A detection method for a feiyanxiao preparation

Abstract

The application belongs to the technical field of medicine detection, and particularly relates to a detection method of Fuyanxiao preparation, which is made of seven medicinal materials of Oxalis corniculata L., Patrinia, Radix Trichosanthis, Radix et Rhizoma Rhei, Cortex Moutan, Atractylodes and Liriodendron, and the detection method is to use HPLC method, acetonitrile as mobile phase A, 0.2% phosphoric acid aqueous solution as mobile phase B for gradient elution to determine the fingerprint and / or characteristic spectrum. The establishment of the detection method is beneficial to comprehensively monitor the product quality, and further helps to guarantee the medicine quality and clinical efficacy.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug detection, and particularly relates to a detection method of Fuyanxiao preparation. BACKGROUND

[0002] At present, gynecological inflammation is a big problem that plagues many female patients. If it is not taken seriously, it will not only lead to the aggravation of inflammation, but also cause a series of complications, and cause greater harm to the body. In the prior art, there are many drugs for treating gynecological inflammation, and Fuyanxiao capsule is an ideal drug among them. However, in the prior art, the content determination item in the quality control method of Fuyanxiao capsule only detects emodin, which is not comprehensive enough, and it is difficult to fully characterize their physical and chemical characteristics. Therefore, the comprehensive improvement of the quality control method of Fuyanxiao preparation has become an active research topic at present.

[0003] Traditional Chinese medicine fingerprint or characteristic spectrum refers to the spectrum of chromatography or spectroscopy of certain types or several types of components commonly shared and characteristic of certain traditional Chinese medicinal materials or Chinese patent medicines. At the present stage, since the effective components of traditional Chinese medicines are mostly not clear, the traditional Chinese medicine fingerprint has important significance for effectively controlling the quality of traditional Chinese medicinal materials or Chinese patent medicines. Japanese Han Fang medicines mainly produced by enterprises have adopted high-performance liquid chromatography fingerprint to control quality in the enterprise since the 1980s. Germany and France found in the joint development of ginkgo leaf extract that the medical effect of ginkgo leaf extract is the overall effect of the substance group obtained by extraction, and the quality control of this whole is also carried out by using high-performance liquid chromatography fingerprint. In recent years, the United States has also explicitly taken the fingerprint as a quality control method for a mixture group. With the in-depth research, people found that as the product of the practice of traditional Chinese medicine theory, traditional Chinese medicine, especially compound traditional Chinese medicine, cannot represent the overall curative effect of any component contained therein. People gradually realized that the current quality standard referring to the quality control mode of western medicine cannot properly reflect the internal quality of traditional Chinese medicine. From the prior quality control mode to a comprehensive, macroscopic and quantifiable identification combined with the determination of the content of main effective components is the development trend.

[0004] Fuyanxiao preparation is a pure traditional Chinese medicine preparation made of Oxalis corniculata L., Patrinia villosa Juss., Trichosanthes kirilowii Maxim., Rheum officinale Baill., Moutan Cortex, Atractylodes lancea (Thunb.) DC. and Lindera aggregata (Sims) Kosterm. according to the medicine habit of Miao nationality and the theory of traditional Chinese medicine, which has the effects of clearing heat and resolving toxin, promoting blood circulation and removing blood stasis, and removing dampness and stopping leucorrhea. It is clinically used for treating female genital inflammation, and its therapeutic effect has been verified clinically. Whether the quality of the medicine can be guaranteed and the content of the effective components in Fuyanxiao preparation is the basis for determining the therapeutic effect of Fuyanxiao preparation. If the internal quality of Fuyanxiao preparation is simply explained by emodin, it has certain one-sidedness. To control the quality of Fuyanxiao preparation, it is not enough to only characterize and control one or two chemical components, and the whole substance group must be controlled. Therefore, in addition to "microscopic analysis", a certain "macroscopic analysis" method should be used to effectively characterize the quality of traditional Chinese medicine as a whole. As a quality control method for Chinese herbal medicines and their extracts, fingerprint has become an international consensus. At present, there are many determination methods for active components such as emodin in emodin, but there is no report on how to establish a macro fingerprint or characteristic spectrum method from a more macroscopic point of view with chemical components in Fuyanxiao preparation as indicators. SUMMARY

[0005] The present application is directed to the deficiencies of the prior art, and provides a fingerprint and characteristic spectrum detection method for Fuyanxiao preparation, which is characterized by that the fingerprint and characteristic spectrum characterization is more comprehensive, objectively and truly reflects the types and quantities of the internal chemical components of the product, and has high similarity compared with the control. The fingerprint and characteristic spectrum of Fuyanxiao preparation are established for the first time, which improves the detection requirements of the quality of the product, so that the measurement results of the quality detection method of Fuyanxiao preparation are more accurate and reliable, the quality control of Fuyanxiao preparation is improved, which is helpful to establish a perfect quality control system of the product, and further helps to guarantee the quality of the medicine and the clinical efficacy.

[0006] Specifically, the present application is realized by the following technical solutions:

[0007] The present application provides a detection method for Fuyanxiao preparation, which is a solid preparation or liquid preparation made of Oxalis corniculata L., Patrinia villosa Juss., Trichosanthes kirilowii Maxim., Rheum officinale Baill., Moutan Cortex, Atractylodes lancea (Thunb.) DC. and Lindera aggregata (Sims) Kosterm. The detection method is to determine the fingerprint and / or characteristic spectrum by high performance liquid chromatography. The high performance liquid chromatography detection conditions are as follows: octadecylsilane bonded silica gel is used as the filler, acetonitrile or methanol is used as the mobile phase A, 0.1%-0.5% phosphoric acid aqueous solution is used as the mobile phase B for gradient elution, the column temperature is 30-40℃, the detection wavelength is 220-240nm, the flow rate is 0.8-1.2ml per minute, and the injection volume is 5-20µL. The gradient elution conditions are as follows:

[0008] 0-12min, mobile phase A 3%→12%, mobile phase B 97%→88%;

[0009] 12-30min, mobile phase A 12%→18%, mobile phase B 88%→82%;

[0010] 30-40min, mobile phase A 18%→20%, mobile phase B 82%→80%;

[0011] 40-52min, mobile phase A 20%→31%, mobile phase B 80%→69%;

[0012] 52-53min, mobile phase A 31%→35%, mobile phase B 69%→65%;

[0013] 53-60min, mobile phase A 35%→40%, mobile phase B 65%→60%;

[0014] 60-75min, mobile phase A 40%→55%, mobile phase B 60%→45%;

[0015] 75-90min, mobile phase A 55%→80%, mobile phase B 45%→20%;

[0016] 90-110min, mobile phase A 80%, mobile phase B 20%.

[0017] Preferably, for the above-mentioned detection method, the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid aqueous solution, the chromatographic column is GL Sciences Inertsil ODS-35um, the column temperature is 35°C, the detection wavelength is 230nm, the flow rate is 1ml / min, and the injection volume is 10µL.

[0018] Preferably, the solid preparation of Fuyanxiao is capsule, tablet, granule, or pill.

[0019] Further, the solid preparation of Fuyanxiao is prepared by extracting and concentrating raw medicinal materials in the following proportions: Oxalis corniculata 300 parts, Patrinia scabiosaefolia 300 parts, Trichosanthes kirilowii 250 parts, Rheum palmatum 300 parts, Moutan cortex 150 parts, Atractylodes lancea 250 parts, and Liriodendron chinense 300 parts, and adding one or more pharmaceutically acceptable excipients.

[0020] Preferably, the fingerprint detection method of the solid preparation of Fuyanxiao is to inject the test solution and the reference solution of the solid preparation of Fuyanxiao into a liquid chromatograph, respectively, to determine according to the high performance liquid chromatography working conditions, and to record the chromatogram.

