Method for determining content of caulis polygoni multiflori in traditional Chinese medicine preparation

Abstract

The invention discloses a method for determining the content of caulis polygoni multiflori in a traditional Chinese medicine preparation, and relates to the field of detection methods of high performance liquid chromatography, the method adopts high performance liquid chromatography for determination, the high performance liquid chromatography comprises selection of a reference substance, a filler and a mobile phase, the reference substance comprises 2, 3, 5, 6-tetramethyl-3-piperidine-2-one, and the filler comprises 2, 3, 5, 6-tetramethyl-3-piperidine-2-one. The mobile phase is prepared from 4, 4 '-tetrahydroxystilbene-2-O-beta-D-glucoside, the filler is prepared from octadecylsilane chemically bonded silica, the mobile phase is prepared from acetonitrile and water, and the volume ratio of the acetonitrile to the water is (14-25): (75-95). By optimizing chromatographic conditions and chromatographic detection conditions, such as composition of a mobile phase and selection of a reference substance and a filling agent, the detection efficiency of 2, 3, 5, 6, 7-tetramethyl-4-piperidine in the gastrodia elata and uncaria granules is improved, and the quality of the gastrodia elata and uncaria granules is improved. According to the present invention, with the method, the THSG concentration in the sample can be accurately determined according to the precision and the sensitivity of the THSG content in the sample, such that the reliable result can be obtained even at the low concentration level.

Description

Technical Field

[0001] The invention relates to the field of high performance liquid chromatography detection methods, and in particular to a method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation. Background Art

[0002] Hypertension (diabetes) is a common endocrine metabolic disease, with persistent increase in systemic arterial blood pressure as the main manifestation. Symptoms of hypertensive patients include dizziness, headache, insomnia, irritability, palpitations, memory loss, limb numbness, chest tightness, shortness of breath, palpitations, etc.

[0003] Among the existing drugs for treating hypertension, Tianma Gou Teng Granule is the best Chinese medicine for clinical treatment of hypertension. Tianma Gou Teng Granule is mainly used to calm the liver and extinguish wind, clear away heat and calm the mind. It is used for headache, dizziness, tinnitus, blurred vision, tremor, insomnia caused by hyperactivity of liver yang; hypertension with the above symptoms. The prescription of Tianma Gou Teng Granule is derived from hundreds of years of precipitation, accumulation and development. It uses authentic all-natural Chinese medicinal materials and is refined by modern pharmaceutical technology. It embodies the experience and hard work of clinical practice of traditional Chinese medicine for a hundred years.

[0004] The currently available Tianma Gou Teng Granules are composed of Tianma, Uncaria, Semen Cassiae, Gardenia, Scutellaria, Achyranthes, Eucommia, Leonurus, Morus, Polygonum multiflorum, and Poria. They have been used clinically for many years and their efficacy is unquestionable. However, for the quality control of Tianma Gou Teng Granules, the current national standard only conducts TLC identification of emodin in Polygonum multiflorum. The detection method is too simple and there is a large gap compared with the current quality control requirements of traditional Chinese medicine products. Therefore, the existing quality control method cannot effectively control the quality of traditional Chinese medicine preparations for the treatment of hypertension, thereby affecting the production and quality assurance of the product.

[0005] In view of this, this application is filed. Summary of the invention

[0006] The purpose of the present invention is to provide a method for determining the content of Polygonum multiflorum in a traditional Chinese medicine preparation, and to solve the problem that the quality of a traditional Chinese medicine preparation for treating hypertension cannot be effectively controlled in the prior art by limiting a reference substance, a filler and a mobile phase in a high performance liquid chromatography.

[0007] The present invention is achieved through the following technical scheme: An embodiment of the present invention provides a method for determining the content of Polygonum multiflorum in a traditional Chinese medicine preparation, comprising determining by high performance liquid chromatography, wherein the high performance liquid chromatography comprises selecting a reference substance, a filler and a mobile phase, the reference substance comprises 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, the filler comprises octadecylsilane bonded silica gel, the mobile phase comprises acetonitrile and water, the volume ratio of the acetonitrile to the water is 14-25:75-95, preferably, the volume ratio of the acetonitrile to the water is 20:95.

