Detection method of active ingredients in Yumai oropharyngeal mixture

Abstract

The present invention belongs to the technical field of quality detection of traditional Chinese medicine, and specifically relates to a method for detecting the content of effective ingredients in Yumai oropharyngeal mixture. The method is: injecting a reference solution and a test solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the contents of gallic acid, corilagin, paeoniflorin and ellagic acid in Yumai oropharyngeal mixture by an external standard method; the chromatographic conditions of the high performance liquid chromatography are: the chromatographic column uses octadecylsilane bonded silica gel as a filler; the mobile phase uses acetonitrile as mobile phase A, and a phosphoric acid aqueous solution with a volume concentration of 0.1% as mobile phase B, and gradient elution; dual wavelength detection: 225-235nm, 245-255nm; the column temperature is: 25°C-35°C; the flow rate is: 0.5-1.5mL / min. The method of the present invention can effectively detect the contents of four components, gallic acid, corilagin, paeoniflorin and ellagic acid, at the same time, and can provide a comprehensive and reliable basis for quality control of Yumai oropharyngeal mixture.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicines, and particularly relates to a method for detecting the content of effective ingredients in Yumai oropharyngeal mixture. Background Art

[0002] Yumai oropharyngeal mixture is a traditional Chinese medicine mixture. The prescription contains multiple Chinese medicinal herbs such as Phyllanthus emblica, Rehmannia glutinosa, Paeonia lactiflora, Ophiopogon japonicus, and Licorice. It has the effect of nourishing yin and reducing fire. It is used to treat burning, pain, local redness and swelling, irritability, dry mouth, yellow and red urine, and recurrent oral ulcers caused by yin deficiency and excessive fire, and virtual fire ascending.

[0003] Red peony root and emblica fruit are the main active ingredients in Yumai oral and pharyngeal mixture, which have the effects of clearing heat and cooling blood, dispersing blood stasis and relieving pain, promoting digestion and strengthening the stomach, and promoting fluid production and relieving cough. Among them, paeoniflorin can relieve fever, anti-inflammatory and anti-ulcer; gallic acid can promote fluid production and quench thirst, stop bleeding and cool blood; corilagin can resist bacteria, kill bacteria and reduce inflammation, and inhibit viruses; ellagic acid can resist oxidation, prevent infection and inhibit ulcers.

[0004] At present, there is no report on the simultaneous detection of gallic acid, corilagin, paeoniflorin and ellagic acid in Yumai oropharyngeal mixture. Therefore, a method with high accuracy, high specificity and good reproducibility that can simultaneously detect the contents of gallic acid, corilagin, paeoniflorin and ellagic acid in Yumai oropharyngeal mixture is established, which provides a basis for evaluating the quality of Yumai oropharyngeal mixture and can more effectively monitor it. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for simultaneously detecting the contents of gallic acid, corilagin, paeoniflorin and ellagic acid in Yumai oropharyngeal mixture.

[0006] The technical solution adopted by the present invention to solve the above technical problems is to provide a method for detecting the content of effective ingredients in Yumai oropharyngeal mixture. The detection method uses high performance liquid chromatography to detect gallic acid, corilagin, paeoniflorin and ellagic acid in Yumai oropharyngeal mixture, injects the reference solution and the test solution into the high performance liquid chromatograph, records the chromatogram, and calculates the content of gallic acid, corilagin, paeoniflorin and ellagic acid in Yumai oropharyngeal mixture by external standard method; the chromatographic conditions of the high performance liquid chromatography are: the chromatographic column uses octadecylsilane bonded silica gel as the filler; the mobile phase uses acetonitrile as mobile phase A, and a phosphoric acid aqueous solution with a volume concentration of 0.1% as mobile phase B, and gradient elution is performed; dual wavelength detection: 225-235nm, 245-255nm; column temperature: 25℃-35℃; flow rate: 0.5-1.5mL / min.

[0007] Furthermore, the gradient elution of the mobile phase is: 0-10 min, 7% mobile phase A, 93% mobile phase B; 10-30 min, 7%-25% mobile phase A, 93%-75% mobile phase B; 30-40 min, 25% mobile phase A, 75% mobile phase B; 40-45 min, 25%-7% mobile phase A, 75%-93% mobile phase B; 45-50 min, 7% mobile phase A, 93% mobile phase B.

