Detection method and application of mussaenda pubescens detoxicating granules

Abstract

The invention relates to the technical field of traditional Chinese medicine quality control and detection, in particular to a detection method and application of mussaenda pubescens leaf detoxification granules. According to the method, a YCM Meteoric Core C18 (150 * 4.6 mm, 2.7 [mu] m) chromatographic column is adopted, the flow velocity is 1.0 mL / min, the column temperature is 30 DEG C, the wavelength is 254 nm, acetonitrile is adopted as a mobile phase A, a 0.05% phosphoric acid aqueous solution is adopted as a mobile phase B, and gradient elution is performed; the method is applied to fingerprint spectrum construction of the mussaenda pubescens leaf detoxification granules and content determination of 12 components, methodology verifies that 19 characteristic peaks of the mussaenda pubescens leaf detoxification granules are effectively detected, effective content determination is carried out on 12 identification peaks, and the method can be well used for quality control of the mussaenda pubescens leaf detoxification granules.

Description

Technical Field

[0001] This application relates to the technical field of quality control and detection of traditional Chinese medicines, and particularly to a detection method for Yuye Jiedu Granules and its application. Background Art

[0002] Yuye Jiedu Granules is a traditional Chinese medicine compound preparation composed of seven herbs, namely Mussaenda pubescens, Lonicera confusa, Ilex asprella, Helicteres angustifolia, Chrysanthemi Flos, Chrysanthemi Indici Flos, and Centella asiatica. Controlling single components or several components of single herbs in traditional Chinese medicine compound preparations can no longer meet the requirements for overall quality control of traditional Chinese medicine compound preparations. Fingerprint / characteristic spectra can reflect the chemical components, distribution characteristics, distribution ratios, content characteristics, potential physiological activities, and pharmacodynamic quantification characteristics of traditional Chinese medicines, which are consistent with the integrity, characteristic, multi-target, and non-linear features that should be reflected in the quality control of traditional Chinese medicines. Currently, there is no content determination index in the quality standard of Yuye Jiedu Granules. It is necessary to develop a detection method that can comprehensively control its quality and apply it to the construction of fingerprint spectra and multi-index content determination of Yuye Jiedu Granules. Summary of the Invention

[0003] To solve the above technical problems, the object of the present invention is to provide a detection method for Yuye Jiedu Granules. The method uses high-performance liquid chromatography to simultaneously detect 19 components in Yuye Jiedu Granules, and 12 of these components are: neochlorogenic acid, gardenoside methyl ester, chlorogenic acid, cryptochlorogenic acid, loganin, secologanin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, and buddleoside; The chromatographic conditions of the detection method are: a C18 chromatographic column, a detection wavelength of 254 nm, acetonitrile as mobile phase A, and a 0.05%-0.1% phosphoric acid aqueous solution as mobile phase B for gradient elution; The gradient elution program is: 0 - 3 min, 3% A; 3 - 25 min, 3 - 16% A; 25 - 30 min, 16% A; 30 - 50 min, 16 - 20% A; 50 - 70 min, 25 - 35% A; Take an appropriate amount of Yuye Jiedu Granules sample with a 50%-70% methanol solution, extract by ultrasonic or reflux, filter, and take the subsequent filtrate to obtain the test solution. Precisely pipette the test solution into the liquid chromatograph and perform the determination according to the above method, record the chromatogram, and that's it.

[0004] Preferably, a 0.05% phosphoric acid aqueous solution is used as mobile phase B. The model of the C18 chromatographic column is YCM MeteoricCore C18, with a specification of 150×4.6 mm, 2.7 μm. The flow rate is 1.0 mL / min, and the column temperature is 30°C.

[0005] Preferably, a sample of Yuye Jiedu Granules is taken with an appropriate amount of 50%-70% methanol solution, ultrasonically extracted for 10 minutes-30 minutes, filtered, and the filtrate is taken to obtain the test solution.

[0006] Preferably, take a sample of Yuye Jiedu Granules and add an appropriate amount of 50% methanol solution, ultrasonically extract for 10 minutes, filter, and take the filtrate to obtain the test solution; the ultrasonic extraction power is 300W, 40kHz.

[0007] The above method is used in the construction of the fingerprint spectrum of Yuye Jiedu Granules. The construction method of the fingerprint spectrum of Yuye Jiedu Granules is as follows: (1) Take the test solution, reference solution and blank solvent of Yuye Jiedu Granules, and perform the test according to the above method, and record the chromatogram; (2) By comparing the peak elution time, the spectra of the reference substance and the test substance, the characteristic peaks are calibrated, and the chromatographic peaks are identified and labeled; (3) Set the reference peak, calculate the relative retention time and relative peak area of ​​other characteristic peaks, and establish the fingerprint of Yuye Detoxification Granules based on the relative retention time.