[0008] Specifically, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside is an important chemical component in Radix Polygoni Multiflori, and has significant pharmacological effects, so it is selected as a reference substance, which helps to ensure the accuracy and reproducibility of the determination. Octadecylsilane bonded silica gel is a reverse phase chromatography filler with good stability and separation effect, which can improve the separation efficiency of various compounds. The ratio of acetonitrile and water in the mobile phase has an important influence on the separation effect. In the embodiment of the present invention, the volume ratio of the mobile phase is 14-25:75-95, which helps to achieve good separation of the effective ingredients in Radix Polygoni Multiflori. The embodiment of the present invention selects reference substances, fillers and mobile phases, and limits the composition and concentration of the mobile phase to establish an accurate and reliable HPLC method, which can achieve efficient separation and accurate determination of the effective ingredients in Radix Polygoni Multiflori, and is used to determine the content of Radix Polygoni Multiflori in traditional Chinese medicine preparations, thereby improving the level of product quality control.

[0009] Furthermore, in the high performance liquid chromatography, the detection wavelength is 320 nm, and the theoretical plate number is calculated based on the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside peak and is greater than or equal to 3,000.

[0010] 320nm was selected as the detection wavelength because it is close to the maximum absorption wavelength of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (THSG), thus providing good sensitivity and selectivity. The theoretical plate number is an important parameter for evaluating the performance of a chromatographic column, reflecting the separation efficiency of the chromatographic column. A theoretical plate number greater than or equal to 3000 indicates that the chromatographic system has a high separation efficiency and can provide clear chromatographic peaks, which is conducive to accurate quantitative analysis. In HPLC analysis, a high theoretical plate number helps to improve the separation degree and reduce the overlap between peaks, thereby improving the accuracy and repeatability of the analysis.

[0011] Furthermore, the preparation process of the reference substance includes adding an extractant to 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, mixing to obtain the reference substance solution, wherein each 1 ml of the reference substance solution contains 75 to 100 μg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

[0012] The preparation of the reference solution is an important step in chromatographic analysis because it can provide a standard with a known concentration for quantitative analysis. Specifically, a certain amount of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference is accurately weighed, and the weighed reference is added to an appropriate amount of extractant. The extractant needs to be able to effectively dissolve the target compound. For example, it can be methanol, a methanol solution with a volume fraction of 65-75%, or an ethanol solution with a volume fraction of 50-55%, etc. As a preferred embodiment of the present invention, the extractant is preferably a 50-55% ethanol solution. The 50-55% ethanol solution can provide a certain stability, which helps THSG to maintain its chemical properties during storage and reduce decomposition. 50-55% ethanol solution can be effectively used for the extraction and dissolution of THSG, and provides a reliable extractant selection for the determination of THSG content in Polygonum multiflorum by high performance liquid chromatography (HPLC). In addition, THSG, as an active ingredient in Polygonum multiflorum, has multiple pharmacological effects such as antioxidant, anti-aging, and anti-tumor, and is one of the important indicators for evaluating the quality of Polygonum multiflorum. By optimizing the selection of extractants, the extraction efficiency and analytical accuracy of THSG in Polygonum multiflorum can be improved, thereby better controlling the quality of traditional Chinese medicine preparations such as Tianma Uncaria granules.

[0013] Furthermore, the high performance liquid chromatography method also includes the preparation of a test sample, and the preparation process of the test sample includes taking a sample to be tested, grinding it, adding an extractant to mix it, weighing it, then heating the mixed liquid under reflux and then cooling it, weighing it again, making up for the lost weight and then mixing it, and finally filtering it, and the filtrate obtained is the test sample solution.

[0014] The sample is ground into fine powder to increase the contact area between the sample and the extractant and improve the extraction efficiency. The mixed liquid is heated and refluxed to accelerate the extraction process. After heating and reflux, the mixture is allowed to cool naturally or placed in a cold water bath for rapid cooling to reduce the volatilization of the solvent and the degradation of the sample. After cooling, the weight of the mixture is weighed to determine the evaporation amount of the solvent, and the weight lost due to evaporation is supplemented to ensure the accuracy of the concentration of the test solution.

