A quality control method for rutin medicine and rutin formula granules

Abstract

The present invention provides a quality control method for a Ruren medicinal material and a Ruren formula granule, the quality control method comprising the following steps: extracting the Ruren medicinal material or the Ruren formula granule to prepare a test solution; mixing protocatechuic acid and an ethanol aqueous solution to prepare a reference solution; respectively detecting the test solution and the reference solution by high performance liquid chromatography to obtain the content of protocatechuic acid in the Ruren medicinal material or the Ruren formula granule and the characteristic spectrum of the Ruren medicinal material or the Ruren formula granule. The present invention constructs an HPLC characteristic spectrum of the Ruren medicinal material and the Ruren formula granule, fully demonstrating the chemical composition characteristics of the Ruren medicinal material and the Ruren formula granule; at the same time, the present invention establishes an HPLC content determination method for protocatechuic acid in the Ruren medicinal material and the Ruren formula granule, which is simple to operate, high in accuracy and good in stability.

Description

Technical Field

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

[0002] Prinsepia uniflora Batal is the dried mature kernel of Prinsepia uniflora Batal or Prinsepia uniflora Batal.var.serrata Rehd, a plant of the Rosaceae family. It tastes sweet and is slightly cold in nature. It enters the liver meridian. It mainly contains a variety of chemical substances such as unsaturated fatty acids, which have the effects of dispelling wind and heat, nourishing the liver and improving eyesight, and calming the mind. It is mainly used to treat symptoms such as red and swollen eyes, canthus ulcers and excessive tears, dark photophobia, and restless sleep. Pharmacological studies have shown that it has antihypertensive and calming effects.

[0003] At present, the legally recorded quality standards only include identification of properties, thin layer identification and water content determination. There is almost no report on the fingerprint identification of Rhizoma Rugosae.

[0004] Linoleic acid in Rhizoma Rugosae can soften human cardiovascular and cerebrovascular system, accelerate blood circulation, lower blood pressure and blood lipid, promote metabolism, and protect cardiovascular and cerebrovascular system. Liu Huiling, Ma Guoxu, Sun Zhonghao, Yang Junshan, Xu Xudong, Determination of linoleic acid content in Rhizoma Rugosae medicinal materials from different origins by reversed phase high performance liquid chromatography-evaporative light scattering detection, Chinese Journal of Pharmaceutical Sciences, 2014, 49(15) discloses a method for determining the content of linoleic acid in Rhizoma Rugosae medicinal materials from different origins, chromatographic column: AichromaBond-AQC8 chromatographic column (4.6mm×250mm, 5um); mobile phase: acetonitrile-0.1% formic acid water (90:10); flow rate: 1.0mL / min; column temperature: 25℃; detector conditions: nebulization temperature 21.0℃, drift tube temperature 40.0℃, carrier gas pressure 145Pa, gain value set to 20.

[0005] The existing technology for studying the components of Rhizoma Rugosae mainly focuses on the detection of linoleic acid in fatty acids, while ignoring the content of other active ingredients, and the method is energy-intensive. There is no relevant technology for identifying the characteristic spectrum of Rhizoma Rugosae medicinal materials and formula particles.

[0006] Therefore, how to provide a quality control method for Rhizoma Rugosae medicinal materials and formula granules, which can not only provide characteristic spectra but also determine the content of other effective ingredients except linoleic acid, has become a problem that needs to be solved urgently. Summary of the invention

[0007] In view of the deficiencies of the prior art, the object of the present invention is to provide a quality control method for Ruren medicinal materials and Ruren formula granules. The present invention provides a method for determining the content of protocatechuic acid in Ruren medicinal materials and Ruren formula granules, and a method for determining the characteristic spectrum of Ruren medicinal materials or Ruren formula granules. The determination method provided by the present method is simple to operate, has high accuracy, good stability, strengthens specific identification and overall quality control, establishes the characteristic spectrum of Ruren medicinal materials and Ruren formula granules, and thus performs comprehensive quality control.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] The present invention provides a quality control method for a Ruo Ren medicinal material and a Ruo Ren formula granule, the quality control method comprising the following steps:

[0010] (1) extracting the medicinal material of Ruo Ren or the Ruo Ren formula granules to prepare a test solution; mixing protocatechuic acid and ethanol aqueous solution to prepare a reference solution;

[0011] (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 protocatechuic acid in the Ruoren medicinal material or the Ruoren formula granules and the characteristic spectrum of the Ruoren medicinal material or the Ruoren formula granules.

[0012] The present invention selects protocatechuic acid from another type of effective organic acid in the radix rutaecarpa in addition to linoleic acid as the test index. Unlike linoleic acid, protocatechuic acid can protect the cardiovascular system by reducing coronary blood vessel and myocardial oxygen consumption, improving myocardial hypoxia tolerance, and increasing heart and peripheral blood vessel pressure.