[0008] A total of 19 characteristic peaks were calibrated in the fingerprint spectrum of Yuye Jiedu Granules, and the reference peak was set as isochlorogenic acid B; The relative Poly times of the 19 characteristic peaks relative to the reference peaks are: peak 1 is 0.3397, peak 2 is 0.3688, peak 3 is 0.4471, peak 4 is 0.4686, peak 5 is 0.5066, peak 6 is 0.5897, peak 7 is 0.6552, peak 8 is 0.6751, peak 9 is 0.8600, peak 10 is 0.8840, peak 11 is 1.0000, peak 12 is 1.0466, peak 13 is 1.0666, peak 14 is 1.2108, peak 15 is 1.3182, peak 16 is 1.5807, peak 17 is 1.6166, peak 18 is 1.6363, peak 19 is 1.7059; The relative peak areas of the 19 characteristic peaks relative to the reference peaks are: peak 1 is 0.6483, peak 2 is 0.0443, peak 3 is 0.0516, peak 4 is 0.9365, peak 5 is 0.7678, peak 6 is 0.0831, peak 7 is 0.1857, peak 8 is 0.1669, peak 9 is 0.3707, peak 10 is 0.1810, peak 11 is 1.0000, peak 12 is 0.5307, peak 13 is 0.1819, peak 14 is 1.1556, peak 15 is 0.0453, peak 16 is 0.0349, peak 17 is 0.5827, peak 18 is 0.0679, and peak 19 is 0.1213; Identify and label 12 chromatographic peaks, namely: peak 1 is neochlorogenic acid, peak 3 is gardenoside methyl ester, peak 4 is chlorogenic acid, peak 5 is cryptochlorogenic acid, peak 6 is loganin, peak 8 is secologanin, peak 9 is luteoloside, peak 11 is isochlorogenic acid B, peak 12 is isochlorogenic acid A, peak 14 is isochlorogenic acid C, peak 16 is luteolin, and peak 17 is buddleoside.

[0009] The reference substance solution includes the reference substances corresponding to the above 12 labeled peaks.

[0010] The application of the above detection method or the fingerprint spectrum of Yuye Jiedu Granules in the determination of the contents of 12 components in Yuye Jiedu Granules. The 12 components are neochlorogenic acid, gardenoside methyl ester, chlorogenic acid, cryptochlorogenic acid, loganin, secologanin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, and buddleoside. The content determination method is as follows: Take the sample of Yuye Jiedu Granules and extract it ultrasonically with 50% methanol solution, filter, and take the subsequent filtrate to obtain the test solution. Take the test solution and determine it according to the above method, and calculate the contents of 12 components by relative retention time and relative peak area.

[0011] The numbers of the 12 components are: peak 1 is neochlorogenic acid, peak 3 is gardenoside methyl ester, peak 4 is chlorogenic acid, peak 5 is cryptochlorogenic acid, peak 6 is loganin, peak 8 is secologanin, peak 9 is luteoloside, peak 11 is isochlorogenic acid B, peak 12 is isochlorogenic acid A, peak 14 is isochlorogenic acid C, peak 16 is luteolin, and peak 17 is buddleoside. The relative retention times and relative peak areas of the 12 components are as described above.

[0012] The linear ranges for the content determination of the 12 components are: for neochlorogenic acid, it is 19.796 - 158.368 μg / ml; for gardenoside methyl ester, it is 0.8176 - 40.8811 μg / ml; for chlorogenic acid, it is 19.9534 - 159.6269 μg / ml; for cryptochlorogenic acid, it is 20.4232 - 163.3856 μg / ml; for loganin, it is 0.8825 - 44.1242 μg / ml; for secologanin, it is 5.7977 - 57.9768 μg / ml; for luteoloside, it is 0.8648 - 43.2425 μg / ml; for isochlorogenic acid B, it is 20.9328 - 167.4624 μg / ml; for isochlorogenic acid A, it is 8.1379 - 81.3792 μg / ml; for isochlorogenic acid C, it is 11.0544 - 154.7616 μg / ml; for luteolin, it is 0.2018 - 20.1845 μg / ml; for buddleoside, it is 8.0978 - 80.9776 μg / ml: The detection method of the present invention has been verified by methodology, achieving effective separation and detection of 19 characteristic peaks of Yuye Jiedu Granules. Compared with the separation and detection of multiple components of single medicinal materials, it can better achieve the comprehensive quality control research of Yuye Jiedu Granules. By detecting a large number of batches of Yuye Jiedu Granule samples with this method, the fingerprint of Yuye Jiedu Granules is constructed, which can more completely characterize the quality attributes of Yuye Jiedu Granule preparations. At the same time, the content determination of 12 identified marker peaks has been studied. After methodology verification, this method can be well used for the content determination of 12 key index components of Yuye Jiedu Granules and comprehensive quality control. This method has simple sample treatment, does not require expensive experimental instruments, does not need to purchase a large number of reference substances for external standard quantification, and can achieve the content determination of 19 characteristic peaks of the key components of each medicinal material in Yuye Jiedu Granules, realizing a better characterization of the quality attributes of Yuye Jiedu Granules, and has important value in production quality control and quality control research. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0014] Figure 1-1 are the fingerprint maps at different detection wavelengths (220nm, 254nm, 280nm); Figure 1-2 are the fingerprint maps at different detection wavelengths (310nm, 340nm, 370nm, 400nm); Figure 2 are the fingerprint maps under different mobile phases; Figure 3 is the result map of the fingerprint peak identification of Yuye Jiedu Granules; Figure 4-1 is the result of the fingerprint peak attribution of Yuye Jiedu Granules of the present invention (solvent blank); Figure 4-2 is the result of the fingerprint peak attribution of Yuye Jiedu Granules of the present invention (test solution, Mussaenda pubescens, Lonicera japonica, Radix Ilicis Asprellae); Figure 4-3 is the result of the fingerprint peak attribution of Yuye Jiedu Granules of the present invention (Sesamum indicum, Chrysanthemum morifolium, Chrysanthemi Indici Flos, Centella asiatica); Figure 5-1HPLC chromatogram for the determination of the contents of 12 components in Yuye Jiedu Granules of the present invention (in the figure, 1 is neochlorogenic acid; 2 is gardenoside methyl ester; 3 is chlorogenic acid; 4 is cryptochlorogenic acid; 5 is loganin; 6 is secologanin; 7 is luteoloside; 8 is isochlorogenic acid B; 9 is isochlorogenic acid A; 10 is isochlorogenic acid C; 11 is luteolin; 12 is buddleoside); Figure 5-2 HPLC chromatogram for the determination of the contents of 12 components in Yuye Jiedu Granules of the present invention. Detailed implementation manners

[0015] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application.