[0008] Furthermore, the reference substance solution is prepared by accurately weighing gallic acid, corilagin, paeoniflorin and ellagic acid reference substances, and adding methanol to prepare a mixed reference substance solution containing 40 μg of each substance per 1 mL.

[0009] Furthermore, the test solution is prepared as follows: accurately measure 5-10 mL of Yumai oropharyngeal mixture sample into a 100 mL volumetric flask, add 80 mL of methanol solution with a volume concentration of 75-85%, ultrasonicate for 30-60 min, the ultrasonic frequency is 33-59 kHz, the power is 250-720 W, cool, make up to volume, shake well, filter to obtain the filtrate, and obtain the solution.

[0010] Preferably, the test solution is prepared as follows: accurately measure 5 mL of Yumai oropharyngeal mixture sample into a 100 mL volumetric flask, add 80 mL of 80% methanol solution by volume, ultrasonicate for 60 min, the ultrasonic frequency is 59 kHz, the power is 720 W, cool, make up to volume, shake well, filter to obtain the filtrate, and obtain the solution.

[0011] Furthermore, the chromatographic conditions of the high performance liquid chromatography method satisfy at least one of the following:

[0012] UV detector dual wavelength detection: 230nm, 250nm.

[0013] Column temperature: 30℃.

[0014] Flow rate: 1.0 mL / min.

[0015] The injection volume was 10 μL.

[0016] Furthermore, the external standard method is to use the concentrations and peak areas of gallic acid, corilagin, paeoniflorin and ellagic acid in the reference solution as reference, and calculate the contents of gallic acid, corilagin, paeoniflorin and ellagic acid in the test sample according to the formula Cx=CR×Ax / AR; wherein CR is the concentration of each reference solution; AR is the chromatographic peak area of ​​gallic acid, corilagin, paeoniflorin and ellagic acid in the mixed reference; Ax is the chromatographic peak area of ​​gallic acid, corilagin, paeoniflorin and ellagic acid in the test sample.

[0017] The beneficial effects of the present invention are:

[0018] The method for detecting the content of effective ingredients in the Yumai oropharyngeal mixture provided by the present invention can simultaneously and effectively detect the contents of four components, namely, gallic acid, corilagin, paeoniflorin and ellagic acid, and has simple detection steps, good linearity, high sensitivity, good repeatability and accuracy, and can provide a comprehensive and reliable quality control basis for the Yumai oropharyngeal mixture.

[0019] Specifically, the linear ranges of gallic acid, corilagin, paeoniflorin and ellagic acid in the content detection method provided by the present invention are 17.751-42.6024 (R2=0.9993), 17.444-41.8656 (R2=0.9991), 22.44-53.856 (R2=0.9991), and 21.07-50.568 (R2=0.9993), respectively, with good linearity; the average recoveries (n=6) of gallic acid, corilagin, paeoniflorin and ellagic acid are 97.56% (RSD=0.42%), 98.42% (RSD=0.92%), 97.16% (RSD=0.54%), and 96.38% (RSD=1.14%), respectively, and the detection results have high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the chromatogram of the test solution of Yumai oropharyngeal mixture in Example 1.

[0021] Figure 2 It is the chromatogram of the mixed reference substance solution in Example 1.

[0022] Figure 3 This is a standard curve diagram of gallic acid for investigating the linear relationship in Example 2.

[0023] Figure 4 This is a standard curve diagram of corilagin for investigating the linear relationship in Example 2.

[0024] Figure 5 This is a standard curve diagram of paeoniflorin for investigating the linear relationship in Example 2.

[0025] Figure 6 This is a standard curve diagram of ellagic acid for investigating the linear relationship in Example 2.

[0026] Figure 7 It is the chromatogram of precision investigation in Example 2. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below through specific examples.