[0021] The test sample solution of the Fuyanxiao solid preparation is prepared as follows: 0.15 g of the Fuyanxiao solid preparation is precisely weighed and placed in a conical flask with a stopper, 20 ml of 50% methanol is precisely added, the weight is determined again, ultrasonic treatment is performed for 10 minutes, the solution is allowed to cool, the weight is determined again, the weight loss is made up with 50% methanol, the solution is shaken uniformly, centrifuged, and the supernatant is taken, thereby obtaining the test sample solution of the Fuyanxiao solid preparation.

[0022] The reference solution is prepared as follows: a proper amount of gallic acid, protocatechuic acid, epicatechin, oroxin, isorhamnetin, paeonol, emodin, chrysophanol, physcion, aloe-emodin, rhein, emodin glucoside, chrysophanol glucoside, and physcion glucoside reference substances are precisely weighed and mixed in 50% methanol to prepare a mixed solution containing 0.1 mg of each reference substance per 1 ml.

[0023] Further, the test sample fingerprint of the Fuyanxiao solid preparation presents chromatographic peaks with the same retention time as the reference material chromatographic peaks, and the similarity between the test sample fingerprint and the control fingerprint is calculated by the Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity is not less than 0.90. The fingerprint is composed of 23 common peaks, wherein peak 1 is the gallic acid peak, peak 2 is the protocatechuic acid peak, peak 3 is the epicatechin peak, peak 11 is the isorhamnetin peak, peak 12 is the oroxin peak, peak 15 is the emodin glucoside peak, peak 16 is the chrysophanol glucoside peak, peak 17 is the physcion glucoside peak, peak 18 is the paeonol peak, peak 19 is the aloe-emodin peak, peak 20 is the rhein peak, peak 21 is the emodin peak, peak 22 is the chrysophanol peak, and peak 23 is the physcion peak.

[0024] More preferably, the characteristic spectrum detection method of the Fuyanxiao solid preparation is that the test sample solution and the reference solution of the Fuyanxiao solid preparation are respectively injected into a liquid chromatograph, the chromatogram is recorded by determining according to the high performance liquid chromatography working conditions.

[0025] The test sample solution of the Fuyanxiao solid preparation is prepared as follows: 0.15 g of the Fuyanxiao solid preparation is precisely weighed and placed in a conical flask with a stopper, 20 ml of 50% methanol is precisely added, the weight is determined again, ultrasonic treatment is performed for 10 minutes, the solution is allowed to cool, the weight is determined again, the weight loss is made up with 50% methanol, the solution is shaken uniformly, centrifuged, and the supernatant is taken, thereby obtaining the test sample solution of the Fuyanxiao solid preparation.

[0026] The reference solution is prepared as follows: a proper amount of gallic acid, protocatechuic acid, epicatechin, oroxin, isorhamnetin, paeonol, emodin, chrysophanol, physcion, aloe-emodin, rhein, emodin glucoside, chrysophanol glucoside, and physcion glucoside reference substances are precisely weighed and mixed in 50% methanol to prepare a mixed solution containing 0.1 mg of each reference substance per 1 ml.

[0027] Further, the characteristic spectrum of the Fuyanxiao solid preparation, the characteristic spectrum of the test sample should have 21 characteristic peaks, except for peaks 3 and 9, the variation range of the relative peak area of each characteristic peak is not more than 2 times, taking the characteristic peak of gallic acid as the S peak, the relative retention time of each characteristic peak to the S peak should be within ±10% of the specified value; the specified value of the relative retention time of each characteristic peak is 1.00 for peak S, 1.73 for peak 2, 2.23 for peak 3, 2.27 for peak 4, 3.11 for peak 5, 3.21 for peak 6, 3.45 for peak 7, 3.83 for peak 8, 4.25 for peak 9, 4.39 for peak 10, 4.50 for peak 11, 6.31 for peak 12, 6.39 for peak 13, 6.75 for peak 14, 6.96 for peak 15, 7.28 for peak 16, 7.70 for peak 17, 8.00 for peak 18, 9.05 for peak 19, 9.91 for peak 20, and 10.27 for peak 21.

[0028] Secondly, the Fuyanxiao liquid preparation is Fuyanxiao standard decoction.

[0029] The preparation method of the Fuyanxiao standard decoction is as follows: 15 g of Galium verum, 15 g of Patrinia, 12.5 g of Trichosanthes, 7.5 g of Radix et Rhizoma Rhei, 7.5 g of Radix Paeoniae Alba, 12.5 g of Atractylodes, and 15 g of Laurus are weighed, 8 times the amount of water is added, and decoction is performed twice, each time for 2 hours, to obtain a first decoction and a second decoction. 100 ml of the first decoction and 100 ml of the second decoction are mixed to obtain the Fuyanxiao standard decoction.

[0030] Preferably, the detection method of the Fuyanxiao standard decoction fingerprint is as follows: the test sample solution and the reference solution of the Fuyanxiao standard decoction are injected into a liquid chromatograph, the chromatogram is recorded by determining according to the high performance liquid chromatography working conditions.

[0031] The preparation of the test sample solution is as follows: 5 ml of the Fuyanxiao standard decoction is accurately measured and placed in a conical flask with a stopper, 5 ml of methanol is accurately added, the weight is determined, ultrasonic treatment is performed for 10 minutes, it is cooled, the weight is determined again, 50% methanol is added to make up for the weight loss, it is shaken uniformly, centrifuged, and the supernatant is obtained to obtain the test sample solution of the Fuyanxiao standard decoction.

[0032] The preparation of the reference solution is as follows: appropriate amounts of gallic acid, protocatechuic acid, epicatechin, isorhamnetin, paeonol, emodin, chrysophanol, emodin methyl ether, aloe emodin, and rhein reference substances are accurately weighed and determined, and methanol is added to prepare a mixed solution containing 0.1 mg of each per 1 ml.

[0033] Further, the reference fingerprint of the Fuyanxiao standard decoction has the same chromatographic peaks as the test sample, and the similarity of the test sample fingerprint and the control fingerprint is not less than 0.80 according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint; the fingerprint is composed of 22 common peaks, wherein peak 1 is gallic acid peak, peak 2 is protocatechuic acid peak, peak 3 is epicatechin peak, peak 12 is isodulcoside peak, peak 17 is paeonol peak, peak 18 is aloe emodin peak, peak 19 is rhein peak, peak 20 is emodin peak, peak 21 is chrysophanol peak, and peak 22 is chrysophanol peak.

[0034] More preferably, the detection method of the characteristic spectrum of the Fuyanxiao standard decoction is to inject the test sample solution and the reference solution of the Fuyanxiao standard decoction into the liquid chromatograph respectively, and determine according to the working conditions of high performance liquid chromatography, and record the chromatogram.

[0035] The test sample solution is prepared as follows: 5 ml of the Fuyanxiao standard decoction is accurately measured and placed in a conical flask with a plug, 5 ml of methanol is accurately added, the weight is determined, ultrasonic treatment is performed for 10 minutes, it is cooled and weighed again, 50% methanol is added to make up for the weight loss, it is shaken uniformly, centrifuged, and the supernatant is obtained, which is the test sample solution of the Fuyanxiao standard decoction.

[0036] The reference solution is prepared as follows: appropriate amounts of gallic acid, protocatechuic acid, epicatechin, isodulcoside, paeonol, emodin, chrysophanol, chrysophanol, aloe emodin, and rhein reference substances are accurately weighed and added to methanol to prepare a mixed solution containing 0.1 mg per 1 ml.

