Method for determining content of anthraquinone components in madder and application

Abstract

The application discloses a method for determining the content of anthraquinone components in Rubia cordifolia, comprising the following steps: preparing a standard solution; drawing a standard curve of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside; preparing a test sample solution; and taking the test sample solution to perform high performance liquid chromatography analysis to determine the content. The method for determining the content of anthraquinone components in Rubia cordifolia can determine the content of the prototype components 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in Rubia cordifolia, and provides a basis for the quality control and evaluation of Rubia cordifolia and its preparations.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of quality analysis and detection of traditional Chinese medicine, and particularly relates to a method for determining the content of anthraquinone components in Rubia Cordifolia L. and application thereof. BACKGROUND

[0002] Rubia Cordifolia L. is recorded in the first volume of Chinese Pharmacopoeia 2020 edition, and the source of Rubia Cordifolia L. is the dry root and rhizome of Rubia Cordifolia L. of Rubiaceae. It is dug in spring and autumn, and the mud and sand are removed and dried. Rubia Cordifolia L. was first recorded in Shennong Bencao Jing and was listed as the top grade. Rubia Cordifolia L. is also known as Qiangen, Lomo, Lomo, Xueshun, Ditusu, Guoshanlong, Fengchegao, Baxiancao, Dixue, and Huoxuedan. Shu Ben·Tu Jing records: “This is the current dye madder. There are many in the east, but not as many as in the west.” Shu Ben·Tu Jing records: “Dyeing fuchsia grass, leaf like jujube leaf, head sharp, stem and leaf are astringent, four or five leaves are opposite nodes, and the vines spread on the grass and wood, the root is purple red.” Bencaogangmu records: “Rubia Cordifolia L. grows in December, and spreads several feet. The stem is hollow with ribs, and has several thorns. Each node has five leaves, and the leaves are rough and astringent, with green faces and green backs. Flowers bloom in July and August, and the fruits are small peppers, with fine seeds in the middle.” Rubia Cordifolia L. has the effects of cooling blood, removing blood stasis, stopping bleeding, and dredging channels. It is used for hemoptysis, epistaxis, metrorrhagia, external trauma bleeding, blood stasis, and joint pain. Modern pharmacological studies have shown that Rubia Cordifolia L. has anti-inflammatory, hemostatic, anticancer, antibacterial, immunosuppressive, vasodilating, antispasmodic, and white blood cell increasing activities, and has been used in clinical practice for many years.

[0003] Studies have found that the main chemical components in Rubia Cordifolia L. include anthraquinone, naphthoquinone, and glycosides, in addition to terpenes, polysaccharides, cyclohexapeptides, Fe, Zn, Mg, and other trace elements. Among them, anthraquinone and naphthoquinone components account for the main components, including hydroxymadder, large-leaf madder, isomadder, 1-hydroxy-2-methylanthraquinone, 1,4-dihydroxy-6-methylanthraquinone, and the like, which have antipyretic, analgesic, anti-inflammatory, antirheumatic, and antitumor pharmacological effects. Rubia Cordifolia L. is distributed all over the country, mainly produced in Weinan, Shaanxi, Luoyang and Gaoxian, Henan, Baoding and Xingtai, Hebei, Shandong, and Penglai; in addition, Hubei, Jiangsu, Zhejiang, Gansu, Liaoning, and Shanxi (autonomous region) also have it.

[0004] The research on the index components of Rubia Cordifolia L. mainly focuses on the hydrolyzed components. The 2020 edition of Chinese Pharmacopoeia takes large-leaf madder and hydroxymadder as the content determination index of Rubia Cordifolia L. The preparation of the test sample adopts cold soaking in methanol overnight, ultrasonic extraction, evaporation of the solvent, hydrolysis with an acid reagent, addition of an alkaline solution, and multiple steps for processing, and the process lacks the research on the original index components. Studies have found that Rubia Cordifolia L. contains a large amount of glycosides with 1,3,6-trihydroxy-2-methylanthraquinone as the aglycone, but there is no report on taking it as the content determination index. Therefore, in order to ensure the quality of Rubia Cordifolia L., it is necessary to study the anthraquinone components of Rubia Cordifolia L. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a method for determining the content of anthraquinone components in Rubia cordifolia, which can determine the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in Rubia cordifolia, and provide a basis for quality control and evaluation of Rubia cordifolia and its preparations.