[0028] Example 1 Detection of the content of active ingredients in Yumai oropharyngeal mixture

[0029] 1.1 Instruments and reagents

[0030] Instruments: Aglient 1260Ⅱ high performance liquid chromatograph, G7114A UV detector, Agilent Technologies, USA; SQP electronic balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.; SB25-12DTD ultrasonic cleaning machine, Ningbo Xinzhi Biotechnology; UPR-Ⅱ-40L ultrapure water machine, Sichuan Youpu Ultrapure Technology.

[0031] Reagents: Methanol (batch number: 2022091902), acetonitrile (batch number: 2022052101), and phosphoric acid (batch number: 2019111501) are all chromatographic grade reagents from Chengdu Cologne Chemicals Co., Ltd.; water is ultrapure water; paeoniflorin reference substance (batch number: 110736-202145); gallic acid reference substance (batch number: 110831-201906, purity 91.5%) were purchased from China Food and Drug Inspection Institutes; corilagin reference substance (batch number: AO2GB143893, purity ≥98%); ellagic acid reference substance (batch number: S24D11G135548, purity ≥98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0032] 1.2 Chromatographic conditions

[0033] Chromatographic column: HPLC column Shim-pack GIST C18 (250mm×4.6mm, 5μm); flow rate 1mL / min; column temperature 30℃; dual wavelength detection: 230nm, 250nm; injection volume 10μL; mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B) gradient elution, the gradient elution program is shown in Table 1.

[0034] Table 1 Mobile phase gradient program

[0035] Time (min) Mobile phase A (acetonitrile) / % Mobile phase B (0.1% phosphoric acid aqueous solution) / % 0~10min 7 93 10~30min 7～25 93～75 30~40min 25 75 40~45min 25～7 75～93 45~50min 7 93

[0036] 1.3 Preparation of reference solution

[0037] Accurately weigh appropriate amounts of gallic acid, corilagin, paeoniflorin and ellagic acid reference substances, and add methanol to prepare mixed reference substance solutions containing 40 μg of each per 1 mL.

[0038] 1.4 Preparation of test solution

[0039] Accurately measure 5 mL of Yumai oropharyngeal mixture sample into a 100 mL volumetric flask, add 80 mL of 80% methanol solution by volume, ultrasonically treat for 60 min, ultrasonic frequency 59 kHz, ultrasonic power 720 W, cool, make up to volume, shake well, filter, and take the filtrate to obtain the test solution.

[0040] 1.5 Determination of the active ingredients of Yumai oropharyngeal mixture

[0041] Accurately pipette 10 μL of the test solution and the mixed reference solution respectively, inject the test solution into the HPLC instrument twice in succession, and obtain the chromatogram. Figure 1 and Figure 2 .

[0042] The theoretical plate number is calculated based on gallic acid, corilagin, paeoniflorin and ellagic acid, and all are not less than 1300. The concentrations and peak areas of gallic acid, corilagin, paeoniflorin and ellagic acid in the reference solution are used as reference, and the contents of gallic acid, corilagin, paeoniflorin and ellagic acid in the test sample are calculated according to the formula Cx=CR×Ax / AR; where CR is the concentration of each reference solution, AR is the chromatographic peak area of ​​gallic acid, corilagin, paeoniflorin and ellagic acid in the mixed reference, and Ax is the chromatographic peak area of ​​gallic acid, corilagin, paeoniflorin and ellagic acid in the test sample. The content determination results are shown in Table 2.

[0043] Table 2 Results of effective components of Yumai oral and pharyngeal mixture / mg / mL

[0044] sample Gallic acid / mg / mL Corila / mg / mL Paeoniflorin / mg / mL Ellagic acid / mg / mL Content 1 6.20 1.63 3.50 0.43 Content 2 6.21 1.63 3.51 0.43 Average content 6.20 1.63 3.50 0.43

[0045] Example 2 Methodology Verification

[0046] 2.1 Investigation of linear relationship

[0047] Accurately measure the reference solution and prepare reference solutions with concentrations of 50%, 80%, 90%, 100%, and 120% corresponding to the original solution. Inject 10 μL of each solution and record the chromatographic peak area. Use the peak area Y as the ordinate and the solution concentration X (μg / mL) as the abscissa for linear regression. The results show (see Table 3) that the linear ranges of gallic acid, corilagin, paeoniflorin, and ellagic acid are 17.751-42.6024 (R2=0.9993), 17.444-41.8656 (R2=0.9991), 22.44-53.856 (R2=0.9991), and 21.07-50.568 (R2=0.9993), respectively, with good linear relationships.

