A kind of red bean formula granule and red bean medicinal material quality control method

Abstract

The present invention provides a quality control method for adzuki bean formula granules and adzuki bean medicinal materials, comprising the following steps: extracting adzuki bean formula granules or adzuki bean medicinal materials to prepare a test solution; mixing uridine and water to prepare a reference solution; respectively detecting the test solution and the reference solution by high performance liquid chromatography to obtain the content of uridine in the adzuki bean formula granules or adzuki bean medicinal materials and the characteristic spectrum of the adzuki bean formula granules or adzuki bean medicinal materials. The present invention provides a method for constructing an HPLC characteristic spectrum of adzuki bean formula granules or adzuki bean medicinal materials and a method for determining the content. The same method can be used as both a characteristic spectrum identification and a content determination method. The method provided by the present invention is highly efficient, simple, and effective, and can strengthen specific identification and content determination for quality control, thereby performing comprehensive quality control on adzuki bean formula granules or adzuki bean medicinal materials.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a red bean formula granule and a quality control method for red bean medicinal materials. Background Art

[0002] The adzuki bean described in the present invention is the dried mature seeds of Vigna umbellata Ohwi et Ohashi or Vigna angularis Ohwi et Ohashi of the leguminous plant. It is sweet and sour in taste, flat in nature and non-toxic. It has the effects of promoting diuresis and relieving swelling, detoxifying and draining pus, and is commonly used clinically for symptoms such as edema, beriberi, limb swelling, jaundice, red urine, rheumatism, heat arthralgia, carbuncle, furuncle, intestinal carbuncle and abdominal pain.

[0003] The current legal quality standards stipulate the identification of properties, microscopic identification, thin layer identification, moisture determination, total ash determination and extracts. There are almost no reports on the fingerprint identification of red bean formula granules, and there are few studies on content determination, mostly on the determination of phenols and flavonoids.

[0004] Muhetar Kaderhazi, Wang Haitao, Tu Pengfei, Jiang Yong, RP-HPLC determination of the content of catechin-7-O-β-D-pyranoglucoside in the Chinese herbal medicine red bean, the published chromatographic conditions are: GracePrevail TM C18 Column (4.6×250mm, 5μm); mobile phase acetonitrile (A)-water solution (B) (6:94); flow rate 1.0mL / min; detection wavelength 230nm; column temperature 30℃; injection volume 10μL, chromatographic analysis time 23min. The content determination spectrum of red bean medicinal material is shown in Figure 1 As shown, A is the catechin-7-O-β-D-pyranoglucoside reference substance, and B is the adzuki bean sample. However, only for adzuki bean medicinal materials, there are differences in the material basis and formula granules.

[0005] Therefore, how to provide a quality control method for adzuki bean formula granules and adzuki bean medicinal materials so that they can simultaneously construct characteristic maps and determine their contents has become a problem that needs to be solved urgently. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a quality control method for adzuki bean formula granules and adzuki bean medicinal materials, which can simultaneously construct a characteristic spectrum and determine the content.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a quality control method for adzuki bean formula granules and adzuki bean medicinal materials, the quality control method comprising the following steps:

[0009] (1) extracting adzuki bean formula granules or adzuki bean medicinal materials to prepare a test solution; mixing uridine and water to prepare a reference solution;

[0010] (2) The test solution and the reference solution obtained in step (1) are respectively detected by high performance liquid chromatography to obtain the content of uridine in the red bean formula granules or red bean medicinal materials and the characteristic spectrum of the red bean formula granules or red bean medicinal materials.

[0011] The present invention selects uridine in nucleosides as an indicator component. Uridine is a related component constituting the nucleic acid of animal cells and can improve the body's antibody level. Animal experiments show that the combination of uridine and inosine can promote myocardial cell metabolism, accelerate protein and nucleic acid biosynthesis and energy production, and promote and improve brain cell metabolism. Diseases that can be treated by uridine also include Parkinson's disease, Alzheimer's disease, bipolar disorder, etc. Animal-based research mainly focuses on the anti-depressant effect of uridine combined with omega-3 fatty acids, and it has been proven that its effect is comparable to that of traditional drugs.