[0006] To solve the above technical problem, the present application provides a method for determining the content of anthraquinone components in Rubia cordifolia, comprising the following steps:

[0007] Preparation of a standard solution comprising 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside reference substance and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside reference substance;

[0008] Precise preparation of standard solutions of different concentrations for high performance liquid chromatography analysis, with sample concentration as the abscissa and peak area as the ordinate to draw standard curves of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside, respectively;

[0009] Taking Rubia cordifolia medicinal materials, Rubia cordifolia standard decoction or Rubia cordifolia formula granules to prepare test sample solutions;

[0010] Taking the test sample solutions for high performance liquid chromatography analysis to determine the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside or 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside;

[0011] The conditions for high performance liquid chromatography analysis include the use of an octadecylsilane-bonded silica gel chromatographic column, with acetonitrile as mobile phase A and formic acid solution as mobile phase B.

[0012] In an embodiment, the formic acid solution has a volume concentration of 0.05%-0.15%;

[0013] The volume ratio of the mobile phase A to the mobile phase B is (25-30):(70-75).

[0014] In an embodiment, the conditions for high performance liquid chromatography analysis further include a sample injection amount of 9-11 μL;

[0015] An Agilent ZORBAX SB-C18 chromatographic column with a size of 150 mm x 4.6 mm and a particle size of 5.0 μm is used.

[0016] The column temperature is 28-32°C.

[0017] The flow rate of the mobile phase is 0.8-1.2 ml / min.

[0018] The UV detection wavelength is 270-280 nm.

[0019] The theoretical plate number calculated based on the 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside peak should be not less than 3000.

[0020] In an embodiment, the Rubia cordifolia medicinal material is used to prepare a test sample solution, including the following steps:

[0021] 0.9-1.1 g of Rubia cordifolia medicinal material powder is precisely weighed and mixed with 20-30 ml of methanol, heated and refluxed for 25-35 min, then cooled, the weight is re-weighed, the lost weight is made up with methanol, shaken, filtered, and the filtrate is obtained.

[0022] In an embodiment, the Rubia cordifolia standard decoction is used to prepare a test sample solution, including the following steps:

[0023] 0.1-0.3 g of Rubia cordifolia standard decoction is precisely weighed and mixed with 20-30 ml of methanol, ultrasonically treated for 25-35 min, then cooled, the weight is re-weighed, the lost weight is made up with methanol, shaken, filtered, and the filtrate is obtained.

[0024] In an embodiment, the Rubia cordifolia formula granules are used to prepare a test sample solution, including the following steps:

[0025] 0.1-0.3 g of Rubia cordifolia formula granules is precisely weighed and mixed with 20-30 ml of methanol, ultrasonically treated for 25-35 min, then cooled, the weight is re-weighed, the lost weight is made up with methanol, shaken, filtered, and the filtrate is obtained.

[0026] In an embodiment, the linear regression equation of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside is y=40.799x+18.992.

[0027] In an embodiment, the linear regression equation of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside is y=35.656x-1.5758.

[0028] Correspondingly, the determination method of the anthraquinone content in Rubia cordifolia is applied in (1), (2), (3), or (4):

[0029] (1) Determination of 1,3,6-trihydroxy-2-methylanthraquinone-3-0-neohesperidoside or 1,3,6-trihydroxy-2-methylanthraquinone-3-0-(6'-0-acetyl)-neohesperidoside in Rubia cordifolia;

[0030] (2) Determination of 1,3,6-trihydroxy-2-methylanthraquinone-3-0-neohesperidoside or 1,3,6-trihydroxy-2-methylanthraquinone-3-0-(6'-0-acetyl)-neohesperidoside in Rubia cordifolia standard decoction;

[0031] (3) Determination of 1,3,6-trihydroxy-2-methylanthraquinone-3-0-neohesperidoside or 1,3,6-trihydroxy-2-methylanthraquinone-3-0-(6'-0-acetyl)-neohesperidoside in Rubia cordifolia dispensing granules;

[0032] (4) Distinguish Rubia cordifolia and counterfeit Rubia akane.

[0033] In one embodiment, if the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-0-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-0-(6'-0-acetyl)-neohesperidoside in medicinal materials or decoction pieces is greater than or equal to 4.0 mg / g, it is Rubia cordifolia medicinal materials or decoction pieces, and if the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-0-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-0-(6'-0-acetyl)-neohesperidoside in medicinal materials or decoction pieces is less than 4.0 mg / g, it is counterfeit Rubia akane medicinal materials or decoction pieces;

[0034] If the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-0-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-0-(6'-0-acetyl)-neohesperidoside in standard decoction is greater than or equal to 15.0 mg / g, it is Rubia cordifolia standard decoction, and if the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-0-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-0-(6'-0-acetyl)-neohesperidoside in standard decoction is less than 15.0 mg / g, it is counterfeit Rubia akane standard decoction;

[0035] If the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the formula granules is greater than or equal to 10.0 mg / g, the formula granules are madder formula granules, and if the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the formula granules is less than 10.0 mg / g, the formula granules are counterfeit large-leaf madder formula granules.