[0016] Example 1: Development of the detection method for Yuye Jiedu Granules and its application in the construction of fingerprint 1.1 Instruments and test drugs 1.1.1 Instruments

[0017] Thermo Ultimate 3000, high performance liquid chromatograph (DAD detector); SB-5200D ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); HH-8 digital display constant temperature water bath (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); ES-502HA electronic balance (Changsha Xiangping Technology Development Co., Ltd.); SQP electronic analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); XSR205DU / A electronic analytical balance (METTLER).

[0018] 1.1.2 Test drugs Yuye Jiedu Granules, produced by Sanjin Group Hunan Sanjin Pharmaceutical Co., Ltd., with a total of 30 batches; acetonitrile, methanol, and phosphoric acid are chromatographically pure, water is purified water, and other reagents are all analytically pure.

[0019] 1.2 Method establishment 1.2.1 Establishment of chromatographic conditions ① Investigation of elution gradient Taking into comprehensive consideration factors such as baseline, number of peaks, and resolution of each chromatographic peak, after gradient exploration, the preliminary method for preparing the test solution is as follows: Take an appropriate amount of this product, grind it finely, take about 5 g, weigh accurately, place it in a stoppered conical flask, accurately add 25 ml of 70% methanol, stopper tightly, weigh, ultrasonically treat (power 300 W, frequency 40 kHz) for 10 minutes, take it out and let it cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0020] The proposed chromatographic conditions are as follows: YCM Meteoric Core C18 (150×4.6 mm, 2.7 μm) chromatographic column, flow rate 1.0 mL / min, column temperature 30 °C, wavelength 254 nm, injection volume 5 μl, using acetonitrile as mobile phase A and 0.05% phosphoric acid aqueous solution as mobile phase B, and perform gradient elution according to the regulations in Table 1.

[0021] Table 1 Fingerprint gradient elution program

[0022] ② Selection of wavelength Take the test solution of this product and compare the chromatograms at wavelengths of 220 nm, 254 nm, 280 nm, 310 nm, 340 nm, 370 nm, and 400 nm. The results are shown in Figure 1-1 and Figure 1-2 . The results show that using a diode array detector (DAD) for full scan at wavelengths of 190 - 400 nm, according to factors such as the number of chromatographic peaks, peak response values and their proportions, peak resolution and tailing factor, and baseline stability, select 254 nm wavelength to determine the fingerprint of Yuye Jiedu Granules.

[0023] ③ Selection of types of mobile phase The types of mobile phase were investigated for acetonitrile - water, acetonitrile - 0.1% formic acid aqueous solution, acetonitrile - 0.1% phosphoric acid aqueous solution, and acetonitrile - 0.05% phosphoric acid aqueous solution. The results are shown in Figure 2 . The results show that when acetonitrile - water is used as the mobile phase, the number of peaks is less and the baseline is not stable; when acetonitrile - 0.1% formic acid aqueous solution is used as the mobile phase, the separation of each chromatographic peak is not good in the time periods of 15 - 20 min and 30 - 35 min; when acetonitrile - 0.1% phosphoric acid aqueous solution and acetonitrile - 0.05% phosphoric acid aqueous solution are used as the mobile phase, the separation effect and peak shape of each chromatographic peak are relatively good. Therefore, select acetonitrile - 0.05% phosphoric acid aqueous solution with a lower acid concentration as the mobile phase.

[0024] 1.2.2 Investigation on the preparation method of the test solution (1)Extraction method Take an appropriate amount of this product, grind it finely, take about 5 g, a total of 2 portions, weigh accurately, place in a stoppered conical flask, accurately add 25 ml of 70% methanol, and treat them by ultrasonic wave (power 300 W, frequency 40 kHz) and heating under reflux (80 °C) for 10 minutes respectively. Take them out and let them cool, then weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0025] Determine the fingerprint according to the established chromatographic conditions, and the results are shown in Table 2.

[0026] The results show that in the fingerprint of the test sample extracted by ultrasonic wave, the asymmetry of the chromatographic peak at the retention time of 12.330 min is 1.10, the asymmetry of the chromatographic peak at 16.957 min is 1.09, the asymmetry of the chromatographic peak at 18.353 min is 1.09, and the asymmetry of the chromatographic peak at 21.313 min is 1.00; While the asymmetry of the corresponding peaks extracted by reflux is 1.13, 1.21, 1.15, and 1.11 in sequence, indicating that in the time period of 5 - 25 min of the fingerprint of the test sample extracted by ultrasonic wave, the peak shape of the chromatographic peak is better than that of reflux extraction. Considering the convenience of operation, the extraction method of this product is selected as ultrasonic extraction.