[0012] In order to more comprehensively evaluate the quality of adzuki bean formula granules, this study used HPLC to establish a characteristic spectrum and content determination method for adzuki bean formula granules. Three batches of adzuki bean formula granules were used to establish a control spectrum, determine the common peak, and use uridine reference substances for comparison, identify the characteristic peak, take uridine as the content measurement peak, and perform characteristic spectrum and content determination methodology verification. The experimental results show that the method provided by the present invention has good repeatability, high accuracy, good durability, and rich information, which can provide a more comprehensive and effective quality control method for standardizing the quality of adzuki bean formula granules. The method provided by the present invention was used to verify three batches of adzuki bean medicinal materials, and the results showed that the method was also applicable, providing an effective quality control method for adzuki bean medicinal materials. In addition, this study used HPLC to establish a uridine content determination method for adzuki bean, took uridine reference substances as the content measurement index, and performed methodology verification. The experimental results show that the method has good repeatability.

[0013] In the present invention, in step (1), the preparation of the test solution specifically includes the following steps: mixing the adzuki bean formula granules or adzuki bean medicinal materials with an ethanol aqueous solution, ultrasonicating, and filtering to obtain the test solution.

[0014] In the present invention, the volume fraction of ethanol in the ethanol aqueous solution is 5-15% (for example, it can be 5%, 7%, 9%, 11%, 13%, 15%, etc.).

[0015] Preferably, the solid-to-liquid ratio of the adzuki bean formula granules and the ethanol aqueous solution is (0.5-1.5):15g / mL, where "0.5-1.5" can be 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, etc.

[0016] Preferably, the solid-to-liquid ratio of the adzuki bean medicinal material and the ethanol aqueous solution is (1.0-2.0):15g / mL, wherein "1.0-2.0" can be 1.0, 1.3, 1.5, 1.7, 1.9, 2.0, etc.

[0017] In the present invention, the power of the ultrasound is 200-300W (for example, it can be 200W, 220W, 240W, 260W, 280W, 300W, etc.), the frequency of the ultrasound is 30-50kHz (for example, it can be 30kHz, 32kHz, 34kHz, 36kHz, 38kHz, 43kHz, 46kHz, 49kHz, 50kHz, etc.), and the time of the ultrasound is 20-50min (for example, it can be 20min, 23min, 26min, 29min, 30min, 33min, 36min, 39min, 43min, 46min, 49min, 50min, etc.).

[0018] In the present invention, in step (1), the solid-to-liquid ratio of uridine to water is (20-40):1 μg / mL, wherein "20-40" can be 20, 23, 26, 29, 33, 36, 39, 40, etc.

[0019] In the present invention, in step (2), the filler used in the chromatographic column in the high performance liquid chromatography detection includes pure water-resistant octadecylsilane bonded silica gel.

[0020] Preferably, in step (2), the column temperature in the HPLC detection is 28-32°C (for example, it may be 28°C, 29°C, 30°C, 31°C, 32°C, etc.).

[0021] In the present invention, in step (2), the detection wavelength in the high performance liquid chromatography detection is 254 nm.

[0022] Preferably, in step (2), the injection volume in the HPLC detection is 8-12 μL (for example, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, etc.).

[0023] In the present invention, methanol is used as mobile phase A, and 0.1%-0.3% (eg, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, etc.) acetic acid aqueous solution is used as mobile phase B in the HPLC detection.

[0024] In the present invention, in step (2), the HPLC detection is performed by gradient elution according to the following table.

[0025] Time (min) Mobile phase A Mobile phase B 0-28 0→2 100→98 28-35 2→4 98→96 35-44 4→8 96→92 44-56 8→12 92→88

[0026] Preferably, in step (2), in the HPLC detection, the flow rate of the mobile phase is 0.5-0.7 mL / min from 0 to 35 min; the flow rate of the mobile phase is (0.5-0.7)→(0.8-1.0) mL / min from 35 to 44 min; and the flow rate of the mobile phase is 0.8-1.0 mL / min from 44 to 56 min.