[0036] The present application has the following beneficial effects:

[0037] The present application first uses high performance liquid chromatography to analyze the content of anthraquinone components in madder, and the determination method of the content of anthraquinone components in madder provided by the present application meets the requirements in terms of specificity, precision, repeatability, stability, intermediate precision, sample recovery rate and durability, and can be well used for the determination of the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside.

[0038] The peak purity of the chromatographic peaks of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the atlas obtained by using the high performance chromatography analysis method provided by the present application is high, and the peak purity is greater than 0.999. The linear regression equation of the content determination of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside is y=40.799x+18.992, and the correlation coefficient r is 1.0000, indicating that the linear relationship between the concentration of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and the peak area is good within the range of 5.932 μg / ml-296.590 μg / ml; the linear regression equation of the content determination of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside is y=35.656x-1.5758, and the correlation coefficient r is 0.9999, indicating that the linear relationship between the concentration of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside and the peak area is good within the range of 6.172 μg / ml-308.602 μg / ml. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The specificity of the madder medicinal material characteristic atlas is investigated;

[0040] Figure 2Purity investigation of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside peak and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside peak;

[0041] Figure 3 Full wavelength scan chart of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside;

[0042] Figure 4 Full wavelength scan chart of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside;

[0043] Figure 5 Comparison chart of total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in Rubia cordifolia and Rubia akane;

[0044] Figure 6 Comparison chart of total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in Rubia cordifolia and Rubia akane;

[0045] Figure 7 Comparison chart of total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in Rubia cordifolia and Rubia akane;

[0046] Figure 8 Comparison chart of total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in Rubia cordifolia and Rubia akane. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be described in further detail below with reference to the drawings and specific embodiments.

[0048] Madder as a commonly used traditional Chinese medicine, the research on quality control is not perfect. The acid hydrolysis sample treatment method is used to determine the content of hydroxyalizarin and alizarin in madder under the madder item of Chinese Pharmacopoeia 2020 edition, which lacks the research on the prototype component. Nowadays, with the increasingly strict requirements of traditional Chinese medicine quality control, it is an inevitable trend to use the prototype component as the quality control index. In order to solve the above technical problems, the present application provides a method for determining the content of anthraquinone components in madder, and the specific implementation manner is as follows:

[0049] 1 Instruments, reagents and reagents

[0050] Instruments: Waters high performance liquid chromatograph (Waters e2695, Waters Technology Co., Ltd.), Agilent high performance liquid chromatograph (1260, Agilent Company), Agilent ZORBAX SB-C18 (4.6mmx150mm, 5um) chromatographic column, Waters Xselect HSS T3 (4.6mmx150mm, 5um) chromatographic column, Merck Purospher STAR RP-18 (4.6mmx150mm, 5um) chromatographic column; one ten thousandth electronic analytical balance (ME204E, Mettler-Toledo Company), one millionth electronic analytical balance (XP26, Mettler-Toledo Company), numerical control ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck Co., Ltd.), electric heating constant temperature water bath (HWS28, Shanghai Yiheng Technology Co., Ltd.).

[0051] Reagents: ethanol (Tianjin Fuyu Fine Chemical Co., Ltd.), methanol (Tianjin Fuyu Fine Chemical Co., Ltd.) analytical pure; formic acid (Komei Co., Ltd.), acetonitrile (Merck Co., Ltd.), methanol (Merck Co., Ltd.) are chromatographic grade, and water is ultrapure water (laboratory self-made).

[0052] Reagents: 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside reference substance (batch number: 13184; source: Shanghai Shidande Standard Technology Service Co., Ltd.; content≥98%); 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside reference substance (batch number: DST220601-360; source: Chengdu Lemaitian Pharmaceutical Technology Co., Ltd.; content≥98%).

[0053] Chinese Pharmacopoeia 2020 edition stipulates that the madder base is the dried root and rhizome of Rubia cordifolia L. of Rubiaceae. The medicinal materials used in this study are identified by Chinese Academy of Chinese Medical Sciences Institute of Chinese Materia Medica. The specific information is shown in Table 1.