[0027] Table 2 Results of the investigation on extraction methods

[0028] (2) Extraction solvent Take an appropriate amount of this product, grind it finely, take about 5 g, a total of 9 portions, weigh accurately, place in a stoppered conical flask, and accurately add 25 ml of anhydrous ethanol, 70% ethanol, water, 10% methanol, 30% methanol, 50% methanol, 70% methanol, 90% methanol, and methanol respectively. Treat them by ultrasonic wave (power 300 W, frequency 40 kHz) for 10 minutes. Take them out and let them cool, then weigh again, make up the lost weight with each extraction solvent, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0029] Determine the fingerprint according to the established chromatographic conditions, and the results are shown in Table 3.

[0030] Table 3 Results of the investigation on extraction solvents

[0031] The results showed that when the test samples were processed, the samples treated with water could not pass through the microporous filter head; the samples treated with 10% methanol and 30% methanol were relatively difficult to pass through the microporous filter head; for the samples treated with absolute ethanol and 70% ethanol, the overall peak shape was not good, with serious tailing or fronting; for the samples treated with 90% methanol and methanol, the chromatographic peak with a retention time of 12.330 min had a poor peak shape, and the peak asymmetry was only 0.7 - 0.8; the overall peak shapes of 50% methanol and 70% methanol were better, and the overall peak area of the samples treated with 50% methanol was slightly larger than that of the samples treated with 70% methanol. Therefore, 50% methanol was selected as the extraction solvent.

[0032] (3)Extraction time Take an appropriate amount of this product, grind it finely, take about 5 g, a total of 3 portions, weigh accurately, place in a stoppered conical flask, accurately add 25 ml of 50% methanol, and ultrasonically treat (power 300 W, frequency 40 kHz) for 10 minutes, 20 minutes, and 30 minutes respectively. Take out and let it cool, then weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the subsequent filtrate to obtain the sample solution.

[0033] Determine the fingerprint according to the established chromatographic conditions, and the results are shown in Table 4.

[0034] Table 4 Results of the investigation on extraction time

[0035] The results showed that ultrasonic treatment for 10 minutes had basically completed the extraction. Considering time and energy savings, the ultrasonic time was selected as 10 minutes.

[0036] (4)Sampling amount (see Table 5)

[0037] Table 5 Results of the investigation on sampling amount

[0038] Take an appropriate amount of this product, grind it finely, take about 2.5 g, 5 g, and 10 g, a total of 3 portions, weigh accurately, place in a stoppered conical flask, accurately add 25 ml of 50% methanol, ultrasonically treat (power 300 W, frequency 40 kHz) for 10 minutes, take out and let it cool, then weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the subsequent filtrate to obtain the sample solution.

[0039] Inject 10 μl of the sample solution with a sampling amount of 2.5 g, inject 5 μl of the sample solution with a sampling amount of 5 g, inject 2.5 μl of the sample solution with a sampling amount of 10 g, and determine the fingerprint according to the established chromatographic conditions for the rest. The results are shown in Table 5.

[0040] The results showed that when the sampling amount was 2.5 g, the extraction was more complete compared with 5 g and 10 g, and the filtration operation during sample treatment was more convenient. Therefore, the sampling amount was selected as 2.5 g, and the injection volume was 10 μl.

[0041] 1.2.3 Determination of the analysis method of the present invention After comprehensive investigation and research, the analysis method for Yuye Jiedu Granules is determined as follows: Chromatographic conditions: YCM Meteoric Core C18 (150×4.6 mm, 2.7 μm) chromatographic column, flow rate 1.0 mL / min, column temperature 30 °C, wavelength 254 nm. Acetonitrile is used as mobile phase A, and 0.05% phosphoric acid aqueous solution is used as mobile phase B. Gradient elution is carried out according to the regulations in Table 6, and rinse and pre-equilibrate for 30 min.

[0042] Table 6 Fingerprint gradient elution program

[0043] Preparation of test solution: Take an appropriate amount of this product, grind it finely, take about 2.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 50% methanol, tightly stopper it, weigh it, ultrasonically treat it for 10 minutes (300 W, 40 kHz), take it out and let it cool, weigh it again, make up the lost weight with 50% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0044] Determination method: Accurately pipette 10 μl of the test solution, inject it into the liquid chromatograph, determine, record the chromatogram, that is obtained.

[0045] 1.3 Application and verification of the detection method of Yuye Jiedu Granules in fingerprint construction 1.3.1 Chromatographic peak identification According to the determined chromatographic method above, the test solution, reference solution (loganin, isorhamnetin, quercetin, kaempferol, rutin, luteolin, chlorogenic acid, buddleoside, asperosaponin VI, L-phenylalanine, D-phenylalanine, madecassoside, madecassic acid, sophocarpine, shanzhiside methyl ester, ilicifolin A, asiatic acid, secologanin, macranthoidin A, acacetin, 1,5-dicaffeoylquinic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, methyl mussaenoside, 8-O-acetylshanzhiside methyl ester) and solvent blank are detected in sequence. The results are shown in Figure 3 .

[0046] By comparing the peak emergence time, the spectra of the reference substance and the test substance, it is determined that the marked peak 1 is neochlorogenic acid, peak 3 is shanzhiside methyl ester, peak 4 is chlorogenic acid, peak 5 is cryptochlorogenic acid, peak 6 is loganin, peak 8 is secologanin, peak 9 is luteoloside, peak 11 is isochlorogenic acid B, peak 12 is isochlorogenic acid A, peak 14 is isochlorogenic acid C, peak 16 is luteolin, and peak 17 is buddleoside.