[0037] Further, the characteristic spectrum of the Fuyanxiao standard decoction has 15 characteristic peaks, and the variation range of the relative peak area of each characteristic peak is not more than 2 times, with the characteristic peak of gallic acid as the S peak, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified value of the relative retention time of each characteristic peak is 1.00 for peak S, 1.67 for peak 2, 2.03 for peak 3, 2.12 for peak 4, 2.15 for peak 5, 3.09 for peak 6, 3.22 for peak 7, 3.99 for peak 8, 4.26 for peak 9, 4.46 for peak 10, 6.27 for peak 11, 6.31 for peak 12, 6.39 for peak 13, 6.80 for peak 14, and 7.18 for peak 15.

[0038] Beneficial effects:

[0039] 1. The present application adopts HPLC to characterize the fingerprint and / or characteristic spectrum of Fuyanxiao preparation. In particular, standard control fingerprint spectrum is separately established for Fuyanxiao solid preparation and Fuyanxiao standard decoction, and similarity standards are respectively established, wherein 23 common peaks are calibrated for Fuyanxiao solid preparation, and comparison is made by combining with the chromatogram of reference solution, and 14 common peaks are determined, which are: gallic acid peak, protocatechuic acid peak, epicatechin peak, isobrucianol peak, oroxylin peak, rhein glucoside peak, chrysophanol glucoside peak, rheochrysidin glucoside peak, paeonol peak, aloe-emodin peak, rheic acid peak, rhein peak, chrysophanol peak, rheochrysidin peak; 22 common peaks are calibrated for Fuyanxiao standard decoction, and comparison is made by combining with the chromatogram of reference solution, and 10 common peaks are determined, which are: gallic acid peak, protocatechuic acid peak, epicatechin peak, isobrucianol peak, paeonol peak, aloe-emodin peak, rheic acid peak, rhein peak, chrysophanol peak, rheochrysidin peak; in addition, 21 characteristic peaks are specified for the characteristic spectrum of Fuyanxiao solid preparation, and it is stipulated that the variation range of the relative peak area of each characteristic peak is not more than 2 times except for peaks 3 and 9, the characteristic peak of gallic acid is taken as S peak, and the relative retention time of each characteristic peak to S peak is calculated, which should be within ±10% of the stipulated value. 15 characteristic peaks are specified for the characteristic spectrum of Fuyanxiao standard decoction, and it is stipulated that the variation range of the relative peak area of each characteristic peak is not more than 2 times, the characteristic peak of gallic acid is taken as S peak, and the relative retention time of each characteristic peak to S peak is calculated, which should be within ±10% of the stipulated value.

[0040] 2. The method of the present application is simple to operate and has high durability, and further indicates the stability of the process and product quality of Fuyanxiao preparation through the fingerprint and characteristic spectrum of multiple batches of samples, which is more suitable for the quality control of Fuyanxiao preparation compared with the current quality standard, and the method is accurate and effective.

[0041] 3. The method of the present application is accurate and effective, has high precision, good reproducibility and excellent stability. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is the control fingerprint spectrum of Fuyanxiao capsules, wherein peak 1 is gallic acid, peak 2 is protocatechuic acid, peak 3 is epicatechin, peak 11 is isobrucianol, peak 12 is oroxylin, peak 15 is rhein glucoside, peak 16 is chrysophanol glucoside, peak 17 is rheochrysidin glucoside, peak 18 is paeonol, peak 19 is aloe-emodin, peak 20 is rheic acid, peak 21 is rhein, peak 22 is chrysophanol, and peak 23 is rheochrysidin

[0043] Figure 2 is the specific chromatogram of the fingerprint spectrum of Fuyanxiao capsules

[0044] Figure 3are 13 batches of Fuyanxiao capsules fingerprint (S1-S13) and R control fingerprint

[0045] Figure 4 are different mobile phase investigation chromatogram, wherein S1 is methanol-0.1% phosphoric acid, S2 is acetonitrile-0.1% phosphoric acid, S3 is acetonitrile-0.2% phosphoric acid, S4 is acetonitrile-1% ice acetic acid, S5 is acetonitrile-1% formic acid

[0046] Figure 5 are Fuyanxiao standard decoction control fingerprint, wherein peak 1 is gallic acid, peak 2 is protocatechuic acid, peak 3 is epicatechin, peak 12 is isorhamnetin, peak 17 is paeonol, peak 18 is aloe emodin, peak 19 is rhein, peak 20 is emodin, peak 21 is chrysophanol, peak 22 is emodin methyl ether

[0047] Figure 6 are Fuyanxiao capsule content characteristic map, peak 1 is gallic acid, peak 2 is protocatechuic acid, peak 3 is epicatechin, peak 9 is isorhamnetin, peak 10 is swertisin, peak 13 is chrysophanol glucoside, peak 16 is paeonol, peak 17 is aloe emodin, peak 18 is rhein, peak 19 is emodin, peak 20 is chrysophanol, peak 21 is emodin methyl ether

[0048] Figure 7 are Fuyanxiao standard decoction characteristic map, peak 1 is gallic acid, peak 2 is protocatechuic acid, peak 3 is epicatechin, peak 8 is isorhamnetin, peak 13 is chrysophanol glucoside, peak 15 is paeonol

[0049] Figure 8 are the main component score maps of 13 batches of Fuyanxiao capsules

[0050] Figure 9 are Fuyanxiao capsule PLS-DA score scatter diagram

[0051] Figure 10 are the VIP value maps of common peaks of 13 batches of Fuyanxiao capsule contents DETAILED DESCRIPTION

[0052] The application provides a fingerprint and / or characteristic map detection method of Fuyanxiao preparation, wherein the Fuyanxiao preparation comprises Fuyanxiao solid preparation and Fuyanxiao liquid preparation, the Fuyanxiao solid preparation is capsule, tablet, granule preparation or pill; and the Fuyanxiao liquid preparation is Fuyanxiao standard decoction.

[0053] Further, the Nvyanxiao solid preparation is prepared by extracting and concentrating 300 parts of Herba Galiums, 300 parts of Herba Patriniae, 250 parts of Radix Trichosanthis, 300 parts of Radix et Rhizoma Rhei, 150 parts of Cortex Moutan, 250 parts of Radix et Rhizoma Atractylodis, and 300 parts of Lignum Cinnamomi, and then adding one or more pharmaceutically acceptable adjuvants, wherein the Nvyanxiao capsule can be prepared by the method disclosed in CN1413694A or CN111714568A, and the tablet, granules, and pills can be prepared by the method disclosed in CN1733095A.

[0054] The preparation method of the Nvyanxiao standard decoction is as follows: 15 g of Herba Galiums, 15 g of Herba Patriniae, 12.5 g of Radix Trichosanthis, 7.5 g of Radix et Rhizoma Rhei, 7.5 g of Cortex Moutan, 12.5 g of Radix et Rhizoma Atractylodis, and 15 g of Lignum Cinnamomi are weighed, 8 times the amount of water is added, and decoction is performed twice, each time for 2 h, to obtain a first decoction liquid and a second decoction liquid; 100 ml of the first decoction liquid and 100 ml of the second decoction liquid are mixed to obtain the Nvyanxiao standard decoction.

[0055] In the prior art, the WS-10265 (ZD-265) 2002 national drug standard discloses a detection method for the Nvyanxiao capsule, but only involves thin-layer identification of paeoniflorin and content determination of emodin, and a perfect fingerprint spectrum has not been established.

[0056] Therefore, the present application tries to find a more suitable fingerprint spectrum and / or characteristic spectrum characterization method for the Nvyanxiao preparation, so that the spectrum reflects rich information, more common peaks, and clear sources of component confirmation and common peaks.

[0057] Example 1: Fingerprint spectrum detection method of Nvyanxiao capsule

[0058] Determination by high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition four general rules 0512).