[0054] Table 1 Information of medicinal material producing area

[0055]

[0056]

[0057] 2 Chromatographic conditions

[0058] Agilent ZORBAX SB-C18 (4.6 mm x 150 mm, 5 μm) column was selected; acetonitrile-0.1% formic acid solution (28:72) was used as mobile phase; detection wavelength was 276 nm; flow rate was 1 ml per minute; column temperature was 30 °C; injection volume was 10 μl.

[0059] 3 Preparation of reference solution

[0060] 2.080 mg of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside reference substance and 2.112 mg of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside reference substance were precisely weighed into a 20 ml volumetric flask, and methanol was added to prepare a mixed solution containing 101.920 μg of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 103.488 μg of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside per 1 ml, thus obtaining the reference solution.

[0061] 4 Preparation of test solution

[0062] The extraction solvent, extraction method and extraction time of the content determination method of Rubia cordifolia were investigated to determine the optimal sample pretreatment method.

[0063] (1) Investigation of extraction solvent

[0064] About 1 g of Rubia cordifolia powder (passed No. 2 sieve, batch number: YG08) was precisely weighed, and 8 groups in parallel, 2 portions in each group, were placed in a conical flask with a plug. 25 ml of methanol, 70% methanol, 50% methanol, 30% methanol, ethanol, 70% ethanol, 50% ethanol and 30% ethanol were precisely added, respectively, the weight was determined, ultrasonic treatment (power 250 W, frequency 40 kHz) was performed for 30 minutes, the temperature was lowered, the weight was determined again, the lost weight was made up with the corresponding solvent, shaken, filtered, and the filtrate was collected. 10 μl was precisely taken and injected into the liquid chromatograph for determination, thus obtaining the test solution. The experimental results are shown in Table 2.

[0065] Table 2 Content determination results of Rubia cordifolia medicinal material with different extraction solvents

[0066]

[0067]

[0068] The experimental results show that: by comparing the contents of 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside determined by different extraction solvents, it is found that the extraction efficiency of methanol and 70% methanol is better, and the extraction efficiency of methanol is the highest, so methanol is selected as the extraction solvent.

[0069] (2) Extraction method investigation

[0070] Take about 1 g of Rubia cordifolia powder (pass through No. 2 sieve, batch number: YG08), accurately weigh, place in a stoppered triangular flask, accurately add 25 ml of methanol, tightly stop, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, heating reflux for 30 minutes, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, and obtain. Accurately take 10 μl, inject into the liquid chromatograph, and determine. The experimental results are shown in Table 3.

[0071] Table 3 Content determination results of Rubia cordifolia medicinal materials by different extraction methods

[0072]

[0073] The experimental results show that: by comparing the contents of 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside determined by ultrasonic treatment and heating reflux, it is found that the extraction efficiency of heating reflux is higher, so heating reflux is selected as the extraction method.

[0074] (3) Extraction time investigation

[0075] Take about 1 g of Rubia cordifolia powder (pass through No. 2 sieve, batch number: YG08), accurately weigh, place in a stoppered triangular flask, accurately add 25 ml of methanol, tightly stop, weigh, heating reflux for 15 min, 30 min, 45 min, and 60 min, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, and obtain. Accurately take 10 μl, inject into the liquid chromatograph, and determine. The experimental results are shown in Table 4.

[0076] Table 4 Content determination results of Rubia cordifolia medicinal materials by different extraction times

[0077]

[0078] The experimental results show that: by comparing the content of 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in madder at different reflux times, when heated and refluxed for 30 minutes, the extraction is complete, therefore, the heating reflux time is selected as 30 minutes.

[0079] (4) Determination of test solution preparation method

[0080] According to the sample pretreatment investigation experiment results, the test solution preparation method can be determined as follows: take about 1 g of madder medicinal material powder (pass through No. 2 sieve), accurately weigh, place in a conical flask with a plug, accurately add 25 ml of methanol, weigh, heat and reflux for 30 minutes, cool, re-weigh, make up the weight loss with methanol, shake well, filter, and take the filtrate, which is obtained.

[0081] Take the madder standard decoction, grind finely, take about 0.2 g, place in a conical flask with a plug, accurately add 25 ml of methanol, ultrasonic treat (power 300 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with methanol, shake well, filter, and take the filtrate, which is obtained.

[0082] Take the madder formula granules, grind finely, take about 0.2 g, place in a conical flask with a plug, accurately add 25 ml of methanol, ultrasonic treat (power 300 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with methanol, shake well, filter, and take the filtrate, which is obtained.