[0015] Further, the sample to be detected is Tianma Uncaria Granules, and the mass ratio of the sample to be detected to the extractant is 0.5 to 1.0: 30 to 50. Specifically, in order to improve the extraction efficiency, according to the mass ratio, an appropriate amount of Tianma Uncaria Granule sample is accurately weighed, and the operating conditions are strictly controlled to ensure the quality and consistency of the test solution, and at the same time ensure that the effective ingredients in the Tianma Uncaria Granules are fully extracted, so as to provide a high-quality test solution for HPLC analysis.

[0016] Furthermore, the preparation method of the Gastrodia elata and Uncaria rhynchophylla granules comprises the following steps:

[0017] Grind the Gastrodia elata into fine powder for later use, so as to facilitate the subsequent mixing and preparation process;

[0018] Add water to Uncaria rhynchophylla, Semen Cassiae, Gardenia jasminoides, Scutellaria baicalensis, Achyranthes bidentata, Eucommia ulmoides, Leonurus japonicus, Morus alba, Polygonum multiflorum and Poria cocos and boil twice, and combine the decoctions;

[0019] The decoction is filtered in turn, the filtrate is concentrated, dextrin and Gastrodia powder are added and mixed, and granules are made and dried to obtain Uncaria rhynchophylla granules. The medicinal liquids obtained from the two decoctions are combined to ensure that the active ingredients are fully collected. Dextrin, as an auxiliary material, can help improve the formation and stability of the granules.

[0020] Furthermore, in the high performance liquid chromatography method, the reference solution and the test solution are respectively injected into the liquid chromatograph, and the amount of Polygonum multiflorum contained in each 1g of the sample to be tested is calculated according to the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside obtained by the test. Specifically, according to the concentration of THSG in the test solution and the preparation ratio of the test sample, the amount of Polygonum multiflorum contained in each gram of Tianma Gou Teng granules is calculated, the chromatogram is analyzed, the retention time of THSG, the shape of the chromatographic peak and the purity are determined, and according to the calculation results, whether the content of Polygonum multiflorum in Tianma Gou Teng granules meets the quality standards is evaluated.

[0021] Furthermore, the heating reflux time is 55 to 65 minutes to ensure sufficient extraction of the active ingredients while avoiding over-extraction or decomposition. By precisely controlling the heating reflux time, the consistency and repeatability of the extraction process can be ensured, thereby obtaining a high-quality test solution, providing a reliable basis for subsequent HPLC analysis.

[0022] Compared with the prior art, the embodiments of the present invention have the following advantages and beneficial effects:

[0023] 1. The embodiments of the present invention improve the precision and sensitivity of the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) in Gastrodia elata Uncaria granules by optimizing the chromatographic conditions and chromatographic detection conditions, such as the composition of the mobile phase, the selection of reference substances and fillers, so that the concentration of THSG in the sample can be accurately determined, and reliable results can be obtained even at low concentration levels.

[0024] 2. This method provides a reliable means of quality control, making the quality of the drug production process and the final product controllable, which helps regulatory agencies and manufacturers to supervise the quality of drugs. By accurately measuring the content of active ingredients, the actual efficacy of drugs can be evaluated to ensure that the drugs used by patients have the expected therapeutic effects. It helps to ensure the quality control of drugs in the production process and ensure that the quality of the final product is safe, uniform and stable. High performance liquid chromatography provides an effective, accurate and reliable analytical means for the quality control of traditional Chinese medicine preparations for the treatment of hypertension, which helps to improve the overall quality of drugs and the safety and effectiveness of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 The chromatogram of the reference substance solution in Example 4 of the present invention;

[0027] Figure 2 The chromatogram of the sample solution in Example 4 of the present invention;

[0028] Figure 3 It is the chromatogram of the negative sample solution in Example 4 of the present invention; Figure 4 This is a diagram of the linear detection situation in the implementation of the present invention. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0030] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The embodiment of the present invention provides a method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation, comprising the following steps:

[0032] S1. Chromatographic conditions and system suitability experiment: octadecylsilane bonded silica gel was used as filler; acetonitrile-water was used as mobile phase, and the detection wavelength was 320nm; the theoretical plate number calculated based on the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside peak should be no less than 3000;