[0048] Table 3 Linear detection results (n=6)

[0049] Reference Regression equation <![CDATA[R 2 ]]> Linear range μg / ml gallic acid y=23.558x-23.611 0.9993 17.751～42.6024 Korilagin y=35.704x-36.006 0.9991 17.444～41.8656 Paeoniflorin y=14.733x-38.138 0.9991 22.44～53.856 Ellagic acid y=107.65x-142.54 0.9993 21.07～50.568

[0050] 2.2 Precision investigation

[0051] Accurately pipette 10 μl of the mixed reference solution and inject it 6 times in succession. The RSDs of the peak areas of gallic acid, corilagin, paeoniflorin, and ellagic acid were 1.32%, 1.34%, 1.38%, and 1.37%, respectively, indicating that the instrument has good precision. See Table 4 and Figure 7 .

[0052] Table 4 Precision test results

[0053] Element Peak area 1 Peak area 2 Peak area 3 Peak area 4 Peak area 5 Peak area 6 average value RSD% gallic acid 824.158 829.617 804.951 820.961 823.258 803.748 817.782 1.32 Korilagin 1222.255 1232.779 1195.339 1219.012 1221.505 1192.105 1213.833 1.34 Paeoniflorin 640.466 646.184 626.404 638.501 640.319 623.999 635.979 1.38 Ellagic acid 4471.237 4512.104 4368.285 4453.143 4467.909 4360.420 4438.850 1.37

[0054] 2.3 Repeatability study

[0055] Six samples of Yumai oropharyngeal mixture were taken respectively, and Yumai oropharyngeal mixture test solutions were prepared respectively according to the method of the present invention. The test was carried out according to the method of the present invention, and 10 μL was injected each time. The peak areas were measured respectively. The RSD values ​​of gallic acid, corilagin, paeoniflorin, and ellagic acid were 0.42%, 0.46%, 0.33%, and 1.68%, respectively. The results show that the method has good repeatability. The results are shown in Table 5.

[0056] Table 5 Repeatability test results / mg / mL

[0057] Element Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 RSD% gallic acid 5.63 5.59 5.59 5.58 5.57 5.56 0.42 Korilagin 1.49 1.48 1.47 1.48 1.47 1.47 0.46 Paeoniflorin 3.67 3.64 3.64 3.64 3.64 3.64 0.33 Ellagic acid 0.31 0.29 0.29 0.30 0.30 0.30 1.68

[0058] 2.4 Stability investigation

[0059] The above chromatographic conditions were used to accurately draw the Yumai oral and pharyngeal mixture test solution, and 10 μL was injected each time. The peak areas were measured at 0, 4, 8, 12, and 24 hours after preparation. The RSDs of the measured values ​​were 0.27%, 0.35%, 0.24%, and 0.43% respectively, based on the peak areas of the chromatographic peaks of gallic acid, corilagin, paeoniflorin, and ellagic acid, indicating that the sample solution was basically stable within 24 hours. The results are shown in Table 6.

[0060] Table 6 Stability test results

[0061] Time / h Gallic acid / peak area Corilagin / Peak Area Paeoniflorin / peak area Ellagic acid / peak area 0 6497.900 2603.636 2602.719 1637.366 4 6517.206 2610.733 2619.782 1630.640 8 6470.082 2587.439 2610.967 1619.644 12 6458.122 2584.363 2608.447 1626.529 24 6517.999 2612.130 2647.993 1623.834 Average peak area within 24 hours 6492.139 2598.831 2612.403 1627.037 RSD% 0.27 0.35 0.24 0.43

[0062] 2.5 Sample recovery rate investigation

[0063] Accurately measure 6 portions of 5 mL of samples with known contents, add 5 mL of mixed reference solution to each portion, prepare Yumai Oropharyngeal Mixture test solution according to the test solution preparation method, and then perform the determination according to the method of the present invention, inject 10 μL each time, measure the peak area, and calculate the recovery rate of the sample. The results are shown in Table 7.

