A kind of Jingyin Granules and its detection method and application of fingerprint spectrum

Abstract

The present invention provides a Jingyin granule and its preparation. The present invention further provides a method for detecting the fingerprint of Jingyin granule. The present invention also provides a method for constructing a standard fingerprint of Jingyin granule and a method for quality detection of the fingerprint of Jingyin granule. In addition, the present invention provides a method for detecting the contents of 5 components in Jingyin granule and a method for screening the fingerprints of multiple medicinal materials in Jingyin granule. The Jingyin granule and the method for detecting its fingerprint provided by the present invention can obtain Jingyin granules with excellent quality, and also establish a high-performance liquid chromatography fingerprint of Jingyin granule, which is beneficial to the overall control of the internal quality of Jingyin granule and provides a scientific experimental basis for better establishing an overall quality control and evaluation system for Jingyin granule.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of traditional Chinese medicine and the quality control and detection of its components, and relates to a Jingyin Granule and a detection method and application of its fingerprint. Specifically, it relates to a Jingyin Granule, a detection method of the fingerprint of Jingyin Granule, a construction method of the standard fingerprint of Jingyin Granule, a quality detection method of the fingerprint of Jingyin Granule, a detection method of the contents of 5 components in Jingyin Granule, and a screening method of the fingerprints of multiple medicinal materials in Jingyin Granule. Background Art

[0002] Jingyin Granule is composed of 9 medicinal herbs, namely Schizonepeta tenuifolia, Lonicera japonica, Ilex purpurea, Houttuynia cordata, Isatis indigotica, Taraxacum mongolicum, Arctium lappa, Saposhnikovia divaricata, and Glycyrrhiza uralensis, and has the effects of dispelling wind and clearing heat, and dispersing lung qi and relieving sore throat. It is clinically mostly used for colds belonging to the syndrome of wind-heat. In the formula, the monarch herb Schizonepeta tenuifolia is pungent and warm, dispersing and relieving exterior syndrome, warm but not dry, entering the lung and liver meridians, and being the main herb for treating wind-warm syndrome; the monarch herb Lonicera japonica clears heat and detoxifies, disperses and penetrates the exterior, and is the essential herb for clearing heat and detoxifying; the ministerial herbs Ilex purpurea, Taraxacum mongolicum, and Isatis indigotica can all clear the heat of the lung and enhance the effect of the monarch herb in clearing heat and detoxifying; the adjuvant herbs Houttuynia cordata and Arctium lappa have the effects of dispersing lung qi and resolving phlegm, and detoxifying and relieving sore throat; the guiding herbs Glycyrrhiza uralensis and Saposhnikovia divaricata detoxify and harmonize the middle-jiao, and harmonize all the herbs.

[0003] The current quality standard of Jingyin Granule only has thin-layer chromatography identification and the content limit of protocatechuic aldehyde, and cannot comprehensively evaluate the overall quality of this preparation. Fingerprint is a modern quality control method of traditional Chinese medicine from the perspective of "total components" according to the characteristics of the overall comprehensive action of multi-components and multi-targets of traditional Chinese medicine, and is more comprehensive in the quality control of drugs with non-single components. The method of combining fingerprint with multi-component content determination can better control the quality of Jingyin Granule. Summary of the Invention

[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a Jingyin Granule and a detection method and application of its fingerprint, which can provide Jingyin Granule with stable quality, adopt the pretreatment under optimized conditions and the high performance liquid chromatography method, establish a detection method of the fingerprint of Jingyin Granule and its application of high performance liquid chromatography fingerprint, and realize the detection of the chemical components of the whole formula in Jingyin Granule, more comprehensively reflect the current situation of each component in Jingyin Granule, and can objectively, comprehensively and accurately evaluate the quality of Jingyin Granule, which is of great significance for controlling the quality of Jingyin Granule and ensuring clinical efficacy.

[0005] To achieve the above purpose and other related purposes, the first aspect of the present invention provides a Jingyin Granule, which is prepared by water extraction from Schizonepeta tenuifolia, Lonicera japonica, Ilex purpurea, Houttuynia cordata, Isatis indigotica, Taraxacum mongolicum, Arctium lappa, Saposhnikovia divaricata, and Glycyrrhiza uralensis, and the content of at least one component in Jingyin Granule, such as protocatechuic acid, caffeic acid, 5-O-methylvisammioside, arctiin or chlorogenic acid, is determined by high performance liquid chromatography (HPLC) method and meets any one or more of the following conditions:

[0006] 1) In the Jingyin granules, the content of protocatechuic acid is 0.074 - 0.092 mg / g;

[0007] 2) In the Jingyin granules, the content of caffeic acid is 0.023 - 0.044 mg / g;

[0008] 3) In the Jingyin granules, the content of 5-O-methylvisammioside is 0.024 - 0.033 mg / g;

[0009] 4) In the Jingyin granules, the content of arctiin is 0.154 - 0.246 mg / g;

[0010] 5) In the Jingyin granules, the content of chlorogenic acid is 0.113 - 0.191 mg / g.

[0011] The second aspect of the present invention provides a preparation of Jingyin granules, comprising the above Jingyin granules.

[0012] Preferably, the Jingyin granules are selected from one of tablets, capsules, granules, pills, powders, oral liquids, injections or topical pharmaceutical preparations.

[0013] The third aspect of the present invention provides a method for detecting the fingerprint of Jingyin granules, comprising the following steps:

[0014] 1) Preparation of the test solution: Add the Jingyin granule sample to the first solvent, ultrasonically extract and centrifuge, take the supernatant to obtain the test solution;

[0015] 2) Preparation of the reference solution: Provide a reference solution, which includes protocatechuic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, prim-O-glucosylcimifugin, liquiritin, rutin, luteolin-7-O-glucoside, isochlorogenic acid B, isochlorogenic acid A,

[0016] 5-O-methylvisammioside, isochlorogenic acid C, arctiin and the first solvent;

[0017] 3) Determination: Use the high performance liquid chromatography (HPLC) method under the same chromatographic conditions to separately determine the test solution and the reference solution, obtain the fingerprint of the test solution and the fingerprint of the reference solution, compare the fingerprint of the test solution with the fingerprint of the reference solution, and assign and locate the target components in the fingerprint of the test solution, so as to obtain the fingerprint of Jingyin granules.

[0018] Preferably, in step 1) or 2), the first solvent is an aqueous methanol solution.

[0019] More preferably, the aqueous methanol solution is an aqueous solution containing 40-60% by volume of methanol, preferably 50%.

[0020] Preferably, in step 1), the ratio of the mass g of the Jingyin granule sample added to the volume mL of the first solvent added is 2.5:20-30, preferably 2.5:25.

[0021] Preferably, in step 1), the Jingyin granule sample needs to be precisely weighed after being added to the first solvent.