[0013] In order to more comprehensively evaluate the quality of Rhizoma Ruoshen, the present invention uses HPLC to establish a characteristic spectrum for Rhizoma Ruoshen medicinal materials, uses 3 batches of Rhizoma Ruoshen medicinal materials to establish a reference spectrum, determines the common peaks, and uses the protocatechuic acid reference substance for comparison, identifies the characteristic peaks, and performs methodological verification. The experimental results show that the method provided by the present invention has good repeatability, good durability, and provides more abundant information, which can provide a more comprehensive and effective quality control method for standardizing the quality of Rhizoma Ruoshen medicinal materials. The method provided by the present method was used to verify 3 batches of Rhizoma Ruoshen formula granules, and the results showed that the method was also applicable, providing an effective quality control method for Rhizoma Ruoshen formula granules. In addition, this study used HPLC to establish a protocatechuic acid content determination method for Rhizoma Ruoshen, using the protocatechuic acid reference substance as the content measurement index, and performed methodological verification. The experimental results show that the method has good repeatability.

[0014] In the present invention, in step (1), the preparation of the test solution specifically comprises the following steps:

[0015] (a) mixing the medicinal material of Ruo Ren or the Ruo Ren formula granules with petroleum ether, ultrasonicating, filtering, and collecting the filter residue;

[0016] (b) mixing the filter residue obtained in step (a) with an ethanol aqueous solution, and refluxing to obtain an extract;

[0017] (c) The extract obtained in step (b) is evaporated to dryness, and then dissolved in methanol to obtain a test solution.

[0018] In the present invention, in step (a), the boiling point of the petroleum ether is 60-90°C (for example, it may be 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, etc.).

[0019] Preferably, in step (a), the solid-to-liquid ratio of the Radix Rhizoma Rhizoma and petroleum ether is (1.0-2.5):50 g / mL, wherein "1.0-2.5" can be 1.0, 1.3, 1.5, 1.7, 1.9, 2, 2.1, 2.3, 2.5, etc.

[0020] Preferably, in step (a), the solid-to-liquid ratio of the Rhizoma Rhizoma formula particles and petroleum ether is (0.05-0.15): 50 g / mL, wherein "0.05-0.15" can be 0.05, 0.07, 0.09, 0.1, 0.13, 0.15, etc.

[0021] Preferably, in step (a), the power of the ultrasound is 200-400 W (for example, it can be 200 W, 230 W, 260 W, 290 W, 300 W, 330 W, 360 W, 390 W, 400 W, etc.), the frequency of the ultrasound is 30-50 kHz (for example, it can be 30 kHz, 33 kHz, 36 kHz, 39 kHz, 40 kHz, 43 kHz, 46 kHz, 49 kHz, 50 kHz, etc.), and the time of the ultrasound is 30-50 min (for example, it can be 30 min, 33 min, 36 min, 39 min, 43 min, 46 min, 49 min, 50 min, etc.).

[0022] In the present invention, in step (b), the volume fraction of ethanol in the ethanol aqueous solution is 30-80% (for example, it can be 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, etc.).

[0023] Preferably, in step (b), the reflux time is 30-70 min (for example, it can be 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, etc.).

[0024] In the present invention, in step (1), the volume fraction of ethanol in the ethanol aqueous solution is 60-80% (for example, it can be 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, etc.).

[0025] Preferably, in step (1), the solid-to-liquid ratio of protocatechuic acid and ethanol aqueous solution is (80-100):1 μg / mL, wherein "80-100" can be 80, 83, 86, 89, 90, 93, 96, 99, 100, etc.

[0026] In the present invention, in step (2), the filler used in the chromatographic column in the high performance liquid chromatography detection includes octadecylsilane bonded silica gel.

[0027] In the present invention, in step (2), the chromatographic column in the HPLC detection has a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0028] 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.).

[0029] In the present invention, in step (2), the flow rate of the mobile phase in the HPLC detection is 0.9-1.1 mL / min (for example, it can be 0.9 mL / min, 0.93 mL / min, 0.96 mL / min, 0.99 mL / min, 1.03 mL / min, 1.06 mL / min, 1.09 mL / min, 1.1 mL / min, etc.).

[0030] Preferably, in step (2), the detection wavelength in the HPLC detection is 254 nm.

[0031] Preferably, in step (2), the injection volume in the HPLC detection is 8-15 μL (for example, it can be 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, etc.).

[0032] In the present invention, in the HPLC detection, methanol is used as mobile phase A, 0.1%-0.3% (for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, etc.) formic acid aqueous solution is used as mobile phase B, and gradient elution is performed according to the following table:

[0033] Time (min) Mobile phase A Mobile phase B 0-10 15→22 85→78 10-15 22→30 78→70 15-30 30→40 70→60 .

[0034] In the present invention, gradient elution is performed according to the following table:

[0035] Time (min) Mobile phase A Mobile phase B 0-10 15→22 85→78 10-15 22→30 78→70 15-30 30→40 70→60 30-45 40→65 60→35 45-60 65 35 .

[0036] In the present invention, the Rushiren medicinal material or Rushiren formula granules have a total of 6 characteristic peaks, which are peak 1, peak 2, peak 3, peak 4, peak 5 and peak 6 in order from short to long in terms of peak elution time, with peak 1 being the S peak, and the relative retention times of peaks 2-6 being within ±10% of the specified values, and the specified values ​​are: 1.62, 2.44, 2.50, 2.82 and 2.95, respectively; wherein peak 1 is protocatechuic acid.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The present invention constructs the HPLC characteristic spectrum of the Ruo Ren medicinal material and the Ruo Ren formula granules, which fully demonstrates the chemical composition characteristics of the Ruo Ren medicinal material and the Ruo Ren formula granules;

[0039] (2) The characteristic spectrum constructed by the present invention can fully reflect the characteristic peak information of the sample, and the method is stable, with high precision and good reproducibility;

[0040] (3) The HPLC characteristic spectrum of the Ruo Ren medicinal material and the Ruo Ren formula granules established in the present invention can be used to identify the Ruo Ren medicinal material and the Ruo Ren formula granules, providing a rapid and reliable detection method for the identification of the Ruo Ren medicinal material and the Ruo Ren formula granules.