[0027] Among them, "0.5-0.7mL / min" can be 0.5mL / min, 0.55mL / min, 0.6mL / min, 0.65mL / min, 0.7mL / min, etc.

[0028] “0.8-1.0 mL / min” can be 0.8 mL / min, 0.85 mL / min, 0.9 mL / min, 0.95 mL / min, 1 mL / min, etc.

[0029] In the present invention, the adzuki bean formula granules or adzuki bean medicinal materials have a total of 4 characteristic peaks, which are peak 1, peak 2, peak 3 and peak 4 in order from short to long in terms of peak elution time, with peak 2 being the S peak, and the relative retention times of peaks 1-4 are within ±10% of the specified values, and the specified values ​​are: 0.27, 1, 2.01 and 2.21, respectively; wherein peak 2 is uridine.

[0030] The content of uridine in adzuki bean formula granules shall not be less than 0.6 mg / g (for example, it may be 0.6 mg / g, 0.7 mg / g, 0.8 mg / g, 0.9 mg / g, 1 mg / g, 1.1 mg / g, 1.2 mg / g, etc.). The content of uridine in adzuki bean medicinal materials shall not be less than 0.1 mg / g (for example, it may be 0.1 mg / g, 0.3 mg / g, 0.5 mg / g, 0.7 mg / g, 0.9 mg / g, 1 mg / g, 1.1 mg / g, etc.).

[0031] The technical problem to be solved by the present invention is as follows: The present invention provides a method for constructing an HPLC characteristic spectrum of red bean formula granules and red bean medicinal materials and a method for determining the content. The same method can be used as both a characteristic spectrum identification and a content determination method. The method provided by the present invention is highly efficient, simple, and effective, and can strengthen the specific identification and content determination quality control, thereby performing comprehensive quality control on the red bean formula granules and red bean medicinal materials.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The present invention constructs the HPLC characteristic spectrum of the red bean formula granules and the red bean medicinal material, which fully demonstrates the chemical composition characteristics of the red bean formula granules and the red bean medicinal material;

[0034] (2) The characteristic spectrum and content determination method constructed by the present invention are highly efficient, stable, precise and reproducible;

[0035] (3) The HPLC characteristic spectrum of the adzuki bean formula granules and adzuki bean medicinal materials established by the present invention can be used to identify the adzuki bean formula granules and adzuki bean medicinal materials, providing a reliable detection method for identifying the adzuki bean formula granules and adzuki bean medicinal materials;

[0036] (4) The HPLC content determination method of the adzuki bean formula granules and adzuki bean medicinal materials established in the present invention can be used to determine the content of uridine in the adzuki bean formula granules and adzuki bean medicinal materials, providing a rapid and reliable detection method for the adzuki bean formula granules and adzuki bean medicinal materials, and can improve the relevant quality standards of the adzuki bean formula granules and adzuki bean medicinal materials to a certain extent, and provide scientific guidance for the better development and utilization of adzuki bean Chinese medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the red bean medicinal material content determination spectrum published in "RP-HPLC determination of catechin-7-O-β-D-pyranoglucoside in the traditional Chinese medicine red bean", where A is the catechin-7-O-β-D-pyranoglucoside reference substance and B is the red bean sample.

[0038] Figure 2 This is the characteristic spectrum of adzuki bean formula granules, where peak 2 is uridine.

[0039] Figure 3 The high performance liquid chromatograms of the reference solution, control solution and adzuki bean formula granule test solution.

[0040] Figure 4 These are the HPLC characteristic spectra of three batches of adzuki bean formula granules, where R is the control characteristic spectrum, S1 is 19041942, S2 is 18026252, and S3 is 20030891.

[0041] Figure 5 These are the HPLC characteristic spectra of three batches of red bean medicinal materials, among which S1 is 202104-03-1, S2 is 202104-03-8, S3 is 202104-03-13, and R is the control characteristic spectrum of red bean medicinal materials.

[0042] Figure 6 This is the HPLC reference characteristic spectrum of red bean medicinal materials.

[0043] Figure 7This is a standard curve of uridine.