[0022] Preferably, in step 1), the time for ultrasonic extraction is 25-35 min, preferably 30 min.

[0023] Preferably, in step 1), the power of the ultrasonic extraction is 150-250 W, and the frequency of the ultrasonic extraction is 30-50 kHz. More preferably, the power of the ultrasonic extraction is 200 W, and the frequency of the ultrasonic extraction is 40 kHz.

[0024] Preferably, in step 1), after the ultrasonic extraction, it needs to be precisely weighed again, and the weight loss is made up with the second solvent.

[0025] More preferably, the second solvent is an aqueous solution containing 65-75% by volume of methanol, preferably 70%.

[0026] Preferably, in step 1), the centrifugation conditions are: the centrifugation time is 5-15 min, preferably 10 min; the centrifugation rate is 5000-7000 rpm, preferably 6000 rpm.

[0027] Preferably, in step 1), the supernatant can be directly taken as the supernatant after centrifugation or obtained by filtration.

[0028] More preferably, the filtration is to take the supernatant through a filter membrane, discard the initial filtrate, and take the subsequent filtrate.

[0029] Most preferably, the filter membrane is a 0.22 μm or 0.45 μm microporous filter membrane.

[0030] Preferably, in step 2), the CAS number of protocatechuic acid is 99-50-3, the CAS number of chlorogenic acid is 327-97-9, the CAS number of caffeic acid is 331-39-5, the CAS number of cryptochlorogenic acid is 905-99-7, the CAS number of prim-O-glucosylcimifugin is 80681-45-4, the CAS number of liquiritin is 551-15-5, the CAS number of rutin is 153-18-4, the CAS number of luteoloside is 5373-11-5, the CAS number of isochlorogenic acid B is 14534-61-3, the CAS number of isochlorogenic acid A is 2450-53-5, the CAS number of 5-O-methylvisamminol is 84272-85-5, the CAS number of isochlorogenic acid C is 57378-72-0, and the CAS number of arctiin is 20362-31-6.

[0031] Preferably, in step 3), when comparing the fingerprint of the test solution with that of the reference solution, based on the known characteristic peaks in the fingerprint of the reference solution, the corresponding characteristic peaks in the fingerprint of the test solution are identified by relative retention time, so as to assign and locate the target components in the fingerprint of the test solution.

[0032] Preferably, in step 3), the chromatographic column in the high performance liquid chromatography is C 18 chromatographic column.

[0033] More preferably, the chromatographic column in the high performance liquid chromatography is Waters CORTECS C 18 chromatographic column (150 mm × 4.6 mm, 2.7 μm).

[0034] Preferably, in step 3), the detector in the high performance liquid chromatography is a photodiode array detector (PDA).

[0035] Preferably, in step 3), the column temperature in the high performance liquid chromatography is 30-40 °C. More preferably, the column temperature in the high performance liquid chromatography is 35 °C.

[0036] Preferably, in step 3), the injection volume in the high performance liquid chromatography is 5-15 μL. More preferably, the injection volume in the high performance liquid chromatography is 10 μL.

[0037] Preferably, in step 3), in the high performance liquid chromatography, the flow rate is 0.5-2.0 mL / min. More preferably, in the high performance liquid chromatography, the flow rate is 1.0 mL / min.

[0038] Preferably, in step 3), in the high performance liquid chromatography, the detection wavelength is 205-215 nm, preferably 210 nm.

[0039] Preferably, in step 3), in the high performance liquid chromatography method, the mobile phase is 0.05 - 0.2% phosphoric acid aqueous solution - acetonitrile, wherein the A phase is 0.05 - 0.2% phosphoric acid aqueous solution and the B phase is acetonitrile; the analysis time is 66 min; gradient elution is performed.

[0040] More preferably, in the high performance liquid chromatography method, the mobile phase is 0.1% phosphoric acid aqueous solution - acetonitrile, wherein the A phase is 0.1% phosphoric acid aqueous solution and the B phase is acetonitrile; the analysis time is 66 min; gradient elution is performed.

[0041] The 0.05 - 0.2% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.05 - 0.2%. The 0.1% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.1%.

[0042] More preferably, the specific program of the gradient elution is as follows:

[0043] 0 - 3 min, the volume ratio of A phase to B phase is 95:5 - 95:5;

[0044] 3 - 16 min, the volume ratio of A phase to B phase is 95:5 - 90:10;

[0045] 16 - 21 min, the volume ratio of A phase to B phase is 90:10 - 88:12;

[0046] 21 - 35 min, the volume ratio of A phase to B phase is 88:12 - 83:17;

[0047] 35 - 55 min, the volume ratio of A phase to B phase is 83:17 - 70:30;

[0048] 55 - 65 min, the volume ratio of A phase to B phase is 70:30 - 55:45;

[0049] 65 - 66 min, the volume ratio of A phase to B phase is 55:45 - 5:95.

[0050] The fourth aspect of the present invention provides a method for constructing a standard fingerprint of Jingyin granules, including: detecting multiple batches of Jingyin granule samples respectively by using the aforementioned detection method for the fingerprint of Jingyin granules, obtaining a common mode control map of the fingerprints of multiple batches of Jingyin granule samples, taking the chromatographic peaks existing in all the maps as common characteristic peaks, determining the relative retention time of the common characteristic peaks, the ratio of the area of each common characteristic peak to the total peak area, and attributing and positioning the index components in the fingerprint of Jingyin granules according to the relative retention time, thereby establishing the standard fingerprint of Jingyin granules.

[0051] Preferably, the number of batches of the Jingyin granule samples for detection is 10 - 20 batches, preferably 12 batches.

[0052] Preferably, in the common mode control chromatogram, the similarity of the fingerprint chromatograms of multiple batches of Jingyin Granules samples is compared by using the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" 2012 version issued by the Pharmacopoeia Commission of the People's Republic of China.

[0053] More preferably, when comparing the similarity of the fingerprint chromatograms of multiple batches of Jingyin Granules samples, multi-point calibration and Mark peak matching are performed when the "time window width" is 0.1 min, and the fingerprint chromatogram and the reference fingerprint chromatogram are generated by the median method.

[0054] More preferably, the similarity of the fingerprint chromatograms of multiple batches of Jingyin Granules samples is ≥ 0.97.

[0055] Preferably, the standard fingerprint chromatogram of Jingyin Granules is as Figure 1 shown. Taking the ratio of the retention time of each common characteristic peak to the retention time of the reference peak as the relative retention time, it includes 16 common characteristic peaks. Taking the 2nd peak as the reference peak S peak, the retention time is 1.000, and the relative retention times of the other 15 common characteristic peaks are in turn: the 1st peak is 0.625, the 3rd peak is 1.080, the 4th peak is 1.517, the 5th peak is 1.609, the 6th peak is 1.739, the 7th peak is 3.268, the 8th peak is 3.561, the 9th peak is 3.808, the 10th peak is 4.008, the 11th peak is 4.543, the 12th peak is 4.624, the 13th peak is 4.662, the 14th peak is 4.767, the 15th peak is 5.216, the 16th peak is 6.105; the relative deviation of the relative retention times of the above 15 common characteristic peaks except the 2nd peak is ≤ ±0.24%.