[0047] 1.3.2 Peak attribution Weigh Mussaenda pubescens, Lonicera confusa, Ilex asprella, Helicteres angustifolia, Chrysanthemum morifolium, Chrysanthemum indicum, Centella asiatica respectively according to the prescription ratio, add 25 ml of water respectively, decoct for 30 minutes, and evaporate the decoction to dryness after filtration. Subsequently, follow the preparation method of the test solution in "1.2.3" to prepare the test samples of each single herb successively. According to the determined chromatographic conditions, inject samples for detection respectively, and the results are shown in Figure 4-1 and Figure 4-2 and Figure 4-3 , Table 7.

[0048] Table 7 Peak attribution results of the fingerprint of Yuye Jiedu Granules

[0049] Peak attribution results of 12 identified chromatographic peaks: Peak 1 (neochlorogenic acid) and Peak 4 (chlorogenic acid) are common peaks of 7 herbs; Peak 3 (gardenoside methyl ester) is the characteristic peak of Mussaenda pubescens; Peak 5 (cryptochlorogenic acid), Peak 11 (isochlorogenic acid B), Peak 12 (isochlorogenic acid A), and Peak 14 (isochlorogenic acid C) are common peaks of Mussaenda pubescens, Lonicera confusa, Ilex asprella, Chrysanthemum morifolium, Chrysanthemum indicum, and Centella asiatica; Peak 6 (loganin) is a common peak of Mussaenda pubescens and Lonicera confusa; Peak 8 (secologanin) is a common peak of Lonicera confusa and Ilex asprella; Peak 9 (luteoloside) is a common peak of Chrysanthemum morifolium, Chrysanthemum indicum, and Centella asiatica; Peak 16 (luteolin) and Peak 17 (linarin) are common peaks of Chrysanthemum morifolium and Chrysanthemum indicum.

[0050] Peak attribution results of other unidentified chromatographic peaks: Peak 2 is a common peak of Lonicera confusa, Helicteres angustifolia, Chrysanthemum morifolium, Chrysanthemum indicum, and Centella asiatica; Peak 7 is a common peak of Lonicera confusa, Ilex asprella, Chrysanthemum morifolium, Chrysanthemum indicum, and Centella asiatica; Peaks 15 and 18 are attributed to Chrysanthemum indicum; Peaks 10, 13, and 19 are common peaks of Chrysanthemum morifolium and Chrysanthemum indicum; Peak 19 is a common peak of Ilex asprella and Helicteres angustifolia.

[0051] 1.3.3 Repeatability test Prepare 6 test solution samples according to the determined analysis method, inject samples for detection successively, and investigate the repeatability. Calibrate 19 characteristic peaks, take Peak 11 (isochlorogenic acid B) as the reference peak, and calculate the relative retention time and relative peak area of other characteristic peaks. The results show that the RSD of the relative retention time of 19 common peaks does not exceed 1.0%, and the RSD of the relative peak area does not exceed 5.0%, indicating that the method has good repeatability.

[0052] 1.3.4 Solution stability test Prepare 1 sample solution according to the determined analysis method, and determine the fingerprint at 0 h, 10 h, 20 h, 40 h, and 50 h respectively to investigate the stability. Calibrate 19 characteristic peaks, take peak 11 (isochlorogenic acid B) as the reference peak, and calculate the relative retention time and relative peak area of other characteristic peaks. The results show that the RSD of the relative retention time of the 19 common peaks does not exceed 1.0%, and the RSD of the relative peak area does not exceed 5.0%, indicating that the solution has good stability within 50 hours.

[0053] 1.3.5 Precision test Prepare 1 sample solution according to the determined analysis method, inject the sample continuously for 6 times, and investigate the precision. Calibrate 19 characteristic peaks, take peak 11 (isochlorogenic acid B) as the reference peak, and calculate the relative retention time and relative peak area of other characteristic peaks. The results are shown in Table 8-9.

[0054] Table 8 Results of relative retention time of fingerprint precision

[0055] The results of relative peak area are shown in Table 9.

[0056] Table 9 Results of relative peak area of fingerprint precision

[0057] The results show that the RSD of the relative retention time of the 19 common peaks does not exceed 1.0%, and the RSD of the relative peak area does not exceed 5.0%, indicating that the instrument precision is good.

[0058] 1.4 Fingerprint analysis of Yuye Jiedu Granules Determine the fingerprint of 30 batches of Yuye Jiedu Granules according to the method described in 1.2.3, calibrate 19 common characteristic peaks, take peak 11 (isochlorogenic acid B) as the reference peak, and calculate the relative retention time and relative peak area of other characteristic peaks. The results are shown in Tables 10-1, 10-2, 11-1, and 11-2.

[0059] Table 10-1 Results of relative retention time of fingerprint of 30 batches of samples

[0060] The results show that the RSD of the relative retention time of the 19 common peaks does not exceed 2.0%, indicating that the relative retention time of Yuye Jiedu Granules is relatively stable and will not vary greatly due to batches. The fingerprint of this product can be established based on the relative retention time.