[0044] Figure 8 This is the HPLC chromatogram of the adzuki bean formula granules obtained by extraction with different solvents. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0046] The sources of instruments, reagents and reagents in the following examples are as follows:

[0047] Instruments: Agilent 1260 high performance liquid chromatograph; Thermo Vanquish Core high performance liquid chromatograph; JA1002 electronic balance (Shanghai Puchun Metrology Instrument Co., Ltd.); ML204T electronic balance (Mettler Toledo Instrument (Shanghai) Co., Ltd.); MSA6 6S-OCE-DM electronic balance (Sartorius Technology Instrument (Beijing) Co., Ltd.); KQ-300DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); DK-S26 electric constant temperature water bath (Zhongyi Guoke (Beijing) Technology Co., Ltd.);

[0048] Chromatographic column: Waters Xselect HSS T3 (4.6×250 mm, 5 μm);

[0049] Uridine reference substance: (batch number: 110879-201703, purity 99.5%, manufacturer: China Food and Drug Inspection Institutes);

[0050] Adzuki bean formula granules: 18026252, 19041942, 20030891;

[0051] Adzuki bean medicinal materials: 202104-03-1, 202104-03-8, 202104-03-13;

[0052] Adzuki bean control medicinal material (batch number: 121725-201802, manufacturer: China Food and Drug Inspection Institute);

[0053] Reagents: methanol (chromatographic grade, batch number: 191614, manufacturer: Thermo Fisher Scientific (China) Co., Ltd.);

[0054] Ethanol (analytical grade, batch number: 20210114, manufacturer: Tianjin Yongda Chemical Reagent Co., Ltd.);

[0055] Water (distilled water, batch number: 20210113H, manufacturer: Guangzhou Watsons Food and Beverage Co., Ltd.);

[0056] Acetic acid (chromatographically pure, batch number: 196835, manufacturer: Thermo Fisher Scientific (China) Co., Ltd.).

[0057] Example 1

[0058] The present embodiment provides a quality control method for adzuki bean formula granules and adzuki bean medicinal materials, and the quality control method comprises the following steps:

[0059] Preparation of reference solution: Take 1.5 g of adzuki bean reference medicinal material, place it in a stoppered conical flask, add 20 mL of precise distilled water, boil for 30 minutes, cool, shake well, filter, and obtain the filtrate.

[0060] Preparation of reference solution: Take an appropriate amount of uridine reference, weigh accurately, and add distilled water to make a reference solution containing 30 μg of uridine per 1 mL.

[0061] Preparation of the test solution of adzuki bean formula granules: Take adzuki bean formula granules (batch number 19041942), grind them into powder, take about 0.5 g, accurately weigh, put it in a stoppered conical flask, accurately add 15 mL of 10% ethanol aqueous solution, stopper it, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 40 minutes, cool it, weigh it again, make up the lost weight with 10% ethanol aqueous solution, shake it well, filter it, and take the filtrate to obtain it.

[0062] Preparation of adzuki bean medicinal material test solution: The only difference from the adzuki bean formula granule test solution is that 0.5g of adzuki bean formula granule powder is replaced with 1.0g of adzuki bean medicinal material powder.

[0063] High performance liquid chromatography was used to detect the reference solution, reference substance solution and test solution (adzuki bean formula granule test solution, adzuki bean medicinal material test solution).

[0064] The chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A, 0.2% acetic acid aqueous solution as mobile phase B; detection wavelength 254 nm; column temperature 30°C; injection volume 10 μL, gradient elution according to the following table:

[0065] Time (min) Mobile phase (A) Mobile phase (B) Flow rate (mL / min) 0～28 0→2 100→98 0.6 28～35 2→4 98→96 0.6 35～44 4→8 96→92 0.6→0.9 44～56 8→12 92→88 0.9

[0066] The characteristic spectrum of red bean formula granules is as follows Figure 2As shown, there are 4 characteristic peaks, which are peak 1, peak 2, peak 3 and peak 4 in order of peak elution time from short to long, with peak 2 being the S peak, and the relative retention times of peaks 1-4 are within ±10% of the specified values, which are 0.27, 1, 2.01 and 2.21 respectively; peak 2 is uridine. The allowable error is ±10%.