[0056] More preferably, in the standard fingerprint chromatogram of Jingyin Granules, 13 fingerprint peaks are located and determined: the 1st peak is protocatechuic acid, the 2nd peak is chlorogenic acid, the 5th peak is caffeic acid, the 6th peak is cryptochlorogenic acid, the 7th peak is prim-O-glucosylcimifugin, the 8th peak is liquiritin, the 9th peak is rutin, the 10th peak is luteoloside, the 11th peak is isochlorogenic acid B, the 13th peak is isochlorogenic acid A, the 14th peak is 5-O-methylvisammioside, the 15th peak is isochlorogenic acid C, the 16th peak is arctiin.

[0057] The fifth aspect of the present invention provides a quality detection method for the fingerprint chromatogram of Jingyin Granules, including: obtaining the fingerprint chromatogram of Jingyin Granules by using the detection method of the fingerprint chromatogram of Jingyin Granules as described above, and comparing the similarity with the standard fingerprint chromatogram of Jingyin Granules obtained by using the construction method of the standard fingerprint chromatogram of Jingyin Granules as described above.

[0058] Preferably, when comparing the similarities, the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2012 Edition) issued by the Pharmacopoeia Commission of the People's Republic of China is used. Preferably, the similarity ≥ 0.97.

[0059] The sixth aspect of the present invention provides a method for detecting the contents of 5 components in Jingyin Granules, comprising the following steps:

[0060] A) Preparation of the test solution: The same as step 1) of the detection method for the fingerprint of Jingyin Granules;

[0061] B) Preparation of the reference solution: Provide a reference solution, which includes protocatechuic acid, caffeic acid, 5-O-methylvisammioside, arctiin, chlorogenic acid and a first solvent;

[0062] C) Determination: Using high performance liquid chromatography with the same chromatographic conditions as those in the detection method for the fingerprint of Jingyin Granules, respectively determine the test solution in step A) and the reference solution in step B), and calculate the contents of the 5 components in the test solution by the external standard method.

[0063] Preferably, in step B), the first solvent is an aqueous methanol solution.

[0064] More preferably, the aqueous methanol solution is an aqueous solution containing 40 - 60% (v / v) methanol, preferably 50%.

[0065] Preferably, in step B), the reference solution is prepared by dissolving protocatechuic acid, caffeic acid, 5-O-methylvisammioside, arctiin, chlorogenic acid in the first solvent and then making up the volume to obtain the reference solution.

[0066] Preferably, in step B), the reference solution is prepared by gradually diluting the prepared reference stock solution. The reference stock solution needs to be sealed and stored for later use.

[0067] More preferably, in the reference stock solution, the concentration of protocatechuic acid is 0.256 mg / mL, caffeic acid 0.203 mg / mL, 5-O-methylvisammioside 0.207 mg / mL, arctiin 0.508 mg / mL, chlorogenic acid 0.824 mg / mL.

[0068] Preferably, in step B), the content range of protocatechuic acid in the reference solution is 0.016 - 0.256 mg / mL, the content range of caffeic acid is 0.013 - 0.203 mg / mL, the content range of 5-O-methylvisammioside is 0.013 - 0.207 mg / mL, the content range of arctiin is 0.032 - 0.508 mg / mL, and the content range of chlorogenic acid is 0.052 - 0.824 mg / mL.

[0069] Preferably, in step C), the five components in the test sample solution are: protocatechuic acid, caffeic acid, 5-O-methylvisammioside, arctiin, and chlorogenic acid.

[0070] Preferably, the external standard method means: respectively pipette a series of different volumes of the reference substance solution described in step B) to prepare a series of solutions with different concentrations, perform injection analysis using a high performance liquid chromatograph, obtain the linear relationship between the contents of the five components in the reference substance solution and the peak areas, use the chromatographic peak area of each component corresponding to its corresponding content, draw the corresponding standard working curve, and calculate the regression equation of each standard working curve. Then detect the test sample solution using a high performance liquid chromatograph, and substitute the chromatographic peak areas of the five components in the obtained test sample solution into the regression equations of the respective standard working curves to obtain the contents of the corresponding components.

[0071] More preferably, in the standard working curve, the peak area is used as the ordinate and the content of each reference substance solution is used as the abscissa.

[0072] The seventh aspect of the present invention provides a method for screening the fingerprint of multiple herbs in Jingyin Granules, including the following steps:

[0073] a) Preparation of single-herb sample solution: Any one or more of the 9 herb samples of Schizonepeta tenuifolia, Lonicera japonica, Ilex purpurea, Houttuynia cordata, Isatis indigotica, Taraxacum mongolicum, Arctium lappa, Saposhnikovia divaricata, and Glycyrrhiza uralensis in Jingyin Granules are prepared according to step 1) of the detection method for the fingerprint of Jingyin Granules to obtain at least one single-herb sample solution respectively;

[0074] b) Determination: Use high performance liquid chromatography (HPLC) with the same chromatographic conditions as in step 3) of the detection method for the fingerprint of Jingyin Granules to determine the single-herb sample solution and obtain the fingerprint of the single-herb sample solution;

[0075] c) Obtaining the reference fingerprint: Use the same steps as the detection method for the fingerprint of Jingyin Granules to obtain the reference fingerprint of Jingyin Granules;

[0076] d) Quality inspection: Compare the fingerprint of the single-herb sample solution with the reference fingerprint of Jingyin Granules, and identify the corresponding characteristic peaks of the single-herb sample solution in the reference fingerprint of Jingyin Granules through the relative retention time, so as to locate and attribute the characteristic peaks in the fingerprint of the single-herb sample solution.

[0077] Preferably, in step d), the present invention locates the characteristic peaks by comparing the fingerprint of the single-herb medicine sample solution with the control fingerprint of Jingyin Granules, analyzes and processes it using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" 2012 version software released by the Pharmacopoeia Commission of the People's Republic of China, and confirms the attribution of the characteristic peaks of each single-herb medicine in Jingyin Granules based on the relative retention time of each characteristic peak on the control fingerprint of Jingyin Granules. The specific results are shown in Figure 2 .

[0078] Preferably, in step d), in the single-herb medicine sample solution, the fingerprint of the Schizonepeta tenuifolia Briq. sample solution includes 6 common fingerprint peaks, and the 6 common fingerprint peaks are peak No. 2, peak No. 3, peak No. 5, peak No. 10, peak No. 14, and peak No. 15.