[0059] 1. Chromatographic conditions

[0060] Instrument model: water se2695 high performance liquid chromatograph, GL Sciences Inertsil ODS-35um (250mm×4.6mm) is used as the chromatographic column, acetonitrile is used as the mobile phase A, and 0.2% phosphoric acid solution is used as the mobile phase B, elution is performed according to the gradient in Table 1 below, the column temperature is 35°C, the flow rate is 1 ml / min, the injection amount is 10 µL, and the detection wavelength is 230 nm.

[0061] Table 1 Elution gradient

[0062]

[0063] 2. Preparation of reference solution

[0064] Take gallic acid, protocatechuic acid, epicatechin, oroxylin, isorhamnetin, paeonol, emodin, chrysophanol, physcion, aloe-emodin, rhein, emodin glucoside, chrysophanol glucoside, physcion glucoside reference substance, accurately weigh, add methanol to prepare a mixed solution containing 0.1 mg per 1 ml.

[0065] 3. Preparation of test solution

[0066] Take 0.15 g of the content of Fuyanxiao capsules, accurately weigh, place in a conical flask with a plug, accurately add 20 ml of 50% methanol, weigh, ultrasonically treat for 10 minutes, cool, re-weigh, make up the weight loss with 50% methanol, shake well, centrifuge (13000 r, 20 min), take the supernatant and filter through a 0.22 μm microporous filter, take the filtrate, and obtain.

[0067] 4. Determination method

[0068] Take 10 μl of each of the reference solution and the test solution, inject into the liquid chromatograph, determine, and record the chromatogram.

[0069] The control fingerprint of Fuyanxiao capsules is shown in Figure 1 .

[0070] The test fingerprint presents chromatographic peaks with the same retention time as the reference chromatographic peaks, the similarity of the test fingerprint and the control fingerprint is not less than 0.90 according to the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, and the fingerprint is composed of 23 common peaks, wherein peak 1 is the gallic acid peak, peak 2 is the protocatechuic acid peak, peak 3 is the epicatechin peak, peak 11 is the isorhamnetin peak, peak 12 is the oroxylin peak, peak 15 is the emodin glucoside peak, peak 16 is the chrysophanol glucoside peak, peak 17 is the physcion glucoside peak, peak 18 is the paeonol peak, peak 19 is the aloe-emodin peak, peak 20 is the rhein peak, peak 21 is the emodin peak, peak 22 is the chrysophanol peak, and peak 23 is the physcion peak.

[0071] Example 2: Methodological verification of the detection method of the fingerprint of Fuyanxiao capsules

[0072] In order to confirm the practicability and accuracy of the detection method of the present application, the specificity, precision, solution stability, repeatability and the like of the detection method are described as follows:

[0073] 1. Specificity test

[0074] Take the test solution of Fuyanxiao capsules prepared according to the test solution preparation method of Example 1, take the test solution and the blank solvent (methanol), inject into the chromatograph according to the chromatographic conditions of Example 1, determine, and record the chromatogram. Figure 2The results show that the blank solvent does not interfere with the determination, indicating that the method has good specificity.

[0075] 2. Precision test

[0076] The test sample solution of Fuyanxiao Capsules was prepared according to the preparation method of test sample solution in Example 1, and was injected for determination according to the chromatographic conditions in Example 1 for 6 times continuously. With Peak 1 (gallic acid) as the reference peak, the RSDs of the retention time and relative peak area of each common peak were calculated, and the results are shown in Table 2. The results show that the RSDs of the retention time of each common peak are all less than 1.0%, and the RSDs of the relative peak area are all less than 5.0%, indicating that the precision of the instrument is good.

[0077] Table 2. Results of precision test.

[0078]

[0079] 3. Stability test

[0080] The test sample solution of Fuyanxiao Capsules was prepared according to the preparation method of test sample solution in Example 1, and was injected for determination according to the chromatographic conditions in Example 1 at 0, 3, 6, 9, 12 and 24 h after sample preparation. With Peak 1 (gallic acid) as the reference peak, the RSDs of the retention time and relative peak area of each common peak were calculated, and the results show that the RSDs of the retention time of each common peak are all less than 1.0%, and the RSDs of the relative peak area are all less than 5.0%, indicating that the sample is stable within 24 h.

[0081] 4. Reproducibility test

[0082] The test sample solution of Fuyanxiao Capsules was prepared according to the preparation method of test sample solution in Example 1, and was injected for determination according to the chromatographic conditions in Example 1. With Peak 1 (gallic acid) as the reference peak, the RSDs of the retention time and relative peak area of each common peak were calculated, and the results show that the RSDs of the retention time of each common peak are all less than 1.0%, and the RSDs of the relative peak area are all less than 5.0%, indicating that the reproducibility of the method is good.

[0083] 5. Establishment of Fuyanxiao fingerprint spectrum

[0084] Thirteen batches of Fuyanxiao Capsules were prepared into test sample solutions according to the preparation method of test sample solution in Example 1, and were injected for fingerprint spectrum detection according to the chromatographic conditions in Example 1. The related results were calculated by using the fingerprint spectrum similarity evaluation software, and the control fingerprint spectrum was generated (see Figure 1 , Figure 3 ). According to the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, the similarity of the test sample fingerprint spectrum and the control fingerprint spectrum was calculated, and the similarity was all above 0.90, the reproducibility of each batch was good, and the preparation process was stable.

[0085] 6. Reference peak identification study

[0086] The mixed reference solution and each single reference solution (0.1 mg / ml) were prepared according to the reference solution preparation method under "Example 1", and analyzed by injection under the chromatographic conditions of "Example 1" to identify the corresponding peaks in the fingerprint. The results showed that under this chromatographic condition: peak 1 was the gallic acid peak, peak 2 was the protocatechuic acid peak, peak 3 was the epicatechin peak, peak 11 was the isorhamnetin peak, peak 12 was the wogonin peak, peak 15 was the physcion glucoside peak, peak 16 was the chrysophanol glucoside peak, peak 17 was the emodin methyl ether glucoside peak, peak 18 was the paeonol peak, peak 19 was the aloe-emodin peak, peak 20 was the rhein peak, peak 21 was the emodin peak, peak 22 was the chrysophanol peak, and peak 23 was the emodin methyl ether peak.

[0087] In addition, the test sample extraction method and chromatographic working conditions were also compared and studied in this study.

[0088] 1. Preparation method of test sample solution

[0089] Method one: 0.15 g of the content of Fuyanxiao capsules was accurately weighed and placed in a conical flask with a stopper. 20 ml of methanol was accurately added, the weight was determined, and ultrasonic treatment was performed for 30 minutes. After cooling, the weight was determined again, the reduced weight was made up with methanol, shaken well, centrifuged (13000 r, 20 min), the supernatant was filtered with a 0.22 µm microporous filter, and the filtrate was obtained.

[0090] Method two: 0.15 g of the content of Fuyanxiao capsules was accurately weighed and placed in a conical flask with a stopper. 20 ml of 50% methanol was accurately added, the weight was determined, and ultrasonic treatment was performed for 10 minutes. After cooling, the weight was determined again, the reduced weight was made up with 50% methanol, shaken well, centrifuged (13000 r, 20 min), the supernatant was filtered with a 0.22 µm microporous filter, and the filtrate was obtained.

[0091] Method three: 0.15 g of the content of Fuyanxiao capsules was accurately weighed and placed in a conical flask with a stopper. 20 ml of 75% methanol was accurately added, the weight was determined, and heating reflux was performed for 30 minutes. After cooling, the weight was determined again, the reduced weight was made up with 75% methanol, shaken well, centrifuged (13000 r, 20 min), the supernatant was filtered with a 0.22 µm microporous filter, and the filtrate was obtained.

[0092] Method four: 0.15 g of the content of Fuyanxiao capsules was precisely weighed and placed in a conical flask with a stopper. 20 ml of 25% methanol was precisely added, the weight was determined, and ultrasonic treatment was performed for 20 minutes. After cooling, the weight was determined again, and the reduced weight was made up with 25% methanol. After shaking and centrifugation (13000 r, 20 min), the supernatant was filtered through a 0.22 μm microporous filter, and the filtrate was obtained.