[0067] The HPLC chromatograms of the reference solution, control solution and adzuki bean formula granule test solution are shown in Figure 3 As shown, it can be seen that there are differences in the basic substances between the adzuki bean formula granules and the adzuki bean medicinal materials, and the peak 2 of the adzuki bean formula granules is uridine.

[0068] Example 2

[0069] Validation of feature profile methodology

[0070] 2.1 Precision experiment

[0071] 2.1.1 Repeatability

[0072] Take 6 portions of adzuki bean formula granules (batch number: 19041942), grind them into powder, and measure them according to the method provided in Example 1 to obtain their characteristic spectrum. Take peak No. 2 as the reference peak, calculate their relative peak area and relative retention time, and calculate the RSD value. The retention time and relative retention time of the repeatability test of adzuki bean formula granules are shown in Table 1 below:

[0073] Table 1

[0074]

[0075] According to the results of repeatability investigation, the RSD value of the relative retention time of each characteristic peak was within 2.0%, indicating that the repeatability of the characteristic spectrum of this method was good.

[0076] 2.1.2 Intermediate precision

[0077] Different analysts used Thermo Vanquish Core high performance liquid chromatograph to conduct intermediate precision test at different times, and took 6 portions of red bean formula granules (batch number: 19041942), and measured them according to the method provided in Example 1 to obtain their characteristic spectrum, and calculated their relative peak area and relative retention time with peak No. 2 as the reference peak, and calculated the RSD value. The retention time and relative retention time of red bean intermediate precision test are shown in Table 2 below:

[0078] Table 2

[0079]

[0080]

[0081] According to the results of the intermediate precision test, the relative retention time RSD of each characteristic peak is within 0.2%. The relative retention time RSD between different instruments is within the range of 2.9% to 8.3%. The experimental results show that different brands of instruments have a slight impact on the characteristic spectrum method.

[0082] 2.1.3 Solution stability

[0083] Take adzuki bean formula granules (batch number: 19041942), and measure according to the method provided in Example 1 at 0h, 2h, 4h, 6h, 8h, 12h, 24h, and 36h to obtain its characteristic spectrum; take peak No. 2 as the reference peak, calculate its relative retention time, and calculate RSD. The retention time and relative retention time of adzuki bean stability study are shown in Table 3 below:

[0084] Table 3

[0085]

[0086]

[0087] The results of the stability experiment show that the chemical composition in the test solution is stable for 36 hours, and the relative retention time RSD of each characteristic peak is in the range of 0.3% to 1.7%, indicating that the analytical method is stable and reliable.

[0088] 2.1.4 Durability test

[0089] 2.1.4.1 Different column temperatures

[0090] The influence of slight changes in column temperature on the detection method was investigated. The characteristic spectrum of the red bean formula granule test solution was detected according to the method provided in Example 1 at 28°C, 30°C and 32°C. The retention time and relative retention time of each peak at different column temperatures are shown in Table 4 below:

[0091] Table 4

[0092]

[0093] From the above data, it can be seen that under different column temperature conditions, the RSD values ​​of the relative retention time are in the range of 0.8 to 2.1%, indicating that this method has good durability to the column temperature.

[0094] 2.2 Verification of three batches (19041942, 18026252, 20030891) of adzuki bean formula granules

[0095] The spectra of the three batches of red bean formula granules were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for data matching, and the control characteristic spectra of the red bean formula granules were established. The HPLC characteristic spectra of the three batches of red bean formula granules are as follows: Figure 4As shown, R is the control characteristic spectrum, S1 is 19041942, S2 is 18026252, and S3 is 20030891.

[0096] The retention time and relative retention time of three batches of adzuki bean formula granules are shown in Table 5 below:

[0097] Table 5

[0098]

[0099] The results showed that the parallel samples of HPLC content determination results of 3 batches of red bean formula granules were stable and the method was reliable, which provided a more scientific basis for the quality control of red bean formula granules. There were 4 characteristic peaks in the HPLC characteristic spectrum of 3 batches of red bean formula granules, among which peak 2 had the same retention time as the uridine reference substance. Peak 2 was taken as the S peak, and the relative retention time of each characteristic peak and the S peak was calculated. The relative retention time was within ±10% of the specified value. The specified values ​​were: 0.28 (peak 1), 1.99 (peak 3), and 2.21 (peak 4).