[0083] 5 Methodology verification

[0084] (1) Specificity investigation

[0085] Take about 1 g of madder medicinal material powder (pass through No. 2 sieve, batch number: YG05), accurately weigh, prepare the test solution according to the method determined in item “4”, and take the 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside mixed control solution and negative sample solution according to the chromatographic conditions in item “2” for injection determination. The results are shown in Figure 1 . Figure 1 The results show that the test sample chromatogram has the same chromatographic peak at the retention time corresponding to the control sample chromatogram, and the negative sample has no interference, indicating that the method has good specificity.

[0086] (2) Peak purity investigation

[0087] Accurately take 10 μl of the test solution in the specificity item, inject into the liquid chromatograph, and perform 190 nm-400 nm scanning detection with a PDA detector according to the chromatographic conditions in item “2”, and calculate the peak purity. The results are shown inFigure 2 , Figure 3 The results show that no impurity peak is detected in the sample of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside, and the peak purity is >0.999, indicating that the peak purity of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside meets the requirements under the chromatographic conditions.

[0088] (3) Linear investigation

[0089] Accurately weigh 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside reference substance 3.122 mg and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside reference substance 3.149 mg into a 10 ml volumetric flask, dissolve with methanol to the mark to obtain a mixed reference substance stock solution, the concentration of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside is 296.59 μg / ml, and the concentration of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside is 308.602 μg / ml.

[0090] Accurately pipette 0.2, 0.5, 1, 2, and 5 ml of the above reference substance stock solution into 10 ml volumetric flasks, add methanol to the mark, shake well, and sequentially inject 10 μl of the sample according to the chromatographic conditions in item “2” along with the reference substance stock solution, and record the chromatographic peak area. Take the peak area as the vertical coordinate (y) and the reference substance concentration as the horizontal coordinate (x), the results are shown in Tables 5 and 6, and the standard curve is drawn.

[0091] Table 5 Linear investigation results of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside

[0092]

[0093] Table 6 Linear investigation results of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside

[0094]

[0095] The analysis shows that the linear regression equation of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside is y = 40.799x + 18.992, the correlation coefficient r = 1.0000, indicating that the concentration of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside has a good linear relationship with the peak area in the range of 5.932 μg / ml-296.590 μg / ml; the linear regression equation of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside is y = 35.656x-1.5758, the correlation coefficient r = 0.9999, indicating that the concentration of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside has a good linear relationship with the peak area in the range of 6.172 μg / ml-308.602 μg / ml.

[0096] (4) Precision examination

[0097] The madder medicinal material (batch number: YG05) was precisely taken to prepare the test sample solution according to the method in item “4”, the sample was repeatedly injected 6 times under the chromatographic conditions in item “2”, the injection volume was 10 μl, the peak areas of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the test sample solution were determined, and the peak area RSD was calculated, and the determination results are shown in Table 7.

[0098] Table 7 Precision examination results

[0099]

[0100] The results show that the same control sample solution is continuously injected for 6 times, the peak area RSD value of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside is 0.71%, the peak area RSD value of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside is 0.65%, both of which are less than 3.0%, indicating that the instrument precision is good.

[0101] (5) Stability examination

[0102] The madder medicinal material (batch number: YG05) was precisely taken to prepare the test sample solution according to the method in item “4”, the sample was repeatedly injected 6 times under the chromatographic conditions in item “2”, the injection volume was 10 μl, the peak areas of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the test sample solution were determined, and the peak area RSD was calculated, and the determination results are shown in Table 7.

[0103] Table 8 Stability Investigation Results

[0104]

[0105]

[0106] The results show that the test solution was sampled at 0 h, 2 h, 4 h, 6 h, 9 h, and 12 h, respectively, and the peak area RSD values of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside were 0.62% and 1.29%, respectively, both less than 3.0%, indicating that the test solution was stable within 12 hours.

[0107] (6) Reproducibility Investigation

[0108] Take about 1 g of Rubia cordifolia powder (pass through No. 2 sieve, batch number: YG05), accurately weigh, and parallelly weigh 6 portions. Prepare the test solution according to the test solution preparation method determined in item “4”. According to the chromatographic condition determination in item “2”, determine the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the test solution, calculate the RSD, and the determination results are shown in Table 9.

[0109] Table 9 Reproducibility Investigation Results

[0110]

[0111] The results show that the same batch of sample was repeatedly determined for 6 times, and the RSD values of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside were 2.72% and 2.61%, respectively. According to the “Guiding Principles for Validation of Analytical Methods for Drug Quality Standards” in the Chinese Pharmacopoeia 2020 edition, when the content of the measured component in the sample is 0.1%-1%, the reproducibility RSD limit is <3%, therefore, the reproducibility of the analysis method is good.