[0079] Preferably, in step d), in the single-herb medicine sample solution, the fingerprint of the Lonicera japonica Thunb. sample solution includes 7 common fingerprint peaks, and the 7 common fingerprint peaks are peak No. 4, peak No. 6, peak No. 9, peak No. 10, peak No. 11, peak No. 13, and peak No. 15.

[0080] Preferably, in step d), in the single-herb medicine sample solution, the fingerprint of the Ilex purpurea Hassk. sample solution includes 7 common fingerprint peaks, and the 7 common fingerprint peaks are peak No. 1, peak No. 2, peak No. 3, peak No. 4, peak No. 5, peak No. 9, and peak No. 15.

[0081] Preferably, in step d), in the single-herb medicine sample solution, the fingerprint of the Houttuynia cordata Thunb. sample solution includes 5 common fingerprint peaks, and the 5 common fingerprint peaks are peak No. 1, peak No. 3, peak No. 4, peak No. 9, and peak No. 13.

[0082] Preferably, in step d), in the single-herb medicine sample solution, the fingerprint of the Isatis indigotica Fort. sample solution includes 3 common fingerprint peaks, and the 3 common fingerprint peaks are peak No. 3, peak No. 5, and peak No. 9.

[0083] Preferably, in step d), in the single-herb medicine sample solution, the fingerprint of the Taraxacum mongolicum Hand.-Mazz. sample solution includes 4 common fingerprint peaks, and the 4 common fingerprint peaks are peak No. 3, peak No. 4, peak No. 5, and peak No. 10.

[0084] Preferably, in step d), in the single-herb medicine sample solution, the fingerprint of the Arctium lappa L. sample solution includes 6 common fingerprint peaks, and the 6 common fingerprint peaks are peak No. 3, peak No. 4, peak No. 6, peak No. 11, peak No. 15, and peak No. 16.

[0085] Preferably, in step d), in the single-herb medicine sample solution, the fingerprint of the Saposhnikovia divaricata (Turcz.) Schischk. sample solution includes 4 common fingerprint peaks, and the 4 common fingerprint peaks are peak No. 3, peak No. 7, peak No. 10, and peak No. 14.

[0086] Preferably, in step d), in the single-herb sample solution, the fingerprint of the licorice sample solution includes 5 common fingerprint peaks, which are peak No. 3, peak No. 7, peak No. 8, peak No. 9, and peak No. 12.

[0087] Among the above-mentioned common fingerprint peaks, peak No. 1 is determined to be protocatechuic acid, peak No. 2 is chlorogenic acid, peak No. 5 is caffeic acid, peak No. 6 is cryptochlorogenic acid, peak No. 7 is prim-O-glucosylcimifugin, peak No. 8 is liquiritin, peak No. 9 is rutin, peak No. 10 is luteoloside, peak No. 11 is isochlorogenic acid B, peak No. 13 is isochlorogenic acid A, peak No. 14 is 5-O-methylvisammioside, peak No. 15 is isochlorogenic acid C, and peak No. 16 is arctiin.

[0088] All the water used in the present invention is purified water.

[0089] As described above, the Jingyin granules provided by the present invention, the detection method and application of its fingerprint can obtain Jingyin granules with excellent quality through the preferred preparation steps and conditions, which have stable quality, low unqualified rate, and good consistency among different batches of Jingyin granules.

[0090] Meanwhile, the present invention uses high performance liquid chromatography to establish the fingerprint of Jingyin granules, which has 16 common peaks, and qualitatively identifies 13 characteristic peaks, highlighting the contributions of different categories of chemical components to the fingerprint of Jingyin granules from different aspects, so as to realize the detection of the chemical components of the whole formula of Jingyin granules and can comprehensively reflect the quality changes of Jingyin granules.

[0091] The method provided by the present invention can also determine the contents of 5 chemical components (protocatechuic acid, caffeic acid, 5-O-methylvisammioside, arctiin, chlorogenic acid) in Jingyin granules. The standard curves of the 5 components determined by this method have good linear relationships within their respective ranges, good repeatability, high accuracy, and high precision.

[0092] The above fingerprint combined with the multi-component simultaneous quantification method makes up for the blank of the detection method of Jingyin granules, has the advantages of simplicity, rapidity, and accuracy, good repeatability, provides a reference basis for realizing the whole-process quality control of the production process, and ensures the safety, effectiveness, and quality controllability of drug use.

[0093] The above method can also confirm the attribution of multiple single-herb chromatographic peaks in Jingyin granules, and the fingerprint reflects the characteristic peaks of Schizonepeta tenuifolia, Lonicera japonica, Ilex purpurea Hassk., Houttuynia cordata Thunb., Isatis indigotica Fortune, Taraxacum mongolicum Hand.-Mazz., Arctium lappa L., Saposhnikovia divaricata (Turcz.) Schischk., and Glycyrrhiza uralensis Fisch. This method can effectively monitor from the source of raw materials, strictly control the quality of raw herbs, and can be used for the quality control of the production process, thus ensuring the safety and effectiveness of clinical drug use. Description of the Drawings

[0094] Figure 1 It shows the common mode fingerprint of 12 batches of Jingyin Granules samples of the present invention.

[0095] Figure 2 It shows the common mode fingerprint of the characteristic peak attribution of 9 medicinal materials of the present invention. Detailed implementation manners

[0096] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention.

[0097] The following illustrates the implementation manners of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0098] The reagents and instruments used in the following examples are as follows:

[0099] 1. Reagents

[0100] Quantitative substances: protocatechuic acid (batch number: 5668, purity 100%), caffeic acid (batch number: 7403, purity 99.7%), 5-O-methylvisammioside (batch number: 6381, purity 99.6%), arctiin (batch number: 5691, purity 98.3%), all purchased from Shanghai Standard Technology Service Co., Ltd.; chlorogenic acid (batch number: A22GB158496, purity 99.4%), purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0101] Qualitative substances: cryptochlorogenic acid (batch number: 3208, purity 98.0%), prim-O-glucosylcimifugin (batch number: 3246, purity 99.6%), liquiritin (batch number: 9722, purity 100.0%), rutin (batch number: 5981, purity 91.7%), luteoloside (batch number: 6567, purity 99.3%), isochlorogenic acid B (batch number: 3089, purity 98.5%), isochlorogenic acid A (batch number: 10639, purity 99.0%), all purchased from Shanghai Standard Technology Service Co., Ltd. Isochlorogenic acid C (batch number: 22070639, purity 98.0%) was purchased from Shanghai Titan Technology Co., Ltd.

[0102] 12 batches of Jingyin Granules samples (batch numbers 200205, 200301, 200302, 200303, 200304, 200305, 220703, 220704, 220705, 220706, 220707, 220708), all provided by Shanghai Shangyao Xingling Pharmaceutical Co., Ltd.