[0100] This method can not only quickly and intuitively identify adzuki bean formula granules, but also simultaneously determine the uridine content in adzuki bean, providing a more scientific basis for the quality control of adzuki bean formula granules.

[0101] 2.3 Verification of three batches (202104-03-1, 202104-03-8, 202104-03-13) of red bean medicinal materials

[0102] Determination method: According to the method provided in Example 1, the characteristic spectrum of the red bean medicinal material test solution is detected, and the spectrum is imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for data matching to establish a control characteristic spectrum of the red bean medicinal material.

[0103] The results of verifying the retention time and relative retention time of three batches of red bean medicinal materials (202104-03-1, 202104-03-8, and 202104-03-13) are shown in Table 6. The HPLC characteristic spectra of the three batches of red bean medicinal materials are as follows Figure 5 As shown (S1 is 202104-03-1, S2 is 202104-03-8, S3 is 202104-03-13, and R is the red bean medicinal material reference characteristic spectrum). Among them, the red bean medicinal material reference characteristic spectrum is the red bean medicinal material reference characteristic spectrum obtained after matching the three batches of red bean medicinal material sample spectra measured in Example 1 with the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". The red bean medicinal material HPLC reference characteristic spectrum is as shown Figure 6 shown.

[0104] Table 6

[0105]

[0106] From Table 6 and Figure 5 The data show that the verification results of three batches of medicinal materials illustrate that the method provided by the present invention is not only applicable to adzuki bean granules, but also can achieve quality control of adzuki bean medicinal materials.

[0107] Example 3

[0108] Validation of assay methodology

[0109] 3.1 Accuracy

[0110] Accuracy refers to the degree to which the result determined by this method is close to the true value or reference value, and is generally expressed as recovery rate (%). Accurately weigh 12.169 mg of uridine (purity: 99.5%) in a 100 mL volumetric flask, add 10% ethanol to dissolve and dilute to the scale to make a mixed reference substance mother solution, from which 10.0 mL, 20.0 mL, and 15.0 mL are accurately measured in 100 mL, 100 mL, and 50 mL volumetric flasks, respectively, and 10% ethanol is added to the scale as reference substance solutions ①, ②, and ③. Take 9 portions of red bean samples (19041942, uridine content is 0.68 mg / g) with known content under the repeatability item, each portion is about 0.5 g, accurately weighed, and each three portions are grouped together, and 15 mL of reference substance solutions ①, ②, and ③ are added respectively. The test method is the same as Example 1, and the recovery rate is calculated according to the following formula. The results are shown in Table 7 below:

[0111]

[0112] Table 7

[0113]

[0114]

[0115] The recovery rate test showed that the recovery rate of uridine ranged from 96.98% to 103.31%, and the average recovery rate was 99.2%, which met the recovery rate requirements of the methodology validation, indicating that the results measured by this method are accurate and reliable.

[0116] 3.2 Precision

[0117] 3.2.1 Repeatability

[0118] Take 6 portions of adzuki bean formula granules (batch number: 19041942), measure according to the method provided in Example 1, calculate the content of uridine, and calculate the RSD value. The results of the uridine content determination of the adzuki bean formula granules are shown in Table 8 below:

[0119] Table 8

[0120]

[0121] The repeatability test showed that the average uridine content in adzuki bean granules was 0.73 mg / g, and the RSD value was 0.5%, which met the repeatability requirements of method validation.

[0122] 3.2.2 Intermediate precision

[0123] Different analysts conducted intermediate precision tests at different times using Thermo Vanquish Core high performance liquid chromatographs. Take 6 portions of adzuki bean granules (batch number: 19041942), grind them into powder, and measure them according to the method provided in Example 1 to calculate the content of uridine and calculate the RSD value. The intermediate precision test of adzuki bean formula granules showed the results of uridine content determination as shown in Table 9 below:

[0124] Table 9

[0125]

[0126] The intermediate precision test measured the average uridine content in adzuki bean granules to be 0.72 mg / g, with an RSD value of 1.9%, which was consistent with the RSD of 1.6% in the repeatability test, meeting the precision requirements of methodological verification.