[0041] (4) The present invention establishes a HPLC method for determining the content of protocatechuic acid in Ruo Ren medicinal materials and Ruo Ren formula granules. The method is simple to operate, highly accurate, and stable, and provides a rapid and reliable detection method for Ruo Ren medicinal materials and Ruo Ren formula granules. It can improve the relevant quality standards of Ruo Ren medicinal materials and formula granules to a certain extent, and provide scientific guidance for better development and utilization of Ruo Ren Chinese medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the content determination and characteristic spectrum of the Radix Ruoshen obtained in Example 1; wherein, peak 1 is protocatechuic acid.

[0043] Figure 2 The content determination and characteristic spectrum of the protocatechuic acid reference substance, Ruoren medicinal material 201001 and Ruoren formula granule KL201001 in Example 1;

[0044] Among them, S1 is the protocatechuic acid reference substance, S2 is the Ruoren medicinal material 201001, and S3 is the Ruoren formula granules KL201001.

[0045] Figure 3 The HPLC characteristic spectra of three batches of Radix Ruoshene;

[0046] Among them, S1 is 201001, S2 is 201006, S3 is 201012, and S4 is the reference characteristic spectrum of the medicinal material Rhizoma Rugosae.

[0047] Figure 4 For the chromatographic column 1 Waters Chromatogram obtained by HSS T3 (4.6*250mm, 5μm); wherein, peak 1 is protocatechuic acid.

[0048] Figure 5 This is a chromatogram obtained using chromatographic column 2 ZORBAX SB-C18 (4.6*250mm, 5μm); wherein peak 1 is protocatechuic acid.

[0049] Figure 6 For the chromatographic column 3 Waters Chromatogram obtained by C18 (4.6*250mm, 5μm); wherein, peak 1 is protocatechuic acid.

[0050] Figure 7 The HPLC characteristic spectra of three batches of Ruo Ren formula granules;

[0051] Among them, S1 is KL201001, S2 is KL201006, S3 is KL201012, and S4 is the control characteristic spectrum of the Ruo Ren formula granules.

[0052] Figure 8 This is the standard curve of protocatechuic acid.

[0053] Fig. 9 This is the characteristic spectrum of the medicinal material Ruiren obtained in Experiment A.

[0054] Fig.10 This is the characteristic spectrum of the medicinal material Ruiren obtained in Experiment B.

[0055] Fig.11 This is the characteristic spectrum of the medicinal material Ruiren obtained in Experiment C. DETAILED DESCRIPTION

[0056] 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.

[0057] The sources of instruments, reagents and reagents in the following examples are as follows:

[0058] Instruments: Waters ACQUITY ArC high performance liquid chromatograph; TUV Detector detector; Empower 3 chromatography workstation; 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.);

[0059] Chromatographic column: Agilent ZORBAX SB-C18 (4.6×250mm, 5μm) column;

[0060] Protocatechuic acid reference substance: (batch number: 110809-201906, purity 97.7%, manufacturer: China Food and Drug Inspection Institute);

[0061] Rhizoma Rugosae formula granules: KL201001, KL201006, KL201012;

[0062] Rhizoma Rugosae: 201001, 201006, 201012;

[0063] Reagents: methanol (chromatographic grade, batch number: I1 106007 032, manufacturer: Merck KGaA (Merck Group, Germany));

[0064] Acetonitrile (chromatographic grade, batch number: JB100130, manufacturer: Merck KGaA (Merck Group, Germany));

[0065] Ethanol (analytical grade, batch number: 20210114, manufacturer: Tianjin Yongda Chemical Reagent Co., Ltd.);

[0066] Water (distilled water, batch number: 20210113H, manufacturer: Guangzhou Watsons Food and Beverage Co., Ltd.);

[0067] Formic acid (chromatographic grade, batch number: 186899, manufacturer: Thermo Fisher Scientific (China) Co., Ltd.);

[0068] Petroleum ether 60-90°C (analytical grade, batch number: 20190929, manufacturer: Sinopharm Chemical Reagent Co., Ltd.).

[0069] The retention times in the following examples are all in minutes (min).