[0033] S2. Preparation of reference solution: accurately weigh an appropriate amount of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference substance, add an extractant to prepare a solution containing 75-100 μg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside per 1 ml, which is the reference solution;

[0034] S3. Preparation of test solution: Grind the product, weigh 0.5-1.0g accurately, add 30-50g of extractant accurately, weigh the weight, heat under reflux for 55-65min, cool, weigh again, make up the lost weight with dilute ethanol, shake well, filter, and take the filtrate, which is the test solution;

[0035] S4. Determination: Accurately aspirate the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine; calculate the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside per 1g of Polygonum multiflorum.

[0036] For ease of understanding, the Chinese medicine preparation in the embodiment of the present invention takes Tianma Uncaria Granules as an example, but the detection method provided in the embodiment of the present invention is not limited to Tianma Uncaria Granules, and other Chinese medicine preparations containing Polygonum multiflorum can also be detected, which is not limited here.

[0037] Content determination experimental process

[0038] 1. Instruments and test drugs

[0039] Instrument: Agilent high performance liquid chromatograph.

[0040] Chromatographic column: phenomenex-C18 column 5μm 4.6×250mm

[0041] Sepax Sappire-C18 column 5μm 4.6×250mm

[0042] Reagents: acetonitrile (chromatographic grade, Thermo Fisher Scientific (China) Co., Ltd.), water is deionized water.

[0043] Reference substance: 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (purchased from China Food and Drug Inspection Institute, batch number: 110844-202317, for content determination).

[0044] Sample: Tianma Gouteng Granules were provided by Chengdu Jiuzhitang Jinding Pharmaceutical Co., Ltd. (Batch No.: 240102, 240103, 240105, 240106, 240109, 240111, 240203, 240301, 240302, 240304.)

[0045] 2. Chromatographic conditions

[0046] 2.1 Selection of detection wavelength: Referring to the content determination item of Polygonum multiflorum in the 2020 edition of the Chinese Pharmacopoeia, the detection wavelength is stipulated to be 320nm.

[0047] 2.2 Selection of mobile phase: Refer to the mobile phases in the 2020 edition of the Chinese Pharmacopoeia under the Radix Polygoni Multiflori and the Tianma Uncaria Granules standard, and select acetonitrile-water (25:75), acetonitrile-water (10:80) and methanol-water (20:80). Although the above mobile phases can obtain good chromatograms, because this product contains many impurities, the peak time of Radix Polygoni Multiflori chromatographic peak is too early, and the baseline separation cannot be achieved, which has a slight impact on the determination results. As a result, acetonitrile-water (20:95) was selected as the mobile phase of this method, and its effect was better. The chromatographic peaks can basically achieve baseline separation, and the separation degree basically meets the requirements.

[0048] 2.3 Chromatographic conditions: The chromatographic conditions of this method were formulated based on the above experiments and with reference to the method for determining the content of Polygonum multiflorum in Part 1 of the 2020 edition of the Chinese Pharmacopoeia.

[0049] Octadecylsilane bonded silica gel was used as filler; acetonitrile-water (20:95) was used as mobile phase; detection wavelength was 320nm, column temperature was 30℃, flow rate was 1ml / min. The theoretical plate number was calculated based on the peak of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside and should be no less than 3000.

[0050] 3. Selection of extraction conditions

[0051] 3.1 Investigation of extraction solvent: Take samples of the same batch number (batch number: 240105), grind them into powder, take about 1g, weigh them accurately, add 50ml of methanol, 70% methanol and dilute ethanol respectively, weigh them, treat them with ultrasound for 60 minutes, let them cool, weigh them again, make up the lost weight with their own solvents, filter them, take the filtrate, and determine the content of Polygonum multiflorum in the samples. The results are shown in Table 1.

[0052] Table 1: Selection of extraction solvents

[0053]

[0054] Table 1 shows that the extraction results using methanol, 70% methanol, and dilute ethanol as solvents showed no significant difference in retention time. The results of the system suitability test showed that dilute ethanol was selected as the extraction solvent.

