the test solution, and the contents of the index components of each batch of Herba Lycopodii of ESM were calculated by linear regression equation. The contents of the measured data were calculated by QAMS method, and the two groups of contents were compared by calculating the relative average deviation (RAD). The results are shown in Table 5, which proves that the QAMS method established in this study has good accuracy and feasibility.

[0030] Table 5 Comparison of content between QAMS method and ESM method

[0031] Example 4 Comparison of the detection method of the present invention with reported methods Comparative literature source: Ma Bokai, Qian Chong, Zhang Mei, et al. Determination of erythrine, formononetin and sophora japonica in the medicinal material Herba Lycopodii by ultra performance liquid chromatography-tandem mass spectrometry [J]. Journal of Analytical Science, 2022, 38(2): 203-208. The comparison with the detection method of the present invention is shown in Table 6.

[0032] Table 6 Comparison of the detection method of the present invention with the method in the literature This method Comparative Literature Method instrument LC-MS / MS LC-MS / MS Analytes Erythrina alkaloids, formononetin, liquiritigenin, calycosin isoflavones, isoliquiritigenin, formononetin, koreanoin Erythrine, formononetin and kaempferol Extraction solvent 50% Methanol 70% Methanol Chromatographic columns <![CDATA[Shimadzu HPLC Shim-pack GIST C 18 (2.1 mm × 100 mm, 2 μm)]]> <![CDATA[WatersC 18 Chromatographic column (2.1mm×100mm, 1.7μm)]]> Elution conditions Methanol (A)-water solution (B), gradient elution: 0-3 min, 25%-60% A; 3-10 min, 60%-90% A; 10-10.1 min, 90%-25% A; 10.1-12 min, 25% A Methanol (A)-0.1% formic acid aqueous solution (B), gradient elution: 0~6min, 10%~90%A; 6~8min, 90%A; 8~8.5min, 90%~10%A; 8.5~10min, 10%A Mass spectrometry conditions Ion source: ESI; ionization mode: positive and negative ion scanning simultaneously. Monitoring mode: multiple reaction monitoring (MRM). Mass spectrometry analysis parameters: mass-to-charge ratios of the parent ions and daughter ions of the seven substances to be tested and their corresponding fragmentation voltages. The ion source is ESI source, the scanning mode is negative ion, and the monitoring mode is multiple reaction monitoring (MRM). The mass spectrometry analysis parameters are the mass-to-charge ratios of the parent ions and daughter ions of the three substances to be tested and their corresponding fragmentation voltages. Experimental Results This method can determine the contents of seven components of Rhizoma Dioscoreae in 12 minutes, and uses glycyrrhizin as the internal standard to establish a one-test-multiple-evaluation method for multiple components, saving the cost of purchasing other reference substances. This method has high precision, good repeatability and high accuracy, and can provide a methodological basis for comprehensively controlling the quality of Rhizoma Dioscoreae. like Figure 2 As shown, it can be used for the analysis and detection of erythrine, formononetin and sophora japonica in the Chinese medicinal material Rhizoma Cynomorii in different growth periods, but it cannot determine the active ingredients such as formononetin and glycyrrhizin.

Claims

1. A method for detecting the quality of Herba Lycopodiellae or its extract, characterized in that: It uses high performance liquid chromatography-mass spectrometry to detect erythrine and flavonoid components in the herb or its extract, wherein the flavonoid components include liquiritigenin, calycosin isoflavones, formononetin, isoliquiritigenin, formononetin, and sophora japonica. The HPLC conditions were: The chromatographic column is Shimadzu HPLC Shim-pack GIST C 18 2.1×100mm, 2μm; mobile phase A is methanol A, mobile phase B is aqueous solution; gradient elution conditions are: 0-3min, 25%-60%A; 3-10min, 60%-90%A; 10-10.1min, 90%-25%A; 10.1~12min, 25%A; volume flow rate: 0.3 mL / min; column temperature: 30 ℃; injection volume: 5 μL; Mass spectrometry conditions: nebulizer gas flow rate 3 L / min, heating gas flow rate 10 L / min, interface temperature 300 °C, desolvation temperature 526 °C, DL temperature 275 °C, heating block temperature 400 °C, drying gas flow rate 10 L / min; ion source ESI; ionization mode: positive and negative ion scanning simultaneously; parent ion, daughter ion, fragmentation voltage CE, and ionization mode of the components to be measured are: Erythrine: m / z 244.8, m / z 141.8, 18 V, negative ions; Licorice root: m / z 255.0, m / z 118.9, 28 V, negative ions; Isoflavones: m / z 283.0, m / z 268.0, 14 V, negative ions; Formononetin: m / z 431.0, m / z 269.1, 16 V, positive ions; Isoliquiritigenin: m / z 255.0, m / z 118.9, 28 V, negative ions; Formononetin: m / z 266.9, m / z 252.0, 25 V, negative ion; Sophora flavescens: m / z 282.9, m / z 254.0, 25 V, negative ions.

2. The quality detection method of the Herba Lycopodii or its extract according to claim 1, characterized in that: It includes the following steps: a. Preparation of test sample: Take 0.5g of dry powder of Radix Codonopsis pilosulae, weigh accurately, place in a stoppered conical flask, add 50mL of 50% methanol accurately, weigh, ultrasonicate for 30min, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, accurately measure 1mL of the filtrate, transfer to a 10mL volumetric flask, add 50% methanol to the mark, shake well, and obtain; b. Preparation of reference solution: accurately weigh reference substances of erythrine, formononetin, licorice, calycosin, isoliquiritigenin, kaempferol, and formononetin, and prepare mixed reference solutions with concentrations of 6000-8000 ng / mL, 30-40 ng / mL, 120-140 ng / mL, 60-80 ng / mL, 70-90 ng / mL, 1400-1600 ng / mL, and 700-900 ng / mL, respectively, with methanol. Dilute the mixed reference solutions into 6 different concentrations, store at 4°C, and set aside. c. Detection according to the method described in claim 1.

3. The quality detection method of the Herba Lysimachiae or its extract according to claim 1 or 2, characterized in that: The amounts of erythrine, formononetin, liquiritigenin, calycosin, isoliquiritigenin, sophora flavescens, and formononetin contained in the Herba Lycopodii are 173.6-331.3 μg / g, 1.113-1.837 μg / g, 1.468-9.127 μg / g, 2.345-6.814 μg / g, 0.7250-2.544 μg / g, 36.43-157.7 μg / g, and 3.229-28.84 μg / g, respectively.

4. A method for determining the contents of seven components in Radix Codonopsis pilosulae or its extract by one-test-multiple-evaluation method, characterized in that: The method adopts the quality detection method described in any one of claims 1 to 3 for detection, takes glycyrrhizin as an internal reference, and calculates the relative correction factors fs / x of erythrinaine, formononetin, isoliquiritigenin, calycosin isoflavone, sophora japonica and formononetin respectively according to the formula fs / x=fs / fx=AxCs / (AsCx); wherein Cs and Cx represent the mass concentrations of the internal reference and the component to be detected respectively, and As and Ax represent the peak area values ​​of the internal reference and the component to be detected respectively; the content of glycyrrhizin is usually determined, and the contents of erythrinaine, formononetin, isoliquiritigenin, calycosin isoflavone, sophora japonica and formononetin in the Radix Eupatorii or its extract are calculated according to the relative correction factors fs / x of erythrinaine, formononetin, isoliquiritigenin, calycosin isoflavone, sophora japonica and formononetin.

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