[0070] Example 1

[0071] This embodiment provides a quality control method for the medicinal material of Ruo Ren and the Ruo Ren formula granules, and the quality control method comprises the following steps:

[0072] Preparation of reference solution: Take an appropriate amount of protocatechuic acid reference substance, weigh accurately, and add 70% ethanol aqueous solution to prepare a reference solution containing 90 μg protocatechuic acid reference substance per 1 mL;

[0073] Preparation of the test solution of the medicinal material Ruo Ren: take 2.0 g of the medicinal material Ruo Ren powder, accurately weigh it, put it in a stoppered conical flask, add 50 mL of petroleum ether (boiling point 60-90°C), stopper it, ultrasonically treat it (power 300 W, frequency 40 kHz) for 40 minutes, filter it, and collect the residue; add 50 mL of 70% ethanol aqueous solution to the residue, weigh the weight, reflux it for 40 minutes, cool it, weigh it again, make up the lost weight with 70% ethanol aqueous solution, shake it well, filter it, and collect the filtrate; take 25 mL of the filtrate, put it in an evaporating dish, evaporate it to dryness, dissolve it with methanol, make up to 5 mL, shake it well, filter it, and take the filtrate to obtain the test solution of the medicinal material Ruo Ren.

[0074] Preparation of the test solution of the formula granules of Ruo Ren: The only difference from the test solution of the medicinal material of Ruo Ren is that 2.0 g of the medicinal material powder of Ruo Ren is replaced by 0.1 g of the formula granules powder of Ruo Ren.

[0075] High performance liquid chromatography was used to test the reference solution, the test solution of the herbal medicine Ruoren and the test solution of the formula granules of Ruoren.

[0076] Chromatographic conditions: Agilent ZORBAX SB-C18, octadecylsilane bonded silica as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A, 0.2% formic acid as mobile phase B; detection wavelength 254 nm; column temperature 30°C; flow rate 1.0 mL min, injection volume 10 μL; theoretical plate number calculated based on protocatechuic acid is not less than 1000.

[0077] Perform gradient elution according to the following table:

[0078] Time (min) Mobile phase A Mobile phase B 0-10 15→22 85→78 10-15 22→30 78→70 15-30 30→40 70→60 30-45 40→65 60→35 45-60 65 35

[0079] The batch number of the Ruo Ren medicinal material is 201001, and the batch number of the Ruo Ren formula granules is KL201001.

[0080] The characteristic spectrum of the medicinal material Ruiren is as follows Figure 1As shown, the sample presents 6 characteristic peaks, among which peak 1 has the same retention time as the protocatechuic acid reference substance. Peak 1 is taken as the S peak, and the relative retention times of peaks 2-6 are calculated; their relative retention times should be within ±10% of the specified values. The specified values ​​are: 1.62 (peak 2), 2.44 (peak 3), 2.50 (peak 4), 2.82 (peak 5), and 2.95 (peak 6).

[0081] Figure 2 These are the characteristic spectra of protocatechuic acid reference substance, Ruoren medicinal material 201001 and Ruoren formula granules KL201001. From the figure, it can be determined that peak 1 is protocatechuic acid.

[0082] The content of protocatechuic acid in Ruoren medicinal material and Ruoren formula granules was determined by external standard method.

[0083] Experimental example

[0084] (I) Study on the characteristic spectrum of Ruo Ren medicinal materials and Ruo Ren formula granules

[0085] Experimental Example 1 Determination of characteristic spectra of different batches of Radix Rutaceae

[0086] Test method: Take different batches of Radix Ruoshen medicinal material samples (batch numbers are 201001, 201006 and 201012 respectively), perform high performance liquid chromatography determination according to the method provided in Example 1, obtain their liquid phase spectrum, import the spectrum into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for data matching, and establish a control characteristic spectrum of Radix Ruoshen medicinal material.

[0087] The retention time and relative retention time of the three batches of Ruo Ren medicinal materials are shown in Table 1. The HPLC characteristic spectra of the three batches of Ruo Ren medicinal materials are shown in Table 1. Figure 3 (S1 is 201001, S2 is 201006, S3 is 201012, and S4 is the reference characteristic spectrum of the medicinal material Ruren). The reference characteristic spectrum of the medicinal material Ruren is the reference characteristic spectrum of the medicinal material Ruren obtained by matching the three batches of Ruren medicinal material sample spectra measured in Example 1 with the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System".

[0088] Table 1

[0089]

[0090] The results showed that 6 characteristic peaks were present in the HPLC characteristic spectrum of 3 batches of Radix Ruoshen, among which peak 1 had the same retention time as the protocatechuic acid reference substance, and peak 1 was taken as the S peak. The relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time was within ±10% of the specified value. The specified values ​​were: 1.62 (peak 2), 2.44 (peak 3), 2.50 (peak 4), 2.82 (peak 5), and 2.95 (peak 6).

[0091] This method can quickly and intuitively identify the medicinal material of Rhizoma Rugosae, and provides a more scientific basis for the quality control of the medicinal material of Rhizoma Rugosae.

[0092] Experimental Example 2 Methodology Verification

[0093] 2.1 Precision experiment

[0094] 2.1.1 Repeatability

[0095] Test method: Take 6 portions of the medicinal material of Ruo Ren (batch number: 201001), perform high performance liquid chromatography determination according to the method provided in Example 1, obtain its characteristic spectrum, take peak 1 as the reference peak, calculate its relative peak area and relative retention time, and calculate RSD. The test results of the retention time and relative retention time of the medicinal material of Ruo Ren are shown in Table 2.

[0096] Table 2

[0097]

[0098] According to the repeatability test results (Table 2), the relative retention time RSD of each characteristic peak is 0.0% to 0.0%, indicating that the repeatability of the characteristic spectrum of this method is good.