[0127] 3.2.3 Solution stability study

[0128] Take adzuki bean formula granules (batch number: 19041942), and measure them according to the method provided in Example 1 at 0h, 2h, 4h, 6h, 8h, 12h, 24h, and 36h, respectively, calculate the content of uridine, and calculate RSD. The results of uridine content determination in the stability study of adzuki bean formula granules are shown in Table 10 below:

[0129] Table 10

[0130]

[0131] From the above data, we can see that the RSD value of the change in uridine peak area is 0.7%, which meets the requirements of system analysis and is relatively stable within 36 hours.

[0132] 3.3 Durability

[0133] 3.3.1 Investigation of different column temperatures

[0134] To investigate the effect of slight changes in column temperature on the detection method, the content of the adzuki bean formula granule test solution was determined at 28°C, 30°C, and 32°C according to the method provided in Example 1, and the changes in the uridine peak area were recorded. The uridine content was calculated, and the RSD was calculated. The results are shown in Table 11 below.

[0135] Table 11

[0136]

[0137] From the above data, it can be seen that the RSD value of the change in uridine peak area is 2.6%, indicating that this method has good durability for column temperatures between 28 and 32°C.

[0138] 3.4 Linearity

[0139] Accurately weigh 9.270 mg of uridine (purity: 97.7%) reference substance in a 200 mL volumetric flask, add 70% ethanol to dissolve and dilute to the mark to prepare reference substance solution ⑤, accurately measure 1.0 mL, 25.0 mL, 50.0 mL, and 2.0 mL therefrom and place them in 20 mL, 250 mL, 250 mL, and 5 mL volumetric flasks, respectively, add 70% ethanol to dilute to the mark, and use them as linear reference substance solutions ①, ②, ③, and ④, respectively. Take the reference substance solutions ① to ⑤ of the above-mentioned concentrations, filter them, accurately draw 10 μL of the subsequent filtrate, inject it into a liquid chromatograph, and determine the peak area of ​​the uridine chromatographic peak according to the method provided in Example 1. Use the peak area of ​​the chromatographic peak as the ordinate and the concentration of the reference substance as the abscissa to observe whether it is linear. Perform linear regression using the least squares method to obtain the linear regression equation for uridine: Y=593.09X+0.0904, R 2 =0.9999, and its linear range is 4.8433-121.0816 μg / mL. The linear results of uridine are shown in Table 12 below:

[0140] Table 12

[0141] serial number ① ② ③ ④ ⑤ Concentration (μg / mL) 4.8433 12.1082 24.2163 60.5408 121.0816 Peak area 2.908 7.1600 14.426 36.346 71.746

[0142] Uridine standard curve Figure 7 As shown, the results showed that uridine had a good linear relationship.

[0143] 3.5 Verification of three batches (19041942, 18026252, 20030891) of adzuki bean formula granules

[0144] Determination method: According to the method provided in Example 1, 10 μL of the reference solution and the test solution of the red bean formula granules were accurately drawn and injected into the liquid chromatograph for determination. The determination results of the content of the red bean formula granules of 3 batches (batch numbers are 19041942, 18026252 and 20030891) are shown in Table 13 below:

[0145] Table 13

[0146]

[0147] 3.6 Verification of three batches (202104-03-1, 202104-03-8, 202104-03-13) of red bean medicinal materials

[0148] Determination method: According to the method provided in Example 1, 10 μL of the reference solution and the red bean medicinal material test solution were accurately drawn, injected into the liquid chromatograph, and determined. The results of the determination of the red bean medicinal material content of 3 batches (batch numbers are 202104-03-1, 202104-03-8 and 202104-03-13) are shown in Table 14 below:

[0149] Table 14

[0150]

[0151] It can be seen from the data in Tables 13 and 14 that the quality control method provided by the present invention is not only applicable to adzuki bean formula granules, but also applicable to adzuki bean medicinal materials.