[0061] Table 10-2 Results of relative retention time of fingerprint of 30 batches of samples (continued table)

[0062] Table 11-1 Results Table of Relative Peak Areas of Fingerprint Chromatograms of 30 Batches of Samples

[0063] Table 11-2 Results Table of Relative Peak Areas of Fingerprint Chromatograms of 30 Batches of Samples (Continued Table)

[0064] Example 2: Method for Determining the Contents of 12 Components in Yuye Jiedu Granules Yuye Jiedu Granules is a traditional Chinese medicine compound preparation composed of seven herbs, namely Mussaenda pubescens, Lonicera hypoglauca, Ilex asprella, Helicteres angustifolia, Chrysanthemi Flos, Chrysanthemi Indici Flos, and Centella asiatica. There are currently no content determination indicators in the quality standard. Based on the above fingerprint chromatogram research, it is proposed to conduct multi-index content determination research on 12 components, namely neochlorogenic acid, methylshanzhiside, chlorogenic acid, cryptochlorogenic acid, loganin, secologanin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, and buddleoside.

[0065] 2.1 Instruments and Reagents 2.1.1 Instruments Thermo Ultimate 3000, high performance liquid chromatograph (DAD detector); SB-5200D ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); HH-8 digital display constant temperature water bath (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); ES-502HA electronic balance (Changsha Xiangping Technology Development Co., Ltd.); SQP electronic analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); XPE105 electronic analytical balance (METTLER).

[0066] 2.1.2 Reagents Yuye Jiedu Granules, produced by Sanjin Group Hunan Sanjin Pharmaceutical Co., Ltd., a total of 30 batches. Acetonitrile, methanol, and phosphoric acid are chromatographically pure, water is pure water, and other reagents are all analytically pure.

[0067] 2.2 Chromatographic Conditions Same as the detection method under item "1.2.3" of Example 1.

[0068] 2.3 Methodological Verification 2.3.1 Specificity Test Preparation of individual reference substance solutions: Weigh appropriate amounts of reference substances of neochlorogenic acid, gardenoside methyl ester, chlorogenic acid, cryptochlorogenic acid, loganin, secologanin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, and buddleoside accurately, and dissolve them in methanol respectively to prepare individual reference substance stock solutions with concentrations of 197.96, 204.41, 199.53, 204.23, 220.62, 193.26, 216.21, 209.33, 203.45, 221.09, 201.84, and 80.98 μg / mL. Pipette appropriate amounts of the individual reference substance stock solutions respectively, add 50% methanol, and dilute to obtain individual reference substance solutions with concentrations of 83.14, 20.44, 83.80, 93.95, 15.44, 32.85, 17.30, 83.73, 32.55, 68.54, 2.02, and 44.54 μg / mL.

[0069] Prepare one test solution, one individual reference substance solution, and a solvent blank (50% methanol) respectively according to the established method. Pipette 10 μL precisely, inject them into a high-performance liquid chromatograph, and determine according to the established chromatographic conditions. The results are shown in Figure 5-1 、 Figure 5-2 。

[0070] The results show that there are no corresponding chromatographic peaks at the positions corresponding to the reference substance chromatographic peaks and the test solution chromatographic peaks in the solvent blank, indicating no interference, and the specificity of this method is good.

[0071] 2.3.2 Precision test Prepare one test solution according to the established method, and repeat the determination 6 times. Record the peak areas of 12 components.

[0072] The results show that the RSDs of the average peak areas of 12 components are between 0.09% and 1.44%; it indicates that the precision of the instrument is good and meets the requirements of quantitative analysis.

[0073] 2.3.3 Solution stability Prepare one test solution according to the established method, and determine it at 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, and 42 h respectively. Record the peak areas of the target substances. The results show that the RSDs of the average peak areas of 12 components are between 0.14% and 1.51%, indicating that the solution stability of the test solution is good within 42 h.

[0074] 2.3.4 Repeatability test According to the established method, prepare 6 test solutions in parallel, determine the peak areas, and calculate the contents of 12 components.

[0075] The results showed that the average content of neochlorogenic acid in this batch of samples was 0.5670 mg / g, with an RSD of 1.85%; the average content of gardenoside methyl ester was 0.1277 mg / g, with an RSD of 1.29%; the average content of chlorogenic acid was 0.5348 mg / g, with an RSD of 2.09%; the average content of cryptochlorogenic acid was 0.5784 mg / g, with an RSD of 2.07%; the average content of loganin was 0.1046 mg / g, with an RSD of 2.66%; the average content of secologanin was 0.1824 mg / g, with an RSD of 2.46%; the average content of luteoloside was 0.1416 mg / g, with an RSD of 2.46%; the average content of isochlorogenic acid B was 0.6257 mg / g, with an RSD of 0.67%; the average content of isochlorogenic acid A was 0.2635 mg / g, with an RSD of 0.61%; the average content of isochlorogenic acid C was 0.5351 mg / g, with an RSD of 0.97%; the average content of luteolin was 0.0096 mg / g, with an RSD of 1.54%; the average content of buddleoside was 0.3803 mg / g, with an RSD of 0.53%. According to the requirements of General Principles 9101 "Guidelines for Validation of Analytical Methods for Drug Quality Standards" in Part IV of the Chinese Pharmacopoeia 2020 Edition, the RSD% of repeatability should be less than 3% when the content of the component to be measured in the sample is 1 mg / g. The test results all met the requirements, indicating that this method had good repeatability.