[0099] 2.1.2 Intermediate precision

[0100] Waters ACQUITY ArC, TUV detector, take 6 portions of Radix Rugosae (batch number: 201001), and perform high performance liquid chromatography according to the method provided in Example 1 to obtain its characteristic spectrum, and calculate its relative peak area and relative retention time with peak 1 as the reference peak, and calculate RSD. The test results of retention time and relative retention time of Radix Rugosae intermediate precision are shown in Table 3 below.

[0101] Table 3

[0102]

[0103]

[0104] According to the results of the intermediate precision investigation (Table 3), the relative retention time RSD of each characteristic peak is within the range of 0.0% to 0.0%. The relative retention time RSD between different instruments is within the range of 0.0% to 0.0%. The experimental results show that the characteristic spectrum method meets the requirements of intermediate precision analysis, and it is recommended to use relative retention time as a standard.

[0105] 2.2 Solution stability study

[0106] Test method: Take the medicinal material of Ruo Ren (batch number: 201001), and perform HPLC determination according to the method provided in Example 1 at 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h, respectively, to obtain its characteristic spectrum, take Peak 1 as the reference peak, calculate its relative retention time, and calculate RSD. The retention time and relative retention time of the stability study of Ruo Ren are shown in Table 4 below.

[0107] Table 4

[0108]

[0109]

[0110] The results of the stability experiment show (Table 4) that the chemical components in the test solution are stable for 24 hours, and the relative retention time RSD of each characteristic peak is 0.0%, indicating that the analytical method is stable and reliable.

[0111] 2.3 Durability test

[0112] 2.3.1 Investigation of different column temperatures

[0113] Test method: To investigate the influence of slight changes in column temperature on the detection method, characteristic spectrum detection was performed on the test solution of Rhizoma Rugosae medicinal materials at 28°C, 30°C and 32°C according to the method provided in Example 1. The test results of the retention time and relative retention time of each peak at different column temperatures are shown in Table 5 below.

[0114] Table 5

[0115]

[0116] The data in Table 5 show that under different column temperature conditions, the relative retention time is within ±10% of the specified value, and the RSD value of the relative retention time is in the range of 0.0 to 2.1%, indicating that the method provided by the present invention has good durability to column temperature.

[0117] 2.3.2 Investigation of different flow rates

[0118] Test method: To investigate the influence of slight changes in flow rate on the detection method, flow rates of 0.9 mL / min, 1.0 mL / min and 1.1 mL / min were used respectively. The characteristic spectrum of the test solution of the medicinal material Rhizoma Rhizoma Rhizoma was detected according to the method provided in Example 1. The test results of the peak retention time and relative retention time at different flow rates are shown in Table 6 below.

[0119] Table 6

[0120]

[0121] It can be seen from the data in Table 6 that under different flow rate conditions, the relative retention time is within ±10% of the specified value, and the RSD value of the relative retention time is in the range of 1.2% to 5.1%, indicating that the method provided by the present invention has good durability to the flow rate.

[0122] 2.3.3 Investigation of different chromatographic columns

[0123] Test method: The characteristic spectrum of the test solution of the medicinal material Rhizoma Rugosae (batch number: 201001) was detected according to the method provided in Example 1. The only difference from Example 1 is that the chromatographic column is different.

[0124] Different chromatographic columns are used respectively, and the specific chromatographic column models are as follows:

[0125] Column 1: Waters C18 (column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm, SN: 03193906413818;

[0126] Chromatographic column 2: ZORBAX SB-C18 (column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm, SN: USCL099141);

[0127] Chromatographic column 3: Waters Xselect HSS T3 (column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm, SN: 01443905012921).

[0128] Taking peak 1 protocatechuic acid (S) as the reference peak, the retention time and relative retention time of peaks 2, 3, 4, 5 and 6 were calculated, and the RSD values ​​were calculated. The results are shown in Table 7.

[0129] Table 7

[0130]

[0131]

[0132] Chromatographic column 1 Waters The chromatogram obtained by HSS T3 (4.6*250mm, 5μm) is as follows Figure 4 The chromatogram obtained using chromatographic column 2 ZORBAX SB-C18 (4.6*250mm, 5μm) is shown in Figure 5 As shown. The chromatographic column used is Waters The chromatogram obtained by C18 (4.6*250mm, 5μm) is as follows Figure 6 shown.

[0133] The results of using different chromatographic columns showed that the RSD values ​​of the relative retention times of the characteristic peaks were

[0134] The range of 1.9% to 5.8% is within ±10% of the specified value, indicating that the analysis method is suitable for chromatographic columns of different models with octadecylsilane bonded silica gel as filler.

[0135] 2.4 Validation of three batches (KL201001, KL201006, KL201012) of rutin formula granules

[0136] Test method: According to the method provided in Example 1, the characteristic spectrum of the test solution of the Ruo Ren formula granules was detected, and the spectrum was imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for data matching to establish a control characteristic spectrum of the Ruo Ren formula granules.

[0137] The results of verification of retention time and relative retention time of three batches of Ruo Ren formula granules (KL201001, KL201006, KL201012) are shown in Table 8 below. The HPLC characteristic spectra of the three batches of Ruo Ren formula granules are shown in Table 8 below. Figure 7 As shown (S1 is KL201001, S2 is KL201006, S3 is KL201012, and S4 is the control characteristic spectrum of the Ruiren Formula Granule). The control characteristic spectrum of the Ruiren Formula Granule is the control characteristic spectrum of the Ruiren Formula Granule obtained by matching the sample spectra of the three batches of the Ruiren Formula Granule measured in Example 1 with the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System".