[0103] Single herbs: Nine herbs, including Schizonepeta tenuifolia Briq., Lonicera japonica Thunb., Ilex chinensis Sims., Houttuynia cordata Thunb., Isatis indigotica Fortune, Taraxacum mongolicum Hand.-Mazz., Arctium lappa L., Saposhnikovia divaricata (Turcz.) Schischk., and Glycyrrhiza uralensis Fisch., were all purchased from Shanghai Qingpu Chinese Herbal Pieces Co., Ltd.

[0104] Methanol and acetonitrile were of chromatographic grade (Merck, Germany); phosphoric acid was of analytical grade (Shanghai Chemical Reagent Co., Ltd., China National Pharmaceutical Corporation); water was ultrapure water prepared by a Direct-Q 3UV ultrapure water machine.

[0105] 2. Instruments

[0106] Waters Acquity ARC high performance liquid chromatograph (Waters); KQ5200DE digital control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); Sartorius BS 124S electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); Sartorius SQPQUINTIX65-1CN one in a hundred thousandth analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); Direct-Q 3UV ultrapure water machine (Millipore, USA).

[0107] Example 1

[0108] 1. Sample pretreatment

[0109] Precisely weigh 2.5 g of Jingyin Granules sample (batch number 200205), add 25 mL of 50% methanol aqueous solution, weigh precisely, perform ultrasonic extraction (power: 200 W, frequency: 40 kHz) for 30 min, weigh precisely again, make up the weight loss with 70% methanol aqueous solution, and then centrifuge at a rate of 6000 rpm for 10 min. Take the supernatant to obtain the test solution 1. # .

[0110] Weigh appropriate amounts of protocatechuic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, prim-O-glucosylcimifugin, liquiritin, rutin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, 5-O-methylvisamminol glucoside, isochlorogenic acid C, and arctiin, place them in 5-mL volumetric flasks respectively, dilute and make up the volume with 50% methanol aqueous solution to prepare the reference solution.

[0111] 2. Chromatographic conditions

[0112] The chromatographic conditions for high performance liquid chromatography were as follows: Chromatographic column: Waters CORTECS C 18Chromatographic column (150 mm × 4.6 mm, 2.7 μm); Detector: Photodiode array detector (DAD); Column temperature: 35 °C; Injection volume: 10 μL; Flow rate: 1.0 ml / min; Detection wavelength: 210 nm; Mobile phase: 0.1% phosphoric acid aqueous solution - acetonitrile, where phase A is 0.1% phosphoric acid aqueous solution and phase B is acetonitrile; Analysis time is 66 min; Gradient elution.

[0113] The specific program of gradient elution is as follows:

[0114] 0 - 3 min, volume ratio of phase A to phase B is 95:5 - 95:5;

[0115] 3 - 16 min, volume ratio of phase A to phase B is 95:5 - 90:10;

[0116] 16 - 21 min, volume ratio of phase A to phase B is 90:10 - 88:12;

[0117] 21 - 35 min, volume ratio of phase A to phase B is 88:12 - 83:17;

[0118] 35 - 55 min, volume ratio of phase A to phase B is 83:17 - 70:30;

[0119] 55 - 65 min, volume ratio of phase A to phase B is 70:30 - 55:45;

[0120] 65 - 66 min, volume ratio of phase A to phase B is 55:45 - 5:95.

[0121] 3. Determination

[0122] The test solution 1 in step 1 above was determined by high - performance liquid chromatography using the chromatographic conditions in step 2 above # and the reference solution to obtain the fingerprint of the test solution 1 # and the fingerprint of the reference solution. Among them, the fingerprint of the test solution 1 # should be compared with the fingerprint of the reference solution. According to the known characteristic peaks in the fingerprint of the reference solution, through the relative retention time, the corresponding characteristic peaks in the fingerprint of the test solution 1 # are identified, so as to locate the index components in the fingerprint of the test solution 1 # and obtain the fingerprint of Jingyin Granules.

[0123] Example 2

[0124] 1. Sample pretreatment

[0125] Accurately weigh 2.5 g of Jingyin Granules sample (batch number 200205), add 30 mL of 45% methanol aqueous solution and weigh accurately. Perform ultrasonic extraction (power: 220 W, frequency: 45 kHz) for 30 min, then weigh accurately again, make up the weight loss with 75% methanol aqueous solution, and centrifuge at a rate of 6500 rpm for 8 min. Take the supernatant to obtain the test solution 2 # 。

[0126] Weigh appropriate amounts of protocatechuic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, prim-O-glucosylcimifugin, liquiritin, rutin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, 5-O-methylvisammioside, isochlorogenic acid C, and arctiin respectively, place them in 5-mL volumetric flasks, add 45% methanol aqueous solution to dilute and make up the volume to prepare the reference substance solution.

[0127] 2. Chromatographic conditions

[0128] The chromatographic conditions for high performance liquid chromatography are as follows: chromatographic column: Waters CORTECS C 18 chromatographic column (150 mm × 4.6 mm, 2.7 μm); detector: photodiode array detector (DAD); column temperature: 32 °C; injection volume: 8 μL; flow rate: 1.5 mL / min; detection wavelength: 212 nm; mobile phase: 0.05% phosphoric acid aqueous solution - acetonitrile, where phase A is 0.05% phosphoric acid aqueous solution and phase B is acetonitrile; analysis time is 66 min; gradient elution.

[0129] The specific program of gradient elution is the same as that in step 2 of Example 1.

[0130] 3. Determination

[0131] Use high performance liquid chromatography under the chromatographic conditions in step 2 above to separately determine the test solution 2 # and the reference substance solution, and obtain the fingerprint of the test solution 2 # and the fingerprint of the reference substance solution. Among them, the fingerprint of the test solution 2 # shall be compared with the fingerprint of the reference substance solution. According to the known characteristic peaks in the fingerprint of the reference substance solution, through the relative retention time, identify the corresponding characteristic peaks in the fingerprint of the test solution 2 # so as to attribute and locate the index components in the fingerprint of the test solution 2 # and obtain the fingerprint of Jingyin Granules.

[0132] Example 3

[0133] The fingerprint detection method of Jingyin Granules established in Example 1 above was used to detect 12 batches of Jingyin Granules (S1-S12), and the fingerprint of the test solution and the fingerprint of the reference solution were obtained. That is, it was imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" 2012 version software released by the Pharmacopoeia Commission of the People's Republic of China. When the "time window width" was 0.1 min, multi-point calibration and Mark peak matching were performed, and the median method was used to generate the fingerprint of the test solution and the fingerprint of the reference solution. The fingerprint of the test product was compared with the reference fingerprint of Jingyin Granules obtained under the same fingerprint detection conditions, and the similarity of the fingerprint of each batch of Jingyin Granules was calculated. The results are shown in Figure 1 .