[0055] 3.2 Investigation of extraction time: Take samples of the same batch number (batch number: 240105), grind them into powder, take about 1g, weigh them accurately, add 50ml of dilute ethanol accurately, weigh them, heat and reflux for 30 and 60 minutes respectively, cool them, weigh them again, make up the lost weight with dilute ethanol, filter them, take the filtrate, and determine the content of Polygonum multiflorum in the samples. The results are shown in Table 2.

[0056] Table 2: Investigation of extraction time

[0057]

[0058] Table 2 shows that when the extraction time is 30 min and 60 min, the content determination result is smaller and the relative deviation is larger when the extraction time is 30 min. Therefore, the heating reflux time is selected as 60 min in this experiment.

[0059] 3.3 Solution preparation

[0060] 3.3.1 Preparation of reference solution: Take an appropriate amount of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference substance, accurately weigh it, and add dilute ethanol to make a solution containing 0.1 mg per 1 ml.

[0061] 3.3.2 Preparation of test solution: Take the product, grind it into powder, take about 1g, weigh it accurately, add 50ml of dilute ethanol accurately, weigh it, heat and reflux for 60 minutes, cool it, weigh it again, make up the lost weight with dilute ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0062] 3.3.3 Preparation of negative sample solution: Take all the herbs except Polygonum multiflorum according to the prescription, prepare the preparation sample according to the above method, and then prepare the negative sample solution according to the test sample solution preparation method in "3.3.2".

[0063] 3.3.4 Determination: Accurately pipette 10μl of each solution under "3.3.1~3.3.3", inject it into the high performance liquid chromatograph, and analyze it according to the chromatographic conditions of "2.3". The obtained chromatogram is as follows, and the chromatogram of the reference solution is as follows: Figure 1 , the chromatogram of the test solution is shown in Figure 2 , the chromatogram of negative sample solution is shown in Figure 3 As can be seen in the figure, the negative sample has no interference at the chromatographic peak of Polygonum multiflorum.

[0064] 3.4 Preparation of standard curve

[0065] Take an appropriate amount of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference substance, accurately weigh it, add dilute ethanol to make a solution containing 0.12 mg per ml, and dilute it with dilute ethanol to make solutions containing 2, 10, 40, 80, 100, and 120 μg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference substance per ml. As the reference solution, accurately pipette 10 μl of the reference solution of different concentrations mentioned above, inject them into the high performance liquid chromatograph respectively, analyze them according to the chromatographic conditions of "2.3", determine the peak area of ​​each, take the reference sample injection amount (μg) as the horizontal coordinate, and the peak area value as the vertical coordinate, and obtain the regression equation: Y=38.573x-9.3711, r=0.9999. The results show that Polygonum multiflorum has good linearity in the range of 2-120 μg, and the results are shown in Table 3 and Figure 4 shown.

[0066] Table 3

[0067] Concentration of reference substance (μg / μ1) Injection volume (μ1) Peak area 2.04 10 77.74 9.96 10 381.94 40.08 10 1512.30 80.04 10 3091.76 99.84 10 3823.05 120.00 10 4633.31

[0068] 3.5 Injection precision test

[0069] Take the sample of the same batch number (batch number: 240105), grind it into powder, take about 1g, weigh it accurately, prepare the test solution according to "4.2", analyze it according to the chromatographic conditions of "2.3", inject the sample continuously for 6 times, and determine the peak area of ​​2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in the sample. The average peak area is 434.08; the RSD of the peak area is 3.2%, which meets the requirements. The results are shown in Table 4.

[0070] Table 4

[0071]

[0072]

[0073] 3.6 Reproducibility test

[0074] Take 6 samples of the same batch number (batch number: 240105), grind them into powder, take about 1g, weigh them accurately, and follow the preparation of the test solution in "4.2" and the chromatographic conditions in "2.3" to determine the content of Polygonum multiflorum in each sample. The average content of Polygonum multiflorum in the samples was 1.2842 mg / g, and the RSD was 4.65%, which met the requirements. The results are shown in Table 5.