[0112] (7) Intermediate Precision Investigation

[0113] Different analysts operated on different dates and different chromatographs, and took about 1 g of the same batch of Rubia cordifolia powder (pass through No. 2 sieve, batch number: YG05), accurately weighed, and parallelly weighed 6 portions. Prepare the test solution according to the test solution preparation method determined in item “4”, and determine according to the chromatographic condition determination in item “2”. The results are shown in Table 10.

[0114] The intermediate precision investigation results in Table 10

[0115]

[0116] The results show that the same batch of samples is operated by different personnel at different times on different instruments, and the 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-neohesperidoside content RSD value is 1.87% for 6 repeated determinations, compared with the 6 data of the repeatability test, the 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-neohesperidoside content RSD value is 2.28%; the 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside content RSD value is 2.00%, compared with the 6 data of the repeatability test, the 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside content RSD value is 2.82%, according to the "Guiding Principles for Verification of Analysis Methods for Drug Quality Standards" in the Chinese Pharmacopoeia 2020 edition, when the content of the measured component in the sample is 0.1% to 1%, the intermediate precision RSD limit is <4%, therefore, different analysts operate on different dates and different chromatographs, and the intermediate precision of the method is good.

[0117] (8) Investigation of sample addition recovery rate

[0118] Precisely weigh 25.102 mg of 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-neohesperidoside reference substance (batch number: 13184; source: Shanghai Shidande Standard Technology Service Co., Ltd.; content ≥98%) and 39.443 mg of 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside reference substance (batch number: DST220601-360; source: Chengdu Lemaitian Pharmaceutical Technology Co., Ltd.; content ≥98%) into a 20 ml volumetric flask, add methanol to prepare a sample addition recovery mother liquor containing 1.23000 mg of 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-neohesperidoside and 1.93271 mg of 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside per 1 ml. Precisely take 1.0 ml, 2.0 ml, and 3.0 ml of the above mother liquor into a stoppered conical flask, 3 replicates for each group, a total of 9 replicates, and dry the solvent; then take about 0.5 g of madder herb powder (passed through a No. 2 sieve, batch number: YG05) into the above 9 conical flasks, prepare the test sample solution according to the method determined in item "4"; determine the amount of 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the test sample solution by injecting the sample under the chromatographic conditions in item "2", and calculate the sample addition recovery rate, the results are shown in Tables 11 and 12.

[0119] Table 11 Rubia medicinal material 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside sample addition recovery rate results

[0120]

[0121] Table 12 Rubia medicinal material 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside sample addition recovery rate results

[0122]

[0123] The results show that the sample addition recovery rate of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside ranges from 96.52% to 99.22%, the average sample addition recovery rate is 97.89%, and the RSD is 0.88%; the sample addition recovery rate of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside ranges from 95.81% to 100.21%, the average sample addition recovery rate is 98.62%, and the RSD is 1.28%; according to the "Guiding Principles for Validation of Analysis Methods for Drug Quality Standards" in the Chinese Pharmacopoeia 2020 edition, when the content of the measured component in the sample is in the range of 0.1%-1%, the recovery rate limit is 92%-105%, indicating that the recovery rate is good.

[0124] (9) Durability investigation

[0125] ① Investigation of different chromatographic columns

[0126] The effects of Agilent ZORBAX SB-C18 (4.6 mm x 150 mm, 5 μm) chromatographic column, Waters Xselect HSS T3 (4.6 mm x 150 mm, 5 μm) chromatographic column, and Merck Purospher STAR RP-18 (4.6 mm x 150 mm, 5 μm) chromatographic columns of different brands on the determination of the contents of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in Rubia medicinal material were compared.

[0127] Rubia medicinal material powder (passed through a No. 2 sieve, batch number: YG05) was taken to prepare the test solution according to the method in item "4", except that the chromatographic column was different, and the other chromatographic conditions were according to the chromatographic conditions in item "2". The experimental results are shown in Table 13.

[0128] Table 13 Durability investigation results of different chromatographic columns

[0129]

[0130]

[0131] The results show that the RSD values of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside determined by using three different brands of chromatographic columns are 1.77% and 2.49% respectively, both of which are less than 3.0%, indicating that the analysis method has good durability on different brands of chromatographic columns.

[0132] ②Different column temperature investigation

[0133] The effects of different column temperatures 28℃, 30℃ and 32℃ on the determination of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in Rubia cordifolia were compared. Rubia cordifolia powder (passed through No. 2 sieve, batch number: YG05) was prepared into test solution according to the method under item “4”, except that the column temperature was 28℃, 30℃ and 32℃ respectively, and other chromatographic conditions were according to the chromatographic conditions under item “2”. The experimental results are shown in Table 14.