[0134] As Figure 1 shown, the similarities of the fingerprints of 12 batches of Jingyin Granules were all greater than 0.97, and there was no increase or decrease in the number of peaks, indicating that the fingerprints of 12 batches of samples had a high similarity and the overall quality was relatively stable. Among them, the retention time of peak 2 was moderate, the resolution was good, and the peak area was stable. Therefore, it was selected as the reference peak (S). It was calculated that the RSD of the relative retention time of the common peaks of 12 batches of Jingyin Granules was <0.24%, indicating that the peak emergence time of 12 batches of Jingyin Granules was stable; the RSD of the peak area of each common peak was 5.30%-21.14%, indicating that there were batch differences in the components contained in 12 batches of Jingyin Granules.

[0135] Taking Figure 1 the chromatographic peaks existing in all as the common characteristic peaks, the relative retention time of the common characteristic peaks, the ratio of the area of each common characteristic peak to the total peak area were determined, and the index components in the fingerprint of the Jingyin Granule sample were attributed and located according to the relative retention time, and the standard fingerprint of Jingyin Granules was established.

[0136] The standard fingerprint of Jingyin Granules used the ratio of the retention time of each common characteristic peak to the retention time of the reference peak as the relative retention time, including 16 common characteristic peaks. Taking peak 2 as the reference peak S, the retention time was 1.000, and the relative retention times of the other 15 common characteristic peaks were as follows: peak 1 was 0.625, peak 3 was 1.080, peak 4 was 1.517, peak 5 was 1.609, peak 6 was 1.739, peak 7 was 3.268, peak 8 was 3.561, peak 9 was 3.808, peak 10 was 4.008, peak 11 was 4.543, peak 12 was 4.624, peak 13 was 4.662, peak 14 was 4.767, peak 15 was 5.216, peak 16 was 6.105; the relative deviations of the relative retention times of the 15 common characteristic peaks except peak 2 were all ≤±0.24%.

[0137] Example 4

[0138] The standard fingerprint of Jingyin Granules obtained according to Example 3 was compared with the fingerprint of the reference substance solution. Based on the known characteristic peaks in the fingerprint of the reference substance solution, the corresponding characteristic peaks in the control fingerprint of Jingyin Granules were identified by relative retention time, and 13 fingerprint peaks were located and determined: Peak 1 is protocatechuic acid, Peak 2 is chlorogenic acid, Peak 5 is caffeic acid, Peak 6 is cryptochlorogenic acid, Peak 7 is prim-O-glucosylcimifugin, Peak 8 is liquiritin, Peak 9 is rutin, Peak 10 is luteoloside, Peak 11 is isochlorogenic acid B, Peak 13 is isochlorogenic acid A, Peak 14 is 5-O-methylvisammioside, Peak 15 is isochlorogenic acid C, and Peak 16 is arctiin.

[0139] Methodological Investigation of the Fingerprint in Example 5

[0140] 1. Precision

[0141] Take the same batch of Jingyin Granules samples (batch number 200205), prepare the test solution (S1) according to the detection method of the fingerprint of Jingyin Granules in Example 1 above and then detect it. Inject samples continuously for 6 times and record the chromatogram. Using Peak 2 (chlorogenic acid) as the reference peak, calculate the relative retention time and relative peak area of each common peak. The results show that the RSD of the relative retention time of each common peak is less than 0.46%, and the RSD of the relative peak area is less than 2.53% (n = 6), indicating that the precision of this method is good. The specific experimental results are shown in Tables 1 and 2.

[0142] Table 1 Evaluation Results of Relative Retention Time

[0143]

[0144]

[0145] Table 2 Evaluation Results of Relative Peak Area

[0146]

[0147] 2. Stability

[0148] Take the same batch of Jingyin Granules samples (batch number 200205), and after standing for 0, 2, 4, 8, 12, and 24 hours respectively, prepare the test solution (S1) according to the detection method of the fingerprint of Jingyin Granules in Example 1 above and then detect it. Record the chromatogram. Using Peak 2 (chlorogenic acid) as the reference peak, calculate the relative retention time and relative peak area of each common peak. The results show that the RSD of the relative retention time of each common peak is less than 0.16%, and the RSD of the relative peak area is less than 2.91% (n = 6), indicating that the test sample is stable within 24 hours. The specific experimental results are shown in Tables 3 and 4.

[0149] Table 3 Evaluation Results of Relative Retention Time

[0150]

[0151]

[0152] Table 4 Evaluation results of relative peak areas

[0153]

[0154] 3. Repeatability

[0155] Take 6 portions of the same batch of Jingyin Granules samples (batch number 200205). After preparing the test solution (S1) according to the detection method of the fingerprint spectrum of Jingyin Granules in Example 1 above, perform detection and record the chromatogram. Using peak 2 (chlorogenic acid) as the reference peak, calculate the relative retention time and relative peak area of each common peak. The results show that the RSD of the relative retention time of each common peak is less than 0.38%, and the RSD of the relative peak area is less than 3.00% (n = 6), indicating that this method has good repeatability. The specific experimental results are shown in Tables 5 and 6.

[0156] Table 5 Evaluation results of relative retention times

[0157]

[0158] Table 6 Evaluation results of relative peak areas

[0159]

[0160]

[0161] Example 6

[0162] 1. Sample pretreatment

[0163] Preparation of the test solution: The same as the preparation process of the test solution in step 1 of Example 1 to obtain the test solution 1*.

[0164] Weigh appropriate amounts of protocatechuic acid, caffeic acid, 5-O-methylvisammioside, arctiin, and chlorogenic acid, and place them in 5 mL volumetric flasks respectively. Add 50% methanol aqueous solution to dilute and make up the volume to prepare the reference substance solution. The reference substance solution is prepared by gradually diluting the reference substance stock solution. In the reference substance stock solution, the concentration of protocatechuic acid is 0.256 mg / mL, caffeic acid 0.203 mg / mL, 5-O-methylvisammioside 0.207 mg / mL, arctiin 0.508 mg / mL, and chlorogenic acid 0.824 mg / mL. The reference substance stock solution needs to be sealed and stored for future use.

[0165] The content range of protocatechuic acid in the reference substance solution is 0.016 - 0.256 mg / mL, the content range of caffeic acid is 0.013 - 0.203 mg / mL, the content range of 5-O-methylvisamminol glycoside is 0.013 - 0.207 mg / mL, the content range of arctiin is 0.032 - 0.508 mg / mL, and the content range of chlorogenic acid is 0.052 - 0.824 mg / mL.

[0166] 2. Chromatographic conditions

[0167] The chromatographic conditions of the high performance liquid chromatography are the same as those of the high performance liquid chromatography in step 2 of Example 1.

[0168] 3. Determination

[0169] Using the external standard method, a series of reference substance solutions with different volumes are respectively pipetted to prepare a series of solutions with different concentrations. The prepared solutions are analyzed by injection using a high performance liquid chromatograph, and a standard working curve is plotted. Then, the obtained test sample solution is analyzed by injection using a high performance liquid chromatograph, and the analysis results are substituted into the standard working curve to obtain the contents of the 5 components in the test sample solution.