[0076] 2.3.5 Linear Range Inject the prepared series of reference substance solutions for detection under the chromatographic conditions described in "2.2", and record the peak areas of the reference substance solutions of each component and their corresponding dilution solutions. Using the concentration as the abscissa (X) and the peak area as the ordinate (Y), draw the standard curves in sequence. The results are shown in Tables 12-1 and 12-2.

[0077] Table 12-1 Results of the Investigation on the Linear Range of 12 Reference Substances

[0078] Table 12-2 Results of the Investigation on the Linear Range of 12 Reference Substances (Continued Table)

[0079] The results showed that the linear relationships of the 12 components were all good within their respective mass concentration ranges.

[0080] 2.3.6 Sample Recovery Rate (Accuracy) Take the reference substance stock solutions of neochlorogenic acid, gardenoside methyl ester, chlorogenic acid, cryptochlorogenic acid, loganin, secologanin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, and buddleoside. Accurately measure 3.584, 0.852, 3.454, 3.544, 0.683, 1.231, 0.787, 3.84, 1.681, 3.029, 0.065, and 5.876 mL respectively in sequence, place them in a stoppered conical flask, and evaporate to dryness in a low-temperature water bath at 50 - 60 °C. There are a total of 6 portions, which are sequentially labeled as test samples No. 1 - 6.

[0081] Take Yuye Jiedu Granules (with known contents of neochlorogenic acid 0.5670 mg / g, gardenoside methyl ester 0.1277 mg / g, chlorogenic acid 0.5348 mg / g, cryptochlorogenic acid 0.5784 mg / g, loganin 0.1046 mg / g, secologanin 0.1824 mg / g, luteoloside 0.1416 mg / g, isochlorogenic acid B 0.6257 mg / g, isochlorogenic acid A 0.2635 mg / g, isochlorogenic acid C 0.5351 mg / g, luteolin 0.0096 mg / g, and buddleoside 0.3803 mg / g) samples, grind them finely, take about 1.25 g, accurately weigh it, and sequentially add it to the test samples No. 1 - 6. Then accurately add 25 mL of 50% methanol in sequence, tightly stopper, weigh, ultrasonically treat for 10 minutes (300 W, 40 kHz), take it out and let it cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the subsequent filtrate as the test solution.

[0082] Determine the content according to the established chromatographic conditions, and calculate the recovery rate according to the following formula. The results show that the recovery rates of the 12 components are between 97.81% and 104.72%, and the RSDs are between 0.52% and 1.81%. According to the requirements of General Principles 9101 "Guidelines for the Validation of Analytical Methods for Drug Quality Standards" in Part IV of the Chinese Pharmacopoeia 2020 Edition, when the content of the component to be measured in the sample is 1 mg / g, the recovery rate limit is 90% - 108%. The detection results of the 12 components all meet the requirements, indicating that this method has good accuracy.

[0083] 2.3.7 Content determination results of multiple batches of Yuye Jiedu Granules Determine the contents of 12 components in 30 batches of Yuye Jiedu Granules according to the detection method under "1.2.3" in Example 1. The results are shown in Tables 13 - 1 and 13 - 2. The results show that among the 12 components, the relatively higher contents are neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid C, and buddleoside, while the contents of gardenoside methyl ester, loganin, and luteolin are relatively lower.

[0084] Table 13 - 1 Content determination results of 12 components in 30 batches of Yuye Jiedu Granules (mg / g)

[0085] Table 13 - 2 Determination Results of 12 Components in 30 Batches of Yuye Jiedu Granules (mg / g) (Continued Table)

[0086] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting Yuye Jiedu granules, wherein the Yuye Jiedu granules are composed of seven Chinese medicinal herbs: Yuye Jinhua, Lonicera japonica, Prunus armeniaca, Sesame, Chrysanthemum, Wild Chrysanthemum, and Centella asiatica, and are characterized by: HPLC was used to simultaneously detect 19 components in Yuye Jiedu Granules, of which 12 components were: neochlorogenic acid, shanzhioside methyl ester, chlorogenic acid, cryptochlorogenic acid, loganin, deoxyloganin, luteolin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, and mongoside; The chromatographic conditions of the detection method are: C18 chromatographic column, detection wavelength 254nm, acetonitrile as mobile phase A, 0.05%-0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution; The gradient elution program is: 0-3 min, 3% A; 3-25 min, 3-16% A; 25-30 min, 16% A; 30-50 min, 16-20% A; 50-70 min, 25-35% A; Take a sample of Yuye Jiedu Granules and add an appropriate amount of 50%-70% methanol solution, perform ultrasonic or reflux extraction, filter, and take the filtrate to obtain the test solution; accurately pipette the test solution into a liquid chromatograph for determination according to the above method, and record the chromatogram.

2. The method according to claim 1, characterized in that: 0.05% phosphoric acid aqueous solution was used as mobile phase B.

3. The method according to claim 1, characterized in that: The C18 chromatographic column model is YCM Meteoric CoreC18, with specifications of 150×4.6 mm and 2.7 μm.

4. The method according to claim 1, characterized in that: Take a sample of Yuye Jiedu Granules and add an appropriate amount of 50%-70% methanol solution, ultrasonically extract for 10 minutes-30 minutes, filter, and take the filtrate to obtain the test solution.

5. The method according to claim 4, characterized in that: Take a sample of Yuye Jiedu Granules and add an appropriate amount of 50% methanol solution, perform ultrasonic extraction for 10 minutes, filter, and take the filtrate to obtain the test solution; the ultrasonic extraction power is 300W, 40kHz.