[0152] Example 4

[0153] Extraction solvent investigation

[0154] The present embodiment provides a quality control method for adzuki bean formula granules, which is different from the embodiment 1 only in that water, 10% methanol aqueous solution, 30% methanol aqueous solution, 10% ethanol aqueous solution, and 30% ethanol aqueous solution are respectively used as extraction solvents in the preparation process of the test sample, and the other steps are the same as the embodiment 1.

[0155] The results of extracting different solvents to investigate the uridine content are shown in Table 15 below:

[0156] Table 15

[0157]

[0158] Figure 8 The following is the HPLC chromatogram of the red bean formula granules obtained by different extraction solvents. The results of the investigation of different extraction solvents show that the content of the detected components is relatively high when water and 10% ethanol are used as samples. Since the samples with water as the extraction solvent have flocculent precipitation after 6 hours, such as abnormal data of water sample-2, the sample is unstable, so 10% ethanol is used as the extraction solvent for the determination of the content of red bean granules and the characteristic spectrum.

[0159] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A quality control method for adzuki bean formula granules and adzuki bean medicinal materials, characterized in that: The quality control method comprises the following steps: (1) extracting adzuki bean formula granules or adzuki bean medicinal materials to prepare a test solution; mixing uridine and water to prepare a reference solution; (2) using high performance liquid chromatography to detect the test solution and the reference solution obtained in step (1) respectively, to obtain the content of uridine in the red bean formula granules or red bean medicinal materials and the characteristic spectrum of the red bean formula granules or red bean medicinal materials; The filler used in the chromatographic column in the high performance liquid chromatography detection includes pure water-resistant octadecylsilane bonded silica gel; the column temperature in the high performance liquid chromatography detection is 28-32°C; The detection wavelength in the HPLC detection is 254 nm; the injection volume in the HPLC detection is 8-12 μL; In the HPLC detection, methanol is used as mobile phase A, and 0.1%-0.3% acetic acid aqueous solution is used as mobile phase B; in the HPLC detection, gradient elution is performed according to the following table; In the high performance liquid chromatography detection, the flow rate of the mobile phase is 0.5-0.7 mL / min from 0 to 35 min; the flow rate of the mobile phase is (0.5-0.7)→(0.8-1.0) mL / min from 35 to 44 min; and the flow rate of the mobile phase is 0.8-1.0 mL / min from 44 to 56 min.

2. The quality control method according to claim 1, characterized in that: In step (1), the preparation of the test solution specifically includes the following steps: mixing the adzuki bean formula granules or adzuki bean medicinal materials with an ethanol aqueous solution, ultrasonicating, and filtering to obtain a test solution.

3. The quality control method according to claim 2, characterized in that: The volume fraction of ethanol in the ethanol aqueous solution is 5-15%.

4. The quality control method according to claim 2, characterized in that: The solid-to-liquid ratio of the adzuki bean formula particles and the ethanol aqueous solution is (0.5-1.5):15g / mL.

5. The quality control method according to claim 2, characterized in that: The solid-to-liquid ratio of the adzuki bean medicinal material and the ethanol aqueous solution is (1.0-2.0): 15g / mL.

6. The quality control method according to claim 2, characterized in that: The power of the ultrasound is 200-300W, the frequency of the ultrasound is 30-50kHz, and the time of the ultrasound is 20-50min.

7. The quality control method according to claim 1, characterized in that: In step (1), the solid-to-liquid ratio of uridine to water is (20-40):1 μg / mL.

8. The quality control method according to claim 1, characterized in that: The adzuki bean formula granules or adzuki bean medicinal materials have a total of 4 characteristic peaks, which are peak 1, peak 2, peak 3 and peak 4 in order from short to long in terms of peak elution time, with peak 2 being the S peak, and the relative retention times of peaks 1 to 4 being within ±10% of the specified values, and the specified values ​​are respectively: 0.27, 1, 2.01 and 2.21; wherein peak 2 is uridine.

Images