[0170] Specifically, a series of reference substance solutions with different volumes are respectively pipetted to prepare a series of solutions with different concentrations. The prepared solutions are analyzed by injection using a high performance liquid chromatograph to obtain the linear relationship between the contents of the 5 components and the peak areas in the reference substance solution. Taking the chromatographic peak area of each component corresponding to its corresponding content, the corresponding standard working curves are plotted, and the regression equations of the standard working curves are calculated. Then, the test sample solution is detected by a high performance liquid chromatograph. The chromatographic peak areas of the 5 components in the obtained test sample solution are respectively substituted into the regression equations of the standard working curves to obtain the contents of the corresponding components.

[0171] Example 7

[0172] 1. Sample pretreatment

[0173] Preparation of the test sample solution: The preparation process of the test sample solution is the same as that in step 1 of Example 2 to obtain the test sample solution 2*.

[0174] Preparation of the reference substance solution: The preparation process of the reference substance solution is the same as that in step 1 of Example 6.

[0175] 2. Chromatographic conditions

[0176] The chromatographic conditions of the high performance liquid chromatography are the same as those of the high performance liquid chromatography in step 2 of Example 2.

[0177] 3. Determination

[0178] The measurement process is the same as that in Step 3 of Example 6.

[0179] Example 8

[0180] Accurately measure the reference substance stock solution in Step 1 of Example 6, and dilute it 0, 2, 4, 6, 8, and 10 times with 50% methanol aqueous solution to obtain a series of reference substance solutions with different mass concentrations. Inject and measure according to the chromatographic conditions in Step 2 of Example 6, and record the chromatogram. Use the reference substance mass concentration as the abscissa (X) and the peak area as the ordinate (Y) to plot the standard curve and perform linear regression calculation to obtain the regression equation, correlation coefficient, and linear range. The specific results are shown in Table 7. It can be seen from Table 7 that the linear relationship is good when injecting samples within the corresponding concentration range, and the correlation coefficient r is not less than 0.9995.

[0181] Table 7 Results of linear investigation of 5 components

[0182]

[0183] Example 9

[0184] 1. Specificity experiment

[0185] Take the Jingyin Granules sample (batch number 200205) and prepare the test solution (S1) according to the method in Step 1 of Example 6 above. At the same time, prepare the reference substance solution according to the method in Step 1 of Example 6 above, inject and measure according to the chromatographic conditions in Step 2 of Example 6 above, and record the chromatogram. The results show that the resolution and peak shape symmetry of the chromatographic peaks of the 5 components to be measured are good compared with the adjacent chromatographic peaks. At the positions corresponding to the reference chromatograms of protocatechuic acid, caffeic acid, 5-O-methylvisammioside, arctiin, and chlorogenic acid, corresponding chromatographic peaks appear in the chromatogram of the test solution.

[0186] 2. Precision

[0187] Prepare the reference substance solution according to the method in Step 1 of Example 6 above, and continuously inject and measure 6 times according to the chromatographic conditions in Step 2 of Example 6 above, and record the chromatogram. Take the 2nd peak (chlorogenic acid) as the reference peak, and calculate the relative retention time and relative peak area of each common peak. The results show that the RSD of the relative retention time of each common peak is less than 3%, and the RSD of the relative peak area is less than 3% (n = 6), indicating that the precision of this method is good.

[0188] 3. Stability

[0189] Prepare the reference substance solution according to the method in Step 1 of Example 6 above. After standing for 0, 2, 4, 8, 12, and 24 hours respectively, inject and measure according to the chromatographic conditions in Step 2 of Example 6 above, and record the chromatogram. Using Peak 2 (chlorogenic acid) as the reference peak, calculate the relative retention time and relative peak area of each common peak. The results show that the RSD of the relative retention time of each common peak is less than 3%, and the RSD of the relative peak area is less than 3% (n = 6), indicating that the test sample is stable within 24 hours.

[0190] 4. Repeatability

[0191] Prepare 6 portions of the reference substance solution according to the method in Step 1 of Example 6 above. Inject and measure according to the chromatographic conditions in Step 2 of Example 6 above, and record the chromatogram. Using Peak 2 (chlorogenic acid) as the reference peak, calculate the relative retention time and relative peak area of each common peak. The results show that the RSD of the relative retention time of each common peak is less than 3%, and the RSD of the relative peak area is less than 3% (n = 6), indicating that this method has good repeatability.

[0192] 5. Recovery rate of added samples

[0193] Take the Jingyin Keli samples with known content, and prepare 9 portions of the test sample solution in parallel according to the method in Step 1 of Example 6 above. At the same time, prepare a mixed reference substance solution with the same component content as the Jingyin Keli samples according to the method in Step 1 of Example 6 above. Add the mixed reference substance solution to the test sample solution at 0.5, 1.0, and 1.5 times the content of the Jingyin Keli samples, and mix well. The results show that the average recovery rate of protocatechuic acid, caffeic acid, 5-O-methylvisammioside, arctiin, and chlorogenic acid is 95% - 105%, and the RSD ≤ 3.0% (n = 9), indicating that the method has good accuracy.

[0194] Example 10

[0195] Take 12 batches of Jingyin Granule samples (batch numbers 200205, 200301, 200302, 200303, 200304, 200305, 220703, 220704, 220705, 220706, 220707, 220708), and prepare test solution (S1 - S12) respectively according to the method in step 1 of the above Example 6. Inject and determine according to the chromatographic conditions in step 2 of the above Example 6, record the chromatogram, and calculate the sample content by the external standard method. The specific experimental results are shown in Table 8. The results in Table 8 show that the contents of protocatechuic acid, caffeic acid, 5-O-methylvisamminol glycoside, arctiin, and chlorogenic acid in 12 batches of Jingyin Granule samples are 0.074 - 0.092 mg / g, 0.023 - 0.044 mg / g, 0.024 - 0.033 mg / g, 0.154 - 0.246 mg / g, and 0.113 - 0.191 mg / g respectively. It can be seen that this method can effectively determine the contents of 5 components in Jingyin Granule samples.

[0196] Table 8 Determination results of the contents of 5 components in 12 batches of Jingyin Granule (n = 3, mg / g)

[0197]

[0198] Example 11

[0199] Adopt the sample pretreatment steps in the above Example 1 to prepare the test solution.

[0200] Respectively take 9 kinds of medicinal materials including Herba Schizonepetae, Flos Lonicerae, Folium Ilicis Purpureae, Herba Houttuyniae, Folium Isatidis, Herba Taraxaci, Fructus Arctii, Radix Saposhnikoviae, and Radix Glycyrrhizae, and adopt the sample pretreatment steps in the above Example 1 to prepare 9 kinds of single-herb sample solutions.