[0093] The results show that the reflux extraction of method three and the ultrasonic extraction have similar chromatographic peaks, but the ultrasonic extraction is simple to operate, so ultrasonic extraction is selected. Comparison of the three ultrasonic extraction methods shows that method two uses 50% methanol for ultrasonic extraction for the shortest time, but the extraction effect is the best, which can retain more index components to a large extent. Therefore, method two is finally determined as the preparation method of the test sample.

[0094] 2. Chromatographic condition research:

[0095] 2.1 Selection of detection wavelength: The test sample solution was scanned at 190-400 nm by DAD detector, and the chromatograms at different wavelengths were investigated. The proportion between the peaks in the chromatogram at 220-240 nm was coordinated, and the effect was better. Especially, the chromatogram at 230 nm provided the largest amount of information, so 230 nm was selected as the detection wavelength of the fingerprint.

[0096] 2.2 Selection of chromatographic column

[0097] In order to make the chemical components of Fuyanxiao capsules achieve the best separation effect, GL Sciences Inertsil ODS-35 μm (250 mm x 4.6 mm) and ZORBAX Eclipse Plus C18 4.6 x 250 nm (Agilent) chromatographic columns were compared for their separation ability of the test sample solution of Fuyanxiao capsules. The results showed that the GL Sciences Inertsil ODS chromatographic column had stronger retention ability and better peak shape for each component, so the GL Sciences Inertsil ODS chromatographic column was selected.

[0098] 2.3 Column temperature investigation

[0099] 30, 35 and 40 ℃ were selected for investigation, and the results showed that the chromatographic peaks were faster under the condition of 40 ℃, and the characteristic peaks could not achieve good separation effect. At 30 ℃, the peaks were slower, and the column temperature was finally determined to be 35 ℃.

[0100] 2.4 Investigation of mobile phase

[0101] (1) Methanol-water and acetonitrile-water were compared, and the baseline was relatively shifted. The addition of acid to the mobile phase was attempted for determination.

[0102] (2) Respectively using methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, acetonitrile-0.2% phosphoric acid, acetonitrile-1% glacial acetic acid, acetonitrile-1% formic acid as mobile phase, the chromatogram obtained is shown in Figure 4 . It can be seen from Figure 4 that: the mobile phase with relatively good resolution is acetonitrile-0.2% phosphoric acid.

[0103] 2.5 Investigation of flow rate

[0104] 0.8ml / min, 1ml / min, 1.2ml / min were investigated respectively. The results showed that when the flow rate was 1.0ml / min, the peak shape and separation were the best.

[0105] Example 3: Fuyanxiao standard decoction fingerprint detection method

[0106] Preparation of Fuyanxiao standard decoction: according to the existing prescription and extraction method of Fuyanxiao capsule standard process, the prescription amount is 1 / 20 of the process prescription, and 15g of Eutrema japonicum, 15g of Herba Patriniae, 12.5g of Radix Bupleuri, 7.5g of Radix et Rhizoma Rhei, 7.5g of Cortex Moutan, 12.5g of Atractylodes, and 15g of Linaloe are added. 8 times the amount of water, decoct twice, each time for 2h, to obtain a decoction and a second decoction. Because the water amount of the two decoctions is the same, the water absorption rate of the first decocted medicinal materials is eliminated, and theoretically the decoction amount obtained twice is consistent. Take 100ml of the first decoction and 100ml of the second decoction, mix to obtain the standard decoction, and get the Fuyanxiao standard decoction.

[0107] Fingerprint detection method of Fuyanxiao standard decoction: determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition four chapters 0512).

[0108] Chromatographic conditions

[0109] Instrument model: water se2695 high performance liquid phase, chromatographic column uses GL Sciences Inertsil ODS-35um (250mm×4.6mm), with acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B, eluted according to the gradient in table 3, column temperature is 35℃; flow rate 1ml / min, sample size 10µL, detection wavelength is 230nm.

[0110] Table 3 elution gradient

[0111]

[0112] 2. Preparation of reference solution

[0113] Take gallic acid, protocatechuic acid, epicatechin, isorhamnetin, paeonol, emodin, chrysophanol, physcion, aloe emodin, rhein reference substance, accurately weighed, add methanol to prepare a mixed solution containing 0.1 mg of each per 1 ml.

[0114] Preparation of test sample solution

[0115] Take 5 ml of Fuyanxiao standard decoction, accurately measure, and place it in a conical flask with a plug. Accurately add 5 ml of methanol, weigh, ultrasonic treat for 10 minutes, cool, weigh again, make up the weight loss with 50% methanol, shake well, centrifuge (13000r, 20min), take the supernatant, and Fuyanxiao standard decoction test sample solution is obtained.

[0116] 4. Determination method

[0117] Accurately take 10 μl of the reference solution and the test sample solution respectively, inject into the liquid chromatograph, determine, and record the chromatogram.

[0118] The control fingerprint of Fuyanxiao standard decoction is shown in Figure 5 .

[0119] The test sample fingerprint presents chromatographic peaks with the same retention time as the reference material chromatographic peaks. According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity of the test sample fingerprint and the control fingerprint should not be less than 0.80; the fingerprint is composed of 22 common peaks, peak 1 is gallic acid peak, peak 2 is protocatechuic acid peak, peak 3 is epicatechin peak, peak 12 is isorhamnetin peak, peak 17 is paeonol peak, peak 18 is aloe emodin peak, peak 19 is rhein peak, peak 20 is emodin peak, peak 21 is chrysophanol peak, and peak 22 is physcion peak.

[0120] Example 4: Methodological verification of the detection method of Fuyanxiao standard decoction fingerprint

[0121] In order to confirm the practicability and accuracy of the detection method of the present application, the specificity, precision, solution stability, repeatability and other aspects of the detection method are described as follows:

[0122] 1. Specificity test

[0123] Take the standard decoction test sample and the blank solvent (methanol), and determine by injecting sample under the chromatographic conditions in item “2.1”, and record the chromatogram. The results show that the blank solvent does not interfere with the determination, indicating that the method has good specificity.

[0124] The standard decoction of Fuyanxiao was prepared according to the preparation method of sample solution in Example 3. The sample solution, blank solvent (methanol) were injected and determined according to the chromatographic conditions in Example 3, and the chromatogram was recorded. The results showed that the blank solvent did not interfere with the determination, indicating that the method had good specificity.

[0125] 2. Precision

[0126] The same standard decoction of Fuyanxiao was prepared according to the preparation method of sample solution in Example 3. The sample solution was injected and determined according to the chromatographic conditions in Example 3 for 6 times in succession. Taking peak No. 1 (gallic acid) as the reference peak, the RSD of the retention time and relative peak area of each common peak was calculated. The results showed that the RSD of the retention time of each common peak was less than 1.0%, and the RSD of the relative peak area was less than 5.0%, indicating that the precision of the instrument was good.

[0127] 3. Reproducibility

[0128] The same standard decoction of Fuyanxiao was prepared according to the preparation method of sample solution in Example 3. Six sample solutions of Fuyanxiao standard decoction were prepared in parallel, and were injected and determined according to the chromatographic conditions in Example 3. Taking peak No. 1 (gallic acid) as the reference peak, the RSD of the retention time and relative peak area of each common peak was calculated. The results showed that the RSD of the retention time of each common peak was less than 1.0%, and the RSD of the relative peak area was less than 5.0%, indicating that the reproducibility of the method was good.