[0064] Table 7 Sample recovery results

[0065]

[0066]

[0067] 2.6 Content determination

[0068] 5 mL of 3 batches of Yumai oropharyngeal mixture was accurately measured, and 2 portions of each batch were measured in parallel. The test solution was prepared according to the method of the present invention, and the test was performed to calculate the contents of gallic acid, corilagin, paeoniflorin and ellagic acid in the samples (see Table 8).

[0069] Table 8 Content measurement results

[0070]

[0071] The method of the present invention can effectively detect the contents of four components, namely gallic acid, corilagin, paeoniflorin and ellagic acid, at the same time, and the detection steps are simple, and the method has good linearity, high sensitivity, good repeatability and accuracy, and can provide a comprehensive and reliable quality control basis for Yumai oropharyngeal mixture. The results show that the linear ranges of the method of the present invention are 17.751-42.6024 (R2=0.9993), 17.444-41.8656 (R2=0.9991), 22.44-53.856 (R2=0.9991), and 21.07-50.568 (R2=0.9993), respectively, with good linearity; the average recoveries (n=6) of gallic acid, corilagin, paeoniflorin, and ellagic acid are 97.56% (RSD=0.42%), 98.42% (RSD=0.92%), 97.16% (RSD=0.54%), and 96.38% (RSD=1.14%), respectively, and the detection results have high accuracy.

Claims

1. A method for detecting the content of active ingredients in Yumai oropharyngeal mixture, wherein the method uses high performance liquid chromatography to detect gallic acid, corilagin, paeoniflorin and ellagic acid in Yumai oropharyngeal mixture, and the method is characterized in that: The reference solution and the test solution were injected into a high performance liquid chromatograph, the chromatogram was recorded, and the contents of gallic acid, corilagin, paeoniflorin and ellagic acid in Yumai oropharyngeal mixture were calculated by external standard method; the chromatographic conditions of the high performance liquid chromatography method were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase was acetonitrile as mobile phase A, and a phosphoric acid aqueous solution with a volume concentration of 0.1% was used as mobile phase B, and gradient elution was performed; dual wavelength detection: 225-235nm, 245-255nm; the column temperature was: 2 5℃~35℃; flow rate: 0.5~1.5mL / min; the gradient elution is: 0~10min, 7% mobile phase A, 93% mobile phase B; 10~30min, 7%~25% mobile phase A, 93%~75% mobile phase B; 30~40min, 25% mobile phase A, 75% mobile phase B; 40~45min, 25%~7% mobile phase A, 75%~93% mobile phase B; 45~50min, 7% mobile phase A, 93% mobile phase B; The test solution is prepared as follows: accurately measure 5-10 mL of Yumai oropharyngeal mixture sample into a 100 mL volumetric flask, add 80 mL of methanol solution with a volume concentration of 75-85%, ultrasonicate for 30-60 min, the ultrasonic frequency is 33-59 kHz, the power is 250-720 W, cool, adjust the volume, shake well, filter to obtain the filtrate, and obtain the solution.

2. The method for detecting the content of the active ingredient in the Yumai oropharyngeal mixture according to claim 1, characterized in that: The reference substance solution is prepared as follows: accurately weigh gallic acid, corilagin, paeoniflorin and ellagic acid reference substances, and add methanol to prepare a mixed reference substance solution containing 40 μg of each substance per 1 mL.

3. The method for detecting the content of the active ingredient in the Yumai oropharyngeal mixture according to claim 1, characterized in that: The test solution is prepared as follows: accurately measure 5 mL of Yumai oropharyngeal mixture sample into a 100 mL volumetric flask, add 80 mL of 80% methanol solution by volume, ultrasonicate for 60 min, the ultrasonic frequency is 59 kHz, the power is 720 W, cool, make up to volume, shake well, filter to obtain the filtrate, and obtain the solution.

4. The method for detecting the content of the active ingredient in the Yumai oropharyngeal mixture according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography method meet at least one of the following: UV detector dual wavelength detection: 230nm, 250nm; Column temperature: 30°C; Flow rate: 1.0 mL / min; The injection volume was 10 μL.

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