[0134] Table 14 Durability investigation results of different column temperatures

[0135]

[0136] The results show that the RSD values of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside determined by using three different brands of chromatographic columns are 1.77% and 2.49% respectively, both of which are less than 3.0%, indicating that the analysis method has good durability on different brands of chromatographic columns.

[0137] ③Different flow rate investigation

[0138] The effects of different flow rates 0.8ml / min, 1.0ml / min and 1.2ml / min on the determination of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in Rubia cordifolia were compared. Rubia cordifolia powder (passed through No. 2 sieve, batch number: YG05) was prepared into test solution according to the method under item “4”, except that the flow rate was 0.8ml / min, 1.0ml / min and 1.2ml / min respectively, and other chromatographic conditions were according to the chromatographic conditions under item “2”. The experimental results are shown in Table 15.

[0139] Table 15 Durability investigation results of different flow rates

[0140]

[0141] The results show that the RSD of the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-0-neohesperidoside is 0.70%, and the RSD of the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-0-(6'-0-acetyl)-neohesperidoside is 1.47%, both less than 3.0%, indicating that the analysis method has good durability within the flow rate ± 0.2 ml / min range.

[0142] (4) Investigation of different mobile phase proportions

[0143] The effects of different mobile phase proportions, acetonitrile-0.1% formic acid (26:74), (28:72), (30:70), respectively, on the determination of the contents of 1,3,6-trihydroxy-2-methylanthraquinone-3-0-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-0-(6'-0-acetyl)-neohesperidoside in Rubia cordifolia were compared.

[0144] Rubia cordifolia powder (passed through a No. 2 sieve, batch number: YG05) was taken to prepare the test sample solution according to the method in item "4", except that the mobile phase proportions were acetonitrile-0.1% formic acid (26:74), (28:72), (30:70), respectively, and the other chromatographic conditions were according to the chromatographic conditions in item "2". The experimental results are shown in Table 16.

[0145] Table 16 Investigation results of different mobile phase proportions

[0146]

[0147]

[0148] The results show that the RSD of the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-0-neohesperidoside is 0.70%, and the RSD of the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-0-(6'-0-acetyl)-neohesperidoside is 1.47%, both less than 3.0%, indicating that the analysis method has good durability within the flow rate ± 0.2 ml / min range.

[0149] (10) Durability summary

[0150] In summary, the validation of the determination and analysis method of madder content, the peak purity analysis of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside chromatographic peaks in the test solution were carried out, and the peak purity was greater than 0.999; the linear regression equation of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside content determination was y=40.799x+18.992, and the correlation coefficient r=1.0000, indicating that the 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside concentration had a good linear relationship with the peak area in the range of 5.932 μg / ml-296.590 μg / ml; the linear regression equation of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside content determination was y=35.656x-1.5758, and the correlation coefficient r=0.9999, indicating that the 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside concentration had a good linear relationship with the peak area in the range of 6.172 μg / ml-308.602 μg / ml; the whole analysis method was investigated for specificity, precision, repeatability, stability, intermediate precision, sample recovery rate and durability, and the results met the requirements, indicating that the established method could be well used for the determination of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside contents.

[0151] 6Sample determination

[0152] Take 18 batches of madder medicinal materials and corresponding 18 batches of decoction pieces, 18 batches of standard decoction and 18 batches of formula granules in Table 1, 3 batches of large leaf madder medicinal materials and corresponding 3 batches of decoction pieces, 3 batches of standard decoction and 3 batches of formula granules, prepare 21 test solution according to the test solution preparation method confirmed in item "4", and determine the above test solution by sample, record the peak area of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the chromatogram of the test solution, calculate the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the test solution by external standard method, and calculate the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside, the results are shown in Table 17, Figures 5-8 .

[0153] Table 17 18 batches of madder samples and 3 batches of counterfeit large leaf madder anthraquinone content determination results

[0154]

[0155] From the above results, the method provided by the present application can be applied to (1), (2), (3) or (4):

[0156] (1) determining the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside or 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in madder medicinal materials;

[0157] (2) determining the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside or 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in madder standard decoction;

[0158] (3) determining the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside or 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in madder dispensing granules;

[0159] (4) distinguishing madder medicinal materials, standard decoction or dispensing granules from counterfeit large leaf madder.