[0075] Table 5

[0076]

[0077] 3.7 Stability test

[0078] Take the sample of the same batch number (batch number: 240105), grind it into powder, take about 1g, weigh it accurately, prepare the test solution according to "4.2", analyze it according to the chromatographic conditions of "2.3", and determine the peak area of ​​Polygonum multiflorum in the sample at 0, 4, 8, 12, and 24 hours respectively. The RSD of the peak area value was 1.7%, which met the requirements. The results are shown in Table 6.

[0079] Table 6

[0080]

[0081] 3.8 Recovery test

[0082] Take the same batch number (batch number: 240105) sample, grind it, take about 0.5g, a total of 6 portions, accurately weigh, put into a round-bottom flask, accurately add 10ml of Polygonum multiflorum reference substance dilute ethanol solution (0.008mg / ml), accurately add 50ml of dilute ethanol, and then follow the "4.2" test solution preparation operation to prepare the test solution for recovery rate, analyze according to the "2.3" chromatographic conditions, calculate the recovery rate, and the average recovery rate is 96.7%, RSD is 4.5%. It meets the requirements, and the results are shown in Table 7.

[0083] Table 7

[0084]

[0085] 3.9 Sample determination

[0086] Take samples of 11 batches of this product, prepare the test solution according to "3.3.2", and determine the content of Polygonum multiflorum in the samples according to the chromatographic conditions in "2.3". The results are shown in Table 8.

[0087] Table 8

[0088]

[0089] The 2020 edition of the Chinese Pharmacopoeia stipulates that the herbal medicine containing 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (C 20 H 22 09) shall not be less than 0.20%. Each 1g of this product contains 268mg of Polygonum multiflorum, and theoretically contains no less than 0.536mg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside. This product is an extract concentrate for medicinal use. Calculated based on the content of 62%, each 1g of this product contains 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (C 20 H 22 09), it should not be less than 0.33mg. Table 8 shows that all 11 batches of samples meet the requirements, and the measured content is 0.9092~1.2751mg / g.

[0090] In general, the method for determining the content of Radix Polygoni Multiflori in the traditional Chinese medicine preparation for treating hypertension provided in the embodiment of the present invention has improved and perfected the quality standard of the traditional Chinese medicine preparation for treating hypertension. Compared with the current standard, the thin layer identification of Radix Polygoni Multiflori is optimized to a method for determining the content of Radix Polygoni Multiflori. This method has high precision and sensitivity, good stability, simple and fast operation, and high recovery rate, which provides an experimental basis for its safe and rational use of the medicine and ensures the safety, uniformity, stability, effectiveness and controllability of the quality of the traditional Chinese medicine preparation for treating hypertension.

[0091] Example 1

[0092] The embodiment of the present invention provides a method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation, comprising the following steps:

[0093] S1. Chromatographic conditions and system suitability experiment: Octadecylsilane bonded silica gel was used as filler; acetonitrile-water was used as mobile phase, the volume ratio of acetonitrile to water was 20:95, and the detection wavelength was 320nm; the theoretical plate number calculated based on the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside peak should be no less than 3000;

[0094] S2. Preparation of reference solution: accurately weigh 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference substance, add an appropriate amount of 70% by volume methanol to make a solution containing 78 μg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside per 1 ml, which is the reference solution;

[0095] S3. Preparation of test solution: Grind the product, weigh 0.8g accurately, add 45ml of 70% methanol by volume, weigh the weight, heat under reflux for 55min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate, which is the test solution;

[0096] S4. Determination: Accurately aspirate 8μ1 of the reference solution and 5μ1 of the test solution, respectively, inject into the liquid chromatograph for determination; calculate the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside per 1g of this product.

[0097] Example 2

[0098] The embodiment of the present invention provides a method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation, comprising the following steps:

[0099] S1. Chromatographic conditions and system suitability experiment: Octadecylsilane bonded silica gel was used as filler; acetonitrile-water was used as mobile phase, the volume ratio of acetonitrile to water was 25:85, and the detection wavelength was 320nm; the theoretical plate number calculated based on the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside peak should be no less than 3000;

[0100] S2. Preparation of reference solution: Accurately weigh 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference substance, add an appropriate amount of methanol to make a solution containing 100 μg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside per 1 ml, which is the reference solution;

[0101] S3. Preparation of test solution: Grind the product, weigh 1.2g accurately, add 50ml of methanol accurately, weigh the weight, heat and reflux for 30min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate, which is the test solution;

[0102] S4. Determination: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph for determination; calculate the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in each 1g of Polygonum multiflorum.