[0138] Table 8

[0139]

[0140] From Table 8 and Figure 7 The data show that the verification results of three batches of granules indicate that the method provided by the present invention is not only applicable to the medicinal material of Rhizoma Rugosae, but can also achieve quality control of the Rhizoma Rugosae formula granules.

[0141] (II) Determination of protocatechuic acid content in Rhizoma Rugosae and Rhizoma Rugosae granules

[0142] Experimental Example 3 Determination of protocatechuic acid content in different batches of Rhizoma Rugosae medicinal materials

[0143] Test method: Take different batches of Ruo Ren medicinal material samples (batch numbers are 201001, 201006 and 201012 respectively), perform high performance liquid chromatography determination according to the method provided in Example 1, and use the external standard method to determine the protocatechuic acid content in Ruo Ren medicinal materials. The results of the protocatechuic acid content determination of 3 batches of Ruo Ren medicinal materials are shown in Table 9 below:

[0144] Table 9

[0145]

[0146] From the data in Table 9, it can be seen that the parallel samples of the HPLC content determination results of the three batches of Rhizoma Rugosae medicinal materials are stable and the method is reliable. The method provided by the present invention can quickly determine the content of protocatechuic acid in Rhizoma Rugosae medicinal materials, providing a more scientific basis for the quality control of Rhizoma Rugosae medicinal materials.

[0147] Experimental Example 4 Methodological Verification

[0148] 4.1 Accuracy

[0149] Test method: 9.270 mg of protocatechuic acid (purity: 97.7%) was accurately weighed in a 200 mL volumetric flask, and 70% ethanol aqueous solution was added to dissolve it and dilute to the mark to prepare a mixed reference substance mother solution. 25.0 mL, 50.0 mL, and 60 mL were accurately measured in 250 mL, 250 mL, and 200 mL volumetric flasks, respectively, and 70% ethanol aqueous solution was added to dilute to the mark as reference substance solutions ①, ②, and ③. 9 portions of the known content of the Rhizoma Rhizoma Rhizoma (201001, protocatechuic acid content of 0.050 mg / g) were taken under the repeatability item, each portion was about 2.0 g, accurately weighed, and 50 mL of the reference substance solution containing 0.2264 mg, 0.4528 mg, and 0.6793 mg of protocatechuic acid was accurately added, respectively. The HPLC determination was carried out according to the method provided in Example 1, and the recovery rate was calculated according to the following formula. The test results of the protocatechuic acid recovery rate are shown in Table 10 below.

[0150]

[0151] Table 10

[0152]

[0153] The recovery rate test (Table 10) showed that the recovery rate of protocatechuic acid ranged from 98.50% to 108.80%, and the average recovery rate was 102.5%, which met the recovery rate requirements of the methodology verification, indicating that the results measured by the method provided by the present invention are accurate and reliable.

[0154] 4.2 Precision experiment

[0155] 4.2.1 Repeatability

[0156] Test method: Take 6 portions of Rushien medicinal material (batch number: 201001), and perform determination according to the method provided in Example 1, calculate the content of protocatechuic acid, and calculate RSD. Repeatability study of Rushien medicinal material The results of the determination of protocatechuic acid content are shown in Table 11 below.

[0157] Table 11

[0158]

[0159] From the data in Table 11, it can be seen that the average content of protocatechuic acid in Rhizoma Rugosae was 0.046 mg / g and the RSD value was 5.8%, which was less than 6%, meeting the repeatability requirements of the methodology validation.

[0160] 4.2.2 Intermediate precision

[0161] Different analysts used another Waters ACQUITY ArC, TUV detector at different times to take 6 portions of Radix Rutaceae (batch number: 201001), and measured according to the method provided in Example 1 to calculate the content of protocatechuic acid and RSD. The results are shown in Table 12.

[0162] Table 12

[0163]

[0164] The intermediate precision test (Table 12) measured the average protocatechuic acid content in Rhizoma Rugosae as 0.052 mg / g, with an RSD value of 1.5%, which was 8.7% compared with the RSD of the repeatability test result, which was less than 11%, meeting the requirements for precision of methodological verification.

[0165] 4.3 Solution stability study

[0166] Test method: Take the medicinal material of Ruo Ren (batch number: 201001), and measure it according to the method provided in Example 1 at 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h, calculate the content of protocatechuic acid, and calculate RSD. The results of the protocatechuic acid content determination of Ruo Ren stability investigation are shown in Table 13 below:

[0167] Table 13

[0168]

[0169] From the data in Table 13, it can be seen that the RSD value of the protocatechuic acid peak area change is 0.7%, which meets the requirements of the system analysis. Therefore, the protocatechuic acid content in the sample is relatively stable within 48 hours.