[0129] 4. Stability

[0130] The same standard decoction of Fuyanxiao was prepared according to the preparation method of sample solution in Example 3. The sample solution was injected and determined according to the chromatographic conditions in Example 3 at 0, 3, 6, 9, 12, and 24 h after sample preparation. Taking peak No. 1 (gallic acid) as the reference peak, the RSD of the retention time and relative peak area of each common peak was calculated. The results showed that the RSD of the retention time of each common peak was less than 1.0%, and the RSD of the relative peak area was less than 5.0%, indicating that the sample was stable within 24 h.

[0131] 5. Establishment of standard decoction fingerprint of Fuyanxiao

[0132] Twelve batches of standard decoction of Fuyanxiao were prepared according to the preparation method of sample solution in Example 3. The fingerprint chromatograms of the unit medicine, negative solution, and standard decoction were imported into the 2012 edition of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" in the format of cdf, and were evaluated. Taking S1 as the reference spectrum, the median, time window width of 0.1, and multi-point correction were used for full peak matching. The system generated a control spectrum R according to the common mode of the chromatograms of multiple batches of samples, as shown in Figure 5The similarity of the fingerprint spectrum and the reference spectrum is shown in Table 4. The similarity is above 0.80, the repeatability of each batch is good, and the preparation process is stable.

[0133] Table 4 Similarity table of 12 batches of Fuyanxiao standard decoction

[0134]

[0135] 6. Chromatographic peak identification and attribution

[0136] Fuyanxiao standard decoction has 22 common peaks at 230 nm wavelength. Through comparison with each unit drug test solution, negative solution and mixed reference solution spectrum, peak 1 is gallic acid, peak 2 is protocatechuic acid, peak 3 is epicatechin, peak 4 is attributed to atractylodes, magnolia, peak 5 is attributed to rhubarb, atractylodes; peak 6 is attributed to peony, magnolia; peaks 8 and 9 are oxalis, peak 10 is baejiangcao, peak 11 is baejiangcao, peak 12 is isoduplin, peak 7 and 13 are peony, peaks 14 and 15 are attributed to rhubarb, peak 16 is peony, peak 17 is danpi phenol, peak 18 is aloe emodin, peak 19 is rhein, peak 20 is emodin, peak 21 is chrysophanol, and peak 22 is emodin methyl ether.

[0137] Example 5: Fingerprint spectrum detection method of Fuyanxiao tablets

[0138] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition four general rules 0512).

[0139] 1. Chromatographic conditions

[0140] Instrument model: water se2695 high performance liquid chromatograph, chromatographic column uses GL Sciences Inertsil ODS-35um (250mm×4.6mm), with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, eluted according to the gradient in the following table 5, column temperature is 35℃; flow rate is 1ml / min, sample amount is 10µL, and detection wavelength is 230nm.

[0141] Table 5 Elution gradient

[0142]

[0143] 2. Preparation of reference solution

[0144] Take gallic acid, protocatechuic acid, epicatechin, oroxylin, isoduplin, danpi phenol, emodin, chrysophanol, emodin methyl ether, aloe emodin, rhein, emodin glucoside, chrysophanol glucoside, emodin methyl ether glucoside reference substance, accurately weigh, add methanol to prepare a mixed solution containing 0.1mg per 1ml.

[0145] 3. Preparation of test solution

[0146] Take 20 pieces of Fuyancao tablets and grind them into powder. Weigh 0.15 g of the powder and accurately place it in a conical flask with a stopper. Precisely add 20 ml of 50% methanol, and accurately weigh it. Ultrasonic treat for 10 minutes, cool, and re-weigh. Make up the weight loss with 50% methanol, shake well, and centrifuge (13000 r, 20 min). Take the supernatant and filter it through a 0.22 µm microporous filter membrane. Take the filtrate, and we have obtained it.

[0147] 4. Determination method

[0148] Take 10 µl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine and record the chromatogram.

[0149] The test fingerprint shows chromatographic peaks with the same retention time as the reference chromatographic peaks. According to the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, the similarity between the test fingerprint and the control fingerprint ( Figure 1 ) is not less than 0.90. The fingerprint is composed of 23 common peaks, of which peak 1 is gallic acid, peak 2 is protocatechuic acid, peak 3 is epicatechin, peak 11 is isorhamnetin, peak 12 is daphnetin, peak 15 is emodin glucoside, peak 16 is chrysophanol glucoside, peak 17 is physcion glucoside, peak 18 is paeonol, peak 19 is aloe-emodin, peak 20 is rhein, peak 21 is emodin, peak 22 is chrysophanol, and peak 23 is physcion.

[0150] Example 6: Detection method of Fuyancao capsule characteristic spectrum

[0151] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition four chapters 0512).

[0152] 1. Chromatographic conditions

[0153] Instrument model: water se2695 high performance liquid chromatograph, chromatographic column uses GL Sciences Inertsil ODS-35um (250mm×4.6mm), with acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B, eluted according to the gradient in the following table 6, column temperature is 35℃; flow rate 1ml / min, injection volume 10µL, detection wavelength is 230nm.

[0154] Table 6 Elution gradient

[0155]

[0156] 2. Preparation of reference solution

[0157] Take gallic acid, protocatechuic acid, epicatechin, oroxylin, isorhamnetin, paeonol, emodin, chrysophanol, physcion, aloe emodin, rhein, emodin glucoside, chrysophanol glucoside, physcion glucoside reference substance, accurately weigh, add methanol to prepare a mixed solution containing 0.1 mg per 1 ml.

[0158] 3. Preparation of test sample

[0159] Take 0.15 g of Fuyanxiao capsule content, accurately weigh, place in a conical flask with a plug, accurately add 20 ml of 50% methanol, weigh, ultrasonic treat for 10 minutes, cool, weigh again, make up the weight loss with 50% methanol, shake well, centrifuge (13000 r, 20 min), take the supernatant and filter through a 0.22 μm microporous filter membrane, take the filtrate, and obtain.

[0160] 4. Assay method

[0161] Take 10 μl of reference solution and test sample solution respectively, inject into the liquid chromatograph, determine, record the chromatogram (Figure 1), and obtain. Figure 6

[0162] There are 21 characteristic peaks in the test sample characteristic chromatogram, except peaks 3 and 9, the change range of the relative peak area of each characteristic peak is not more than 2 times, take the characteristic peak of gallic acid as the S peak, calculate the relative retention time of each characteristic peak to the S peak, and the relative retention time is within ±10% of the specified value; the specified value of the relative retention time of each characteristic peak is 1.00 for peak S, 1.73 for peak 2, 2.23 for peak 3, 2.27 for peak 4, 3.11 for peak 5, 3.21 for peak 6, 3.45 for peak 7, 3.83 for peak 8, 4.25 for peak 9, 4.39 for peak 10, 4.50 for peak 11, 6.31 for peak 12, 6.39 for peak 13, 6.75 for peak 14, 6.96 for peak 15, 7.28 for peak 16, 7.70 for peak 17, 8.00 for peak 18, 9.05 for peak 19, 9.91 for peak 20, and 10.27 for peak 21.

[0163] Example 7: Methodology verification of Fuyanxiao capsule characteristic chromatogram detection method

[0164] In order to confirm the practicability and accuracy of the detection method of the present application, the specificity, precision, solution stability, repeatability and other aspects of the detection method are described as follows:

[0165] 1. Specificity test

[0166] ​Fu Yanxiao Capsules test sample solution was prepared according to the preparation method of test sample solution in "Example 6", and the test sample solution, blank solvent (methanol) were injected according to the chromatographic conditions in "Example 6" for determination, and the chromatogram was recorded. The results showed that the blank solvent did not interfere with the determination, indicating that the method had good specificity.

[0167] 2. Precision test

[0168] Fu Yanxiao Capsules test sample solution was prepared according to the preparation method of test sample solution in "Example 6", and the test sample solution, blank solvent (methanol) were injected according to the chromatographic conditions in "Example 6" for determination, and the chromatogram was recorded. The results showed that the blank solvent did not interfere with the determination, indicating that the method had good specificity.