[0160] Further, as shown in Figures 5-8 if the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the medicinal material or decoction piece is greater than or equal to 4.0 mg / g, it is madder medicinal material or decoction piece, and if the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the medicinal material or decoction piece is less than 4.0 mg / g, it is counterfeit large leaf madder medicinal material or decoction piece;

[0161] if the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the standard decoction is greater than or equal to 15.0 mg / g, it is madder standard decoction, and if the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the standard decoction is less than 15.0 mg / g, it is counterfeit large leaf madder standard decoction;

[0162] If the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the formula granules is greater than or equal to 10.0 mg / g, the formula granules are madder formula granules, and if the total content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in the formula granules is less than 10.0 mg / g, the formula granules are counterfeit large-leaf madder formula granules.

[0163] In summary, the present application first uses high performance liquid chromatography to analyze the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in madder, and the entire analysis method meets the requirements of specificity, precision, repeatability, stability, intermediate precision, sample recovery rate and durability, and can provide a basis for quality control and evaluation of madder and its preparations.

[0164] The above is a preferred embodiment of the application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the application, and these improvements and refinements are also considered to be within the scope of protection of the application.

Claims

1. A method for determining the content of anthraquinone components in Rubia cordifolia, characterized in that, It comprises the following steps: Prepare a standard solution comprising 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-neohesperidoside control and 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside control; Prepare different concentrations of standard solution for high performance liquid chromatography analysis, and draw the standard curve of 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside respectively with sample concentration as the abscissa and peak area as the ordinate. Take the madder medicinal material, madder standard decoction or madder formula granules, and extract them with methanol to prepare the test solution. Take the test solution for high performance liquid chromatography analysis to determine the content of 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside. The conditions of high performance liquid chromatography analysis include: using acetonitrile as mobile phase A and formic acid solution as mobile phase B. The volume concentration of the formic acid solution is 0.05%-0.15%. The volume ratio of the mobile phase A to the mobile phase B is (25-30):(70-75). An Agilent ZORBAX SB-C18 chromatographic column with a size of 150mm*4.6mm*5.0μm is used. The ultraviolet detection wavelength is 270nm-280nm.

2. The method for determining the anthraquinone content in Rubia cordifolia as described in claim 1, characterized in that, The conditions of high performance liquid chromatography analysis also include: the sample injection amount is 9μL-11μL. The column temperature is 28°C-32°C. The flow rate of the mobile phase is 0.8mL / min-1.2mL / min. The theoretical plate number calculated according to the peak of 1,3,6-trihydroxy-2-methyl anthraquinone-3-O-(6'-O-acetyl)-neohesperidoside should not be less than 3000.

3. The method for determining the anthraquinone content in Rubia cordifolia as described in claim 1, characterized in that, The preparation of the test solution from madder medicinal material comprises the following steps: Precisely weigh 0.9g-1.1g of madder medicinal material powder and mix it with 20mL-30mL of methanol, heat it to reflux for 25min-35min, cool it down, weigh it again, make up the weight loss with methanol, shake it well, filter it, and take the filtrate, which is the test solution.

4. The method for determining the anthraquinone content in Rubia cordifolia as described in claim 1, characterized in that, The preparation of the test solution from madder standard decoction comprises the following steps: Precisely weigh 0.1g-0.3g of madder standard decoction and mix it with 20mL-30mL of methanol, ultrasonically treat it for 25min-35min, cool it down, weigh it again, make up the weight loss with methanol, shake it well, filter it, and take the filtrate, which is the test solution.

5. The method for determining the anthraquinone content in Rubia cordifolia as described in claim 1, characterized in that, The preparation of the test solution from madder formula granules comprises the following steps: Precisely weigh 0.1g-0.3g of madder formula granules and mix it with 20mL-30mL of methanol, ultrasonically treat it for 25min-35min, cool it down, weigh it again, make up the weight loss with methanol, shake it well, filter it, and take the filtrate, which is the test solution.

6. The method for determining the anthraquinone content in Rubia cordifolia as described in claim 1, characterized in that, The linear regression equation of the 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside is y=40.799x+18.

992.

7. The method for determining the anthraquinone content in Rubia cordifolia as described in claim 1, characterized in that, The linear regression equation of the 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside is y=35.656x-1.5758.

8. The use of the method for determining the content of anthraquinones in Rubia cordifolia according to any one of claims 1-7 in (1), (2) or (3): (1) determining the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in Rubia cordifolia medicinal materials; (2) determining the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in Rubia cordifolia standard decoction; (3) determining the content of 1,3,6-trihydroxy-2-methylanthraquinone-3-O-neohesperidoside and 1,3,6-trihydroxy-2-methylanthraquinone-3-O-(6'-O-acetyl)-neohesperidoside in Rubia cordifolia dispensing granules.

Images