[0170] 4.4 Durability test

[0171] 4.4.1 Investigation of different column temperatures

[0172] Test method: The influence of slight changes in column temperature on the detection method was investigated. The content of the test solution of the medicinal material of Rhizoma Rhizoma Rhizoma was determined according to the method provided in Example 1 at 28°C, 30°C and 32°C respectively. The change of the peak area of ​​protocatechuic acid was recorded, the content of protocatechuic acid was calculated, and the RSD was calculated. The results of the protocatechuic acid content determination at different column temperatures are shown in Table 14 below:

[0173] Table 14

[0174]

[0175]

[0176] It can be seen from the data in Table 14 that the RSD value of the protocatechuic acid peak area change is 1.3%, indicating that the method provided by the present invention has good durability for column temperatures between 28 and 32°C.

[0177] 4.4.2 Investigation of different flow rates

[0178] Test method: To investigate the influence of slight changes in flow rate on the detection method, flow rates of 0.9 mL / min, 1.0 mL / min and 1.1 mL / min were used respectively. The content of the test solution of the medicinal material of Rhizoma ...

[0179] Table 15

[0180]

[0181] It can be seen from the data in Table 15 that the RSD value of the protocatechuic acid peak area change is 1.3%, indicating that the method provided by the present invention has good durability for flow rates between 0.9 and 1.1 mL / min.

[0182] 4.4.3 Investigation of different chromatographic columns

[0183] Test method: The content of the test solution of the medicinal material of Rhizoma Rugosae (batch number: 201001) was determined according to the method provided in Example 1, the change of the peak area of ​​protocatechuic acid was recorded, the content of protocatechuic acid was calculated, and the RSD was calculated. The only difference from Example 1 is that the chromatographic column is different.

[0184] Different chromatographic columns are used respectively, and the specific chromatographic column models are as follows:

[0185] Column 1: Waters C18 (column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm, SN: 03193906413818;

[0186] Chromatographic column 2: ZORBAX SB-C18 (column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm, SN: USCL099141);

[0187] Chromatographic column 3: Waters Xselect HSS T3 (column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm, SN: 01443905012921).

[0188] The results of protocatechuic acid content determination in Rhizoma Rugosae using different chromatographic columns are shown in Table 16 below:

[0189] Table 16

[0190]

[0191] As shown in Table 16, the test results of different types of chromatographic columns show that the average content of protocatechuic acid is 0.055 mg / g, and the RSD value is 5.9%, indicating that this method is suitable for chromatographic columns with different types of octadecylsilane bonded silica gel as fillers.

[0192] 4.5 Linear

[0193] Test method: Accurately weigh 9.270 mg of protocatechuic acid (purity: 97.7%) reference substance in a 200 mL volumetric flask, add 70% ethanol aqueous solution to dissolve and dilute to scale to make reference substance solution ⑤, accurately measure 1.0 mL, 25.0 mL, 50.0 mL, and 2.0 mL in 20 mL, 250 mL, 250 mL, and 5 mL volumetric flasks, add 70% ethanol aqueous solution to dilute to scale, and use them as linear reference substance solutions ①, ②, ③, and ④, respectively. Take the above-mentioned reference substance solutions ① to ⑤, filter them, accurately take 10 μL of the filtrate, inject it into the liquid chromatograph, and determine the peak area of ​​the protocatechuic acid chromatographic peak according to the method provided in Example 1. The peak area of ​​the chromatographic peak is used as the ordinate and the concentration of the reference substance is used as the abscissa to observe whether it is linear. The linear regression equation of protocatechuic acid is obtained by the least square method: Y = 36,360,706.5344X-1,034.7458, R 2 =1.0000, and its linear range is 2.2642~45.2840μg / mL.

[0194] The linear results of protocatechuic acid are shown in Table 17 below, and the standard curve of protocatechuic acid is as follows Figure 8 shown.

[0195] Table 17

[0196]

[0197] From Table 17 and Figure 8 The data show that the protocatechuic acid content measured by the method provided by the present invention has good linearity.

[0198] 4.6 Validation of three batches (KL201001, KL201006, KL201012) of rutabaga formula granules

[0199] Test method: The content of the test solution of the Ruo Ren formula granules was determined according to the method provided in Example 1. The results of the protocatechuic acid content determination of three batches of Ruo Ren formula granules are shown in Table 18 below:

[0200] Table 18

[0201]

[0202] From the data in Table 18, it can be seen that the verification results of three batches of granules show that this method is not only applicable to medicinal materials, but also can achieve quality control of formula granules.

[0203] (III) Comparative experiment of different elution gradients

[0204] Experiment A

[0205] Experimental method: Using HSS T3 (4.6*250mm, 5μm) chromatographic column, acetonitrile as mobile phase A, water as mobile phase B, elution gradient detection as shown in Table 19, detection wavelength of 254nm; column temperature of 30℃; flow rate of 1.0mL / min.

[0206] The test sample is the test solution of Radix Rhizoma Rhizoma (batch number 201001), and the other test steps are the same as those in Example 1.

[0207] Table 19

[0208] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～35 15→45 85→54 35～36 46→85 54→15 36～40 85 15

[0209] The characteristic spectrum of the medicinal material Ruo Ren obtained in Experiment A is as follows Fig. 9 As shown, the chromatogram obtained by this method has small chromatographic peaks, poor peak shape, and poor separation of some peaks.