[0169] Table 7 Precision test results table

[0170]

[0171] 3. Stability test

[0172] Fu Yanxiao Capsules test sample solution was prepared according to the preparation method of test sample solution in "Example 6", and the test sample solution, blank solvent (methanol) were injected according to the chromatographic conditions in "Example 6" for determination, and the chromatogram was recorded. The results showed that the blank solvent did not interfere with the determination, indicating that the method had good specificity.

[0173] Table 8 Stability test results table

[0174]

[0175] 4. Reproducibility test

[0176] Fu Yanxiao Capsules test sample solution was prepared according to the preparation method of test sample solution in "Example 6", and the test sample solution, blank solvent (methanol) were injected according to the chromatographic conditions in "Example 6" for determination, and the chromatogram was recorded. The results showed that the blank solvent did not interfere with the determination, indicating that the method had good specificity.

[0177] Table 9 Reproducibility test results table

[0178]

[0179] 5. Establishment of characteristic chromatogram of Fuyanxiao Capsules

[0180] Thirteen batches of Fuyanxiao Capsules were prepared into test sample solutions according to the preparation method of test sample solution in Example 6, and were injected for determination under the chromatographic conditions in Example 6. The peaks corresponding to the reference substance of gallic acid with better separation and moderate peak area were taken as peaks to calculate the retention time and change amplitude of each characteristic peak and S peak. The results are shown in Table 10.

[0181] Table 10 Change amplitude of retention time of characteristic chromatogram of 13 batches of Fuyanxiao Capsules

[0182]

[0183] From Table 10, it can be seen that the change amplitude of the relative peak area of peaks 3 and 9 exceeds 2 times, and the change amplitudes of the remaining chromatographic peaks all do not exceed 2 times. The RSD value of the specified retention time does not exceed 5%, and the relative retention time should be within ±10% of the specified value. The specified value is 1.00 (peak S), 1.73 (peak 2), 2.23 (peak 3), 2.27 (peak 4), 3.11 (peak 5), 3.21 (peak 6), 3.45 (peak 7), 3.83 (peak 8), 4.25 (peak 9), 4.39 (peak 10), 4.50 (peak 11), 6.31 (peak 12), 6.39 (peak 13), 6.75 (peak 14), 6.96 (peak 15), 7.28 (peak 16), 7.70 (peak 17), 8.00 (peak 18), 9.05 (peak 19), 9.91 (peak 20), and 10.27 (peak 21).

[0184] From the experimental data, it is limited that the change amplitude of each chromatographic peak except peaks 3 and 9 does not exceed 2 times. The RSD value of the specified retention time does not exceed 5%, and the relative retention time should be within ±10% of the specified value.

[0185] Example 8: Detection method of characteristic chromatogram of Fuyanxiao standard decoction

[0186] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition Part IV General Rule 0512).

[0187] 1. Chromatographic conditions

[0188] Instrument model: water se2695 high performance liquid chromatograph, chromatographic column GL Sciences Inertsil ODS-35um (250mm×4.6mm) was used, acetonitrile was used as mobile phase A, and 0.2% phosphoric acid solution was used as mobile phase B, elution was carried out according to the gradient in Table 11 below, column temperature was 35℃, flow rate was 1ml / min, injection amount was 10µL, and detection wavelength was 230nm.

[0189] Table 11 Elution gradient

[0190]

[0191] 2. Preparation of reference solution

[0192] Take gallic acid, protocatechuic acid, epicatechin, isomalloto, paeonol, emodin, chrysophanol, physcion, aloe emodin, and rhein reference substances, accurately weigh, add methanol to prepare a mixed solution containing 0.1 mg per 1 ml.

[0193] 3. Preparation of test sample

[0194] Take 5 ml of Fuyanxiao standard decoction, accurately measure, place in a conical flask with a plug, accurately add 5 ml of methanol, weigh, ultrasonically treat for 10 minutes, cool, weigh again, make up the weight loss with 50% methanol, shake well, centrifuge, take the supernatant and filter through a 0.22 μm microporous filter membrane, take the filtrate, and obtain.

[0195] 4. Determination method

[0196] Take 10 μl of the reference solution and the test sample solution respectively, inject into the liquid chromatograph, determine, record the chromatogram (Figure 1), and obtain. Figure 7

[0197] There are 15 characteristic peaks in the test sample characteristic chromatogram, the change range of the relative peak area of each characteristic peak is not more than 2 times, taking the characteristic peak of gallic acid as the S peak, the relative retention time of each characteristic peak to the S peak is calculated, and the relative retention time is within ±10% of the specified value; the specified value of the relative retention time of each characteristic peak is 1.00 for peak S, 1.67 for peak 2, 2.03 for peak 3, 2.12 for peak 4, 2.15 for peak 5, 3.09 for peak 6, 3.22 for peak 7, 3.99 for peak 8, 4.26 for peak 9, 4.46 for peak 10, 6.27 for peak 11, 6.31 for peak 12, 6.39 for peak 13, 6.80 for peak 14, and 7.18 for peak 15.

[0198] Example 9: Analysis of main components of Fuyanxiao preparation

[0199] The main component analysis of Fuyanxiao capsules was carried out, taking the relative area of the common peaks in the characteristic chromatograms of 13 batches of samples as variables, using SIMCA-13.0 statistical software for PCA analysis, 10 principal components were extracted from 21 variables, and the model explanation rate parameter R 2 was 0.995, and the prediction ability parameter Q 2 was 0.576, indicating that the extracted principal components could explain 99.5% of the original variables, and the prediction ability of the model was 57.6%. The PCA scores of 13 batches of samples are shown in Table 2. Figure 8 ​​

[0200] To further distinguish samples and determine the differential markers affecting sample quality, PLC-DA analysis was performed using SIMCA 13.0 statistical software, and the results showed that R 2 X (X matrix solidity parameter, should be higher than 0.5) was 0.644, R 2 Y (model stability parameter should be higher than 0.5) was 0.911, Q 2 value (prediction ability parameter, the closer to 1, the better the prediction ability) was 0.828, indicating that the model stability and prediction ability were strong, and the PLS-DA score plot was shown in Figure 9 Variable importance projection value (VIP) is an important indicator for screening differential compounds, the higher the VIP value, the greater the influence on the difference between groups, and VIP>1 was used as the threshold, 7 common peaks were screened out according to the VIP value in descending order as follows: peak 13, peak 19, peak 20, peak 11, peak 9, peak 2, and peak 10. The results are shown in Figure 10

[0201] In the production process, attention should be paid to peak 13 chrysophanol glucoside, peak 19 emodin, peak 20 chrysophanol, peak 11 (belonging to patrinia), peak 9 isorhamnetin, peak 2 protocatechuic acid, and peak 10 wilfordine.​