6. The method according to claim 1, characterized in that: Flow rate: 1.0 mL / min, column temperature: 30 °C.

7. Application of the method according to any one of claims 1 to 6 in the construction of a fingerprint of Yuye Jiedu Granules, wherein the method for constructing the fingerprint of Yuye Jiedu Granules is: (1) Take the test solution, reference solution and blank solvent of Yuye Jiedu Granules, perform detection according to the method described in any one of claims 1 to 6, and record the chromatogram; (2) By comparing the peak elution time, the spectra of the reference substance and the test substance, the characteristic peaks are calibrated, and the chromatographic peaks are identified and labeled; (3) Set the reference peak, calculate the relative retention time and relative peak area of ​​other characteristic peaks, and establish the fingerprint of Yuye Detoxification Granules based on the relative retention time.

8. The use according to claim 7, characterized in that: A total of 19 characteristic peaks were calibrated in the fingerprint of Yuye Jiedu Granules, and the reference peak was set as isochlorogenic acid B; The relative Poly times of the 19 characteristic peaks relative to the reference peaks are: peak 1 is 0.3397, peak 2 is 0.3688, peak 3 is 0.4471, peak 4 is 0.4686, peak 5 is 0.5066, peak 6 is 0.5897, peak 7 is 0.6552, peak 8 is 0.6751, peak 9 is 0.8600, peak 10 is 0.8840, peak 11 is 1.0000, peak 12 is 1.0466, peak 13 is 1.0666, peak 14 is 1.2108, peak 15 is 1.3182, peak 16 is 1.5807, peak 17 is 1.6166, peak 18 is 1.6363, peak 19 is 1.7059; The relative peak areas of the 19 characteristic peaks relative to the reference peaks are: peak 1 is 0.6483, peak 2 is 0.0443, peak 3 is 0.0516, peak 4 is 0.9365, peak 5 is 0.7678, peak 6 is 0.0831, peak 7 is 0.1857, peak 8 is 0.1669, peak 9 is 0.3707, peak 10 is 0.1810, peak 11 is 1.0000, peak 12 is 0.5307, peak 13 is 0.1819, peak 14 is 1.1556, peak 15 is 0.0453, peak 16 is 0.0349, peak 17 is 0.5827, peak 18 is 0.0679, and peak 19 is 0.1213; Twelve chromatographic peaks were identified and labeled: Peak 1 was neochlorogenic acid, Peak 3 was jasmine glycoside methyl ester, Peak 4 was chlorogenic acid, Peak 5 was cryptochlorogenic acid, Peak 6 was loganin, Peak 8 was off-oxyloganin, Peak 9 was luteolin, Peak 11 was isochlorogenic acid B, Peak 12 was isochlorogenic acid A, Peak 14 was isochlorogenic acid C, Peak 16 was luteolin, and Peak 17 was monanthraniin.

9. Application of the Yuye Jiedu Granule fingerprint constructed according to claim 8 in the determination of the contents of 12 components in Yuye Jiedu Granules, calculating the contents of the 12 components by relative retention time and relative peak area; The 12 components: peak 1 is neochlorogenic acid, peak 3 is jasmine glycoside methyl ester, peak 4 is chlorogenic acid, peak 5 is cryptochlorogenic acid, peak 6 is loganin, peak 8 is off-oxyloganin, peak 9 is luteolin, peak 11 is isochlorogenic acid B, peak 12 is isochlorogenic acid A, peak 14 is isochlorogenic acid C, peak 16 is luteolin, and peak 17 is monanthus glycoside; Taking isochlorogenic acid B as the reference peak, the relative retention times of the 12 components relative to the reference peak are: peak 1 is 0.3397, peak 3 is 0.4471, peak 4 is 0.4686, peak 5 is 0.5066, peak 6 is 0.5897, peak 8 is 0.6751, peak 9 is 0.8600, peak 11 is 1.0000, peak 12 is 1.0466, peak 14 is 1.2108, peak 16 is 1.5807, and peak 17 is 1.6166; The relative peak areas of the 12 components relative to the reference peaks are: peak 1 is 0.6483, peak 3 is 0.0516, peak 4 is 0.9365, peak 5 is 0.7678, peak 6 is 0.0831, peak 8 is 0.1669, peak 9 is 0.3707, peak 11 is 1.0000, peak 12 is 0.5307, peak 14 is 1.1556, peak 16 is 0.0349, and peak 17 is 0.5827.

10. The use according to claim 9, wherein the linear range of the content determination of the 12 components is: Neochlorogenic acid was 19.796-158.368 μg / ml, shanzhioside methyl ester was 0.8176-40.8811 μg / ml, chlorogenic acid was 19.9534-159.6269 μg / ml, cryptochlorogenic acid was 20.4232-163.3856 μg / ml, loganin was 0.8825-44.1242 μg / ml, deoxylognain was 5.7977-57.9768 μg / ml, luteolin was 0.8648-43.2425 μg / ml, isochlorogenic acid B was 20.9328-167.4624 μg / ml, isochlorogenic acid A was 8.1379-81.3792 μg / ml, isochlorogenic acid C was 11.0544~154.7616 μg / ml, luteolin was 0.2018~20.1845 μg / ml, and montmorillonine was 8.0978~80.9776 μg / ml.

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