[0210] Experiment B

[0211] Experimental methods: HSS T3 (4.6*250mm, 5μm) chromatographic column, with methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, elution gradient detection as shown in Table 20, detection wavelength of 254nm; column temperature of 30℃; flow rate of 1.0mL / min.

[0212] The test sample is the test solution of Radix Rhizoma Rhizoma (batch number 201001), and the other test steps are the same as those in Example 1.

[0213] Table 20

[0214] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 12→35 88→65 5～25 35→60 65→40 25～45 60→65 40→35 45～48 65→75 35→25 48～50 75 25

[0215] The characteristic spectrum of the medicinal material Ruiren obtained in Experiment B is as follows Fig.10 As shown in the figure, the chromatogram obtained by this method has slightly tailing peaks and poor separation.

[0216] Experiment C

[0217] Experimental method: ZORBAX SB-C18 (4.6*250mm, 5μm) chromatographic column, methanol as mobile phase A, 0.2% formic acid as mobile phase B, elution gradient detection according to Table 21, detection wavelength of 254nm; column temperature of 30°C; flow rate of 1.0mL / min.

[0218] The test sample is the test solution of Radix Rhizoma Rhizoma (batch number 201001), and the other test steps are the same as those in Example 1.

[0219] Table 21

[0220] Time (min) Mobile phase (A) Mobile phase (B) 0～70 10→65 90→35 70～80 65 35

[0221] The characteristic spectrum of the medicinal material Ruo Ren obtained in Experiment C is as follows Fig.11 As shown, the separation time is relatively long using this method.

[0222] The characteristic spectrum of the medicinal material of Rhizoma Rugosae provided by the present invention ( Figure 1 ) and Experiment A( Fig. 9 ), Experiment B( Fig.10 ), Experiment C( Fig.11 ) can be seen from the comparison that the chromatogram provided by the present invention has a good peak shape, the peak separation effect is good, and the chromatogram obtained by the method provided by the present invention has good reproducibility.

[0223] 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 the medicinal material of Rhizoma Rugosae and the Rhizoma Rugosae formula granules, characterized in that: The quality control method comprises the following steps: (1) extracting the medicinal material of Ruo Ren or the Ruo Ren formula granules to prepare a test solution; mixing protocatechuic acid and ethanol aqueous solution to prepare a reference solution; The preparation of the test solution specifically comprises the following steps: (a) mixing the medicinal material of Ruo Ren or the Ruo Ren formula granules with petroleum ether, ultrasonicating, filtering, and collecting the filter residue; (b) mixing the filter residue obtained in step (a) with an ethanol aqueous solution, and refluxing to obtain an extract; (c) evaporating the extract obtained in step (b) to dryness, and then dissolving it with methanol to obtain a test solution; The volume fraction of ethanol in the ethanol aqueous solution is 60-80%; (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 protocatechuic acid in the Ruoren medicinal material or the Ruoren formula granules and the characteristic spectrum of the Ruoren medicinal material or the Ruoren formula granules; The filler used in the chromatographic column in the high performance liquid chromatography detection includes octadecylsilane bonded silica gel; In the HPLC detection, methanol was used as mobile phase A, and 0.1%-0.3% formic acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the following table: 。 2. The quality control method according to claim 1, characterized in that: In step (a), the boiling point of the petroleum ether is 60-90°C.

3. The quality control method according to claim 1, characterized in that: In step (a), the solid-to-liquid ratio of the Radix Rhizoma Rhizoma and petroleum ether is (1.0-2.5):50 g / mL.

4. The quality control method according to claim 1, characterized in that: In step (a), the solid-to-liquid ratio of the Rhizoma Rhizoma Rhizoma formula particles and petroleum ether is (0.05-0.15): 50 g / mL.

5. The quality control method according to claim 1, characterized in that: In step (a), the power of the ultrasound is 200-400 W, the frequency of the ultrasound is 30-50 kHz, and the time of the ultrasound is 30-50 min.

6. The quality control method according to claim 1, characterized in that: In step (b), the reflux time is 30-70 min.

7. The quality control method according to claim 1, characterized in that: In step (1), the solid-to-liquid ratio of the protocatechuic acid to the ethanol aqueous solution is (80-100):1 μg / mL.

8. The quality control method according to claim 1, characterized in that: In step (2), the column temperature in the HPLC detection is 28-32°C.

9. The quality control method according to claim 1, characterized in that: In step (2), the flow rate of the mobile phase in the high performance liquid chromatography detection is 0.9-1.1 mL / min.

10. The quality control method according to claim 1, characterized in that: In step (2), the detection wavelength in the high performance liquid chromatography detection is 254 nm.

11. The quality control method according to claim 1, characterized in that: In step (2), the injection volume in the HPLC detection is 8-15 μL.

12. The quality control method according to claim 1, characterized in that: Perform gradient elution according to the following table:

13. The quality control method according to claim 1, characterized in that: The Rushien medicinal material or Rushien formula granules have a total of 6 characteristic peaks, which are peak 1, peak 2, peak 3, peak 4, peak 5 and peak 6 in order from short to long in terms of peak elution time, with peak 1 being the S peak, and the relative retention times of peaks 2-6 being within ±10% of the specified values, and the specified values ​​are: 1.62, 2.44, 2.50, 2.82 and 2.95, respectively; wherein peak 1 is protocatechuic acid.

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