Claims

1. A method for detecting a gynecological anti-inflammatory preparation, characterized in that, The gynecological anti-inflammatory preparation is a solid or liquid preparation made from Oxalis corniculata, Patrinia scabiosaefolia, Trichosanthes kirilowii, Rheum palmatum, Paeonia suffruticosa, Atractylodes lancea, and Lindera strychnifolia. The detection method uses high-performance liquid chromatography (HPLC) to determine the fingerprint and / or characteristic chromatograms. The HPLC detection conditions are: using octadecylsilane-bonded silica gel as the packing material, acetonitrile or methanol as mobile phase A, and 0.1%-0.5% phosphoric acid aqueous solution as mobile phase B for gradient elution; column temperature is 30-40℃; detection wavelength is 220-240nm; flow rate is 0.8-1.2ml / min; injection volume is 5-20µL. The gradient elution conditions are: From 0 to 12 minutes, mobile phase A decreased from 3% to 12%, and mobile phase B decreased from 97% to 88%. Over 12-30 minutes, mobile phase A decreased from 12% to 18%, and mobile phase B decreased from 88% to 82%. 30-40 min, mobile phase A 18%→20%, mobile phase B 82%→80%; 40-52 min, mobile phase A 20%→31%, mobile phase B 80%→69%; 52-53 min, mobile phase A 31%→35%, mobile phase B 69%→65%; 53-60 min, mobile phase A 35%→40%, mobile phase B 65%→60%; 60-75 min, mobile phase A 40%→55%, mobile phase B 60%→45%; 75-90 min, mobile phase A 55%→80%, mobile phase B 45%→20%; 90-110 min, mobile phase A 80%, mobile phase B 20%; The solid preparation for treating gynecological inflammation is in capsule form; wherein... Preparation of solid preparation test solution: Take 0.15g of the contents of Fuyanxiao capsules, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of 50% methanol, weigh it, sonicate for 10 minutes, cool it, weigh it again, make up the reduced weight with 50% methanol, shake well, centrifuge, and take the supernatant to obtain the Fuyanxiao solid preparation test solution. Preparation of reference solution for solid dosage form: Take appropriate amounts of gallic acid, protocatechuic acid, epicatechin, sennain, isochaetin, paeonol, rhein, chrysophanol, rhein methyl ether, aloe-emodin, rhein, rhein glucoside, chrysophanol glucoside, and rhein methyl ether glucoside, accurately weigh them, and add methanol to prepare a mixed solution containing 0.1 mg of each per ml; The liquid preparation for treating gynecological inflammation is a standard decoction of gynecological inflammation treatment; wherein... Preparation of liquid preparation test solution: Take 5ml of Fuyanxiao standard decoction, measure accurately, place in a stoppered conical flask, add 5ml of methanol accurately, weigh, sonicate for 10 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, centrifuge, take the supernatant, and the Fuyanxiao standard decoction test solution is obtained. Preparation of reference solution for liquid formulation: Take appropriate amounts of gallic acid, protocatechuic acid, epicatechin, isochaetin, paeonol, rhein, chrysophanol, rhein methyl ether, aloe-emodin, and rhein, accurately weigh them, and add methanol to prepare a mixed solution containing 0.1 mg of each per 1 ml.

2. The detection method according to claim 1, characterized in that, The mobile phase A was acetonitrile, the mobile phase B was 0.2% phosphoric acid aqueous solution, the chromatographic column was GL Sciences Inertsil ODS-35um, the column temperature was 35℃, the detection wavelength was 230nm, the flow rate was 1ml / min, and the injection volume was 10µL.

3. The detection method according to claim 1, characterized in that, The solid preparation for treating gynecological inflammation is prepared by extracting and concentrating the following raw materials in parts by weight, and then adding one or more pharmaceutically acceptable excipients: 300 parts of Oxalis corniculata, 300 parts of Patrinia scabiosifolia, 250 parts of Trichosanthes kirilowii, 300 parts of Rheum palmatum, 150 parts of Paeonia suffruticosa, 250 parts of Atractylodes lancea, and 300 parts of Lindera strychnifolia.

4. The detection method according to claim 1, characterized in that, The preparation method of the standard decoction for treating gynecological inflammation is as follows: Weigh 15g of Oxalis corniculata, 15g of Patrinia scabiosaefolia, 12.5g of Trichosanthes kirilowii, 7.5g of Rheum palmatum, 7.5g of Paeonia suffruticosa, 12.5g of Atractylodes lancea, and 15g of Lindera strychnifolia. Add 8 times the amount of water and decoct twice, 2 hours each time, to obtain the first decoction and the second decoction. Mix 100ml of the first decoction with 100ml of the second decoction to obtain the standard decoction for treating gynecological inflammation.

5. The detection method according to any one of claims 1-3, characterized in that, The detection method involves injecting the test solution of the solid preparation of Fuyanxiao and the reference solution of the solid preparation into a liquid chromatograph, measuring them according to the working conditions of high performance liquid chromatography, and recording the chromatogram.

6. The detection method according to claim 5, characterized in that, The detection method is a fingerprint chromatographic detection method. In the fingerprint chromatogram, the fingerprint chromatogram of the solid dosage form test sample shows a chromatographic peak with the same retention time as the reference peak. According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint chromatogram, the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram shall not be less than 0.

90. The fingerprint chromatogram consists of 23 common peaks, of which peak 1 is gallic acid peak, peak 2 is protocatechuic acid peak, peak 3 is epicatechin peak, peak 11 is isochaetin peak, peak 12 is chrysophanol peak, peak 15 is emodin glucoside peak, peak 16 is chrysophanol glucoside peak, peak 17 is emodin methyl ether glucoside peak, peak 18 is paeonol peak, peak 19 is aloe-emodin peak, peak 20 is rhein peak, peak 21 is emodin peak, peak 22 is chrysophanol peak, and peak 23 is emodin methyl ether peak.

7. The detection method according to claim 5, characterized in that, The detection method is a characteristic chromatogram detection method. In the characteristic chromatogram, the solid dosage form test sample should have 21 characteristic peaks. Except for peaks 3 and 9, the variation in the relative peak area of ​​each characteristic peak should not exceed 2 times. Taking the characteristic peak of gallic acid as peak S, the relative retention time of each characteristic peak and peak S is calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​for the relative retention time of each characteristic peak are: peak S = 1.00, peak 2 = 1.73, and peak 3 = 2.

23. Peak 4 is 2.27, Peak 5 is 3.11, Peak 6 is 3.21, Peak 7 is 3.45, Peak 8 is 3.83, Peak 9 is 4.25, Peak 10 is 4.39, Peak 11 is 4.50, Peak 12 is 6.31, Peak 13 is 6.39, Peak 14 is 6.75, Peak 15 is 6.96, Peak 16 is 7.28, Peak 17 is 7.70, Peak 18 is 8.00, Peak 19 is 9.05, Peak 20 is 9.91, and Peak 21 is 10.

27.

8. The detection method according to any one of claims 1-4, characterized in that, The detection method involves injecting the test solution of the gynecological anti-inflammatory liquid preparation and the reference solution of the liquid preparation into a liquid chromatograph, measuring them according to the working conditions of high performance liquid chromatography, and recording the chromatogram.

9. The detection method according to claim 8, characterized in that, The detection method is a fingerprint chromatographic detection method. In the fingerprint chromatogram, the fingerprint chromatogram of the liquid preparation test sample shows a chromatographic peak with the same retention time as the reference chromatographic peak. According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint chromatogram, the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram shall not be less than 0.

80. The fingerprint chromatogram consists of 22 common peaks, of which peak 1 is gallic acid peak, peak 2 is protocatechuic acid peak, peak 3 is epicatechin peak, peak 12 is isovitanol peak, peak 17 is paeonol peak, peak 18 is aloe-emodin peak, peak 19 is rhein peak, peak 20 is emodin peak, peak 21 is chrysophanol peak, and peak 22 is emodin methyl ether peak.

10. The detection method according to claim 8, characterized in that, The detection method is a characteristic spectrum detection method. In the characteristic spectrum, the liquid preparation test sample should have 15 characteristic peaks. The variation range of the relative peak area of ​​each characteristic peak should not exceed 2 times. Taking the characteristic peak of gallic acid as the S peak, the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​for the relative retention time of each characteristic peak are: peak S = 1.00, peak 2 = 1.67, peak 3 = 2.03, peak 4 = 2.12, peak 5 = 2.15, peak 6 = 3.09, peak 7 = 3.22, peak 8 = 3.99, peak 9 = 4.26, peak 10 = 4.46, peak 11 = 6.27, peak 12 = 6.31, peak 13 = 6.39, peak 14 = 6.80, and peak 15 = 7.18.

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