[0103] Example 3

[0104] The embodiment of the present invention provides a method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation, comprising the following steps:

[0105] S1. Chromatographic conditions and system suitability experiment: Octadecylsilane bonded silica gel was used as filler; acetonitrile-water was used as mobile phase, the volume ratio of acetonitrile to water was 20:95, and the detection wavelength was 320nm; the theoretical plate number calculated based on the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside peak should be no less than 3000;

[0106] S2. Preparation of reference solution: accurately weigh 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference substance, add appropriate amount of dilute ethanol by volume to make a solution containing 82 μg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside per 1 ml, which is the reference solution;

[0107] S3. Preparation of test solution: Grind the product, weigh 1g accurately, add 50ml of 52.9% dilute ethanol by volume, weigh the weight, heat under reflux for 60min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate, which is the test solution;

[0108] S4. Determination: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph for determination; calculate the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside per 1g of Polygonum multiflorum.

[0109] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation, characterized in that: The method comprises the steps of using high performance liquid chromatography to carry out determination, wherein the high performance liquid chromatography comprises the steps of selecting a reference substance, a filler and a mobile phase, wherein the reference substance comprises 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, the filler comprises octadecylsilane bonded silica gel, and the mobile phase comprises acetonitrile and water, wherein the volume ratio of the acetonitrile to the water is 14-25:75-95.

2. The method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation according to claim 1, characterized in that: In the high performance liquid chromatography, the detection wavelength is 320 nm, and the theoretical plate number is calculated based on the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside peak and is greater than or equal to 3000.

3. The method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation according to claim 2, characterized in that: The preparation process of the reference substance comprises adding an extractant to 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, and mixing to obtain the reference substance solution, wherein each 1 ml of the reference substance solution contains 75 to 100 μg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

4. The method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation according to claim 2, characterized in that: The high performance liquid chromatography method also includes the preparation of a test sample, and the preparation process of the test sample includes taking a sample to be tested, grinding it, adding an extractant to mix it, then heating the mixed liquid to reflux and then cooling it, then weighing it, making up the lost weight and then mixing it, and finally filtering it. The filtrate obtained is the test sample solution.

5. The method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation according to claim 4, characterized in that: The sample to be detected is Gastrodia elata and Uncaria rhynchophylla granules, and the mass ratio of the sample to be detected to the extractant is 0.5-1.0:30-50.

6. The method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation according to claim 3 or 4, characterized in that: The extractant comprises one of methanol, a methanol solution with a volume fraction of 65-75%, and an ethanol solution with a volume fraction of 50-55%.

7. The method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation according to claim 5, characterized in that: The preparation method of the Gastrodia elata and Uncaria rhynchophylla granules comprises the following steps: Grind Gastrodia elata into fine powder for later use; Add water to Uncaria rhynchophylla, Semen Cassiae, Gardenia jasminoides, Scutellaria baicalensis, Achyranthes bidentata, Eucommia ulmoides, Leonurus japonicus, Morus alba, Polygonum multiflorum and Poria cocos and boil twice, and combine the decoctions; The decoction is filtered, the filtrate is concentrated, dextrin and gastrodia powder are added and mixed, and the mixture is made into granules and dried to obtain Uncaria rhynchophylla granules.

8. The method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation according to any one of claims 1 to 7, characterized in that: The high performance liquid chromatography method also includes injecting the reference solution and the test solution into the liquid chromatograph respectively, and calculating the amount of Polygonum multiflorum per 1g of the sample to be tested based on the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside content obtained by the test.

9. The method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation according to claim 4, characterized in that: The heating reflux time is 55 to 65 minutes.

10. The method for determining the content of Radix Polygoni Multiflori in a traditional Chinese medicine preparation according to claim 1, characterized in that: In the fluidity, the volume ratio of acetonitrile to water is 20:95.

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