[0201] Adopt steps 2 and 3 in the detection method of the fingerprint spectrum of Jingyin Granule in Example 1 to determine the test solution and 9 kinds of single-herb sample solutions respectively, and obtain the fingerprint spectra of the test solution and 9 kinds of single-herb sample solutions respectively. Import the obtained fingerprint spectra of the test solution and 9 kinds of single-herb sample solutions into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" software version 2012 released by the National Pharmacopoeia Commission for analysis and processing. At the same time, obtain the reference fingerprint spectrum of Jingyin Granule by using the detection method of the fingerprint spectrum of Jingyin Granule in Example 1. Compare the fingerprint spectra of the test solution and 9 kinds of single-herb sample solutions with the reference fingerprint spectrum of Jingyin Granule respectively, and identify the corresponding characteristic peaks of 9 kinds of single-herb sample solutions in the reference fingerprint spectrum of Jingyin Granule through the relative retention time, so as to attribute and locate the characteristic peaks in the fingerprint spectra of 9 kinds of single-herb sample solutions. The specific results are shown in Figure 2 .

[0202] From Figure 2It can be seen that in the sample solutions of single herbs, the specific conditions of the common fingerprint peaks in the fingerprint spectra of the sample solutions of 9 herbs, namely Schizonepeta tenuifolia Briq., Lonicera japonica Thunb., Ilex chinensis Sims., Houttuynia cordata Thunb., Isatis indigotica Fort., Taraxacum mongolicum Hand.-Mazz., Arctium lappa L., Saposhnikovia divaricata (Turcz.) Schischk., and Glycyrrhiza uralensis Fisch., are as follows. Specifically, it is determined that peak 1 comes from Ilex chinensis Sims. and Houttuynia cordata Thunb., peak 2 comes from Schizonepeta tenuifolia Briq. and Ilex chinensis Sims., peak 3 comes from Schizonepeta tenuifolia Briq., Ilex chinensis Sims., Houttuynia cordata Thunb., Isatis indigotica Fort., Taraxacum mongolicum Hand.-Mazz., Arctium lappa L., Saposhnikovia divaricata (Turcz.) Schischk., and Glycyrrhiza uralensis Fisch., peak 4 comes from Lonicera japonica Thunb., Ilex chinensis Sims., Houttuynia cordata Thunb., Taraxacum mongolicum Hand.-Mazz., and Arctium lappa L., peak 5 comes from Schizonepeta tenuifolia Briq., Ilex chinensis Sims., Isatis indigotica Fort., and Taraxacum mongolicum Hand.-Mazz., peak 6 comes from Lonicera japonica Thunb. and Arctium lappa L., peak 7 comes from Saposhnikovia divaricata (Turcz.) Schischk. and Glycyrrhiza uralensis Fisch., peak 8 comes from Glycyrrhiza uralensis Fisch., peak 9 comes from Lonicera japonica Thunb., Ilex chinensis Sims., Houttuynia cordata Thunb., Isatis indigotica Fort., and Glycyrrhiza uralensis Fisch., peak 10 comes from Schizonepeta tenuifolia Briq., Lonicera japonica Thunb., Taraxacum mongolicum Hand.-Mazz., and Saposhnikovia divaricata (Turcz.) Schischk., peak 11 comes from Lonicera japonica Thunb. and Arctium lappa L., peak 12 comes from Glycyrrhiza uralensis Fisch., peak 13 comes from Lonicera japonica Thunb. and Houttuynia cordata Thunb., peak 14 comes from Schizonepeta tenuifolia Briq. and Saposhnikovia divaricata (Turcz.) Schischk., peak 15 comes from Schizonepeta tenuifolia Briq., Lonicera japonica Thunb., Ilex chinensis Sims., and Arctium lappa L., and peak 16 comes from Arctium lappa L.

[0203] It can be seen that the chemical characteristic peaks of the 9 herbs in Jingyin Granules are well reflected in this fingerprint spectrum and their attributions are confirmed.

[0204] In summary, the Jingyin Granules provided by the present invention and the detection method and application of its fingerprint spectrum can obtain Jingyin Granules with excellent quality, and also establish a high-performance liquid chromatography fingerprint spectrum of Jingyin Granules, which is beneficial to the overall control of the internal quality of Jingyin Granules and provides a scientific experimental basis for better establishing an overall quality control evaluation system for Jingyin Granules. Therefore, the present invention overcomes various shortcomings in the prior art and has high industrial utilization value.

[0205] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A detection method for the fingerprint spectrum of Jingyin Granules, comprising the following steps: 1) Preparation of the test solution: Add the Jingyin Granules sample to the first solvent, perform ultrasonic extraction and centrifugation, take the supernatant to obtain the test solution; 2) Preparation of the reference solution: Provide a reference solution, which includes protocatechuic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, prim-O-glucosylcimifugin, liquiritin, rutin, luteoloside, isochlorogenic acid B, isochlorogenic acid A, 5-O-methylvisamminol glycoside, isochlorogenic acid C, arctiin, and the first solvent; 3) Determination: Use high performance liquid chromatography under the same chromatographic conditions to separately determine the test solution and the reference solution, obtain the fingerprint spectrum of the test solution and the fingerprint spectrum of the reference solution, compare the fingerprint spectrum of the test solution with the fingerprint spectrum of the reference solution, and conduct attribution and positioning of the target components in the fingerprint spectrum of the test solution, so as to obtain the fingerprint spectrum of Jingyin Granules; In step 1) or 2), the first solvent is an aqueous methanol solution; In step 3), in the determination conditions of the high performance liquid chromatography, the chromatographic column is a Waters CORTECS C 18 chromatographic column, 150 mm × 4.6 mm, 2.7 μm; the detector is a photodiode array detector; the mobile phase is 0.05 - 0.2% phosphoric acid aqueous solution - acetonitrile, wherein, phase A is 0.05 - 0.2% phosphoric acid aqueous solution, and phase B is acetonitrile; gradient elution; The specific program of the gradient elution is as follows: 0 - 3 min, the volume ratio of phase A to phase B is 95:5 - 95:5; 3 - 16 min, the volume ratio of phase A to phase B is 95:5 - 90:10; 16 - 21 min, the volume ratio of phase A to phase B is 90:10 - 88:12; 21 - 35 min, the volume ratio of phase A to phase B is 88:12 - 83:17; 35 - 55 min, the volume ratio of phase A to phase B is 83:17 - 70:30; 55 - 65 min, the volume ratio of phase A to phase B is 70:30 - 55:45; 65 - 66 min, the volume ratio of phase A to phase B is 55:45 - 5:

95.

2. The detection method for the fingerprint spectrum of Jingyin Granules according to claim 1, characterized in that, in step 1), it includes any one or more of the following conditions: A1) In step 1), the mass ratio of the added Jingyin Granules sample to the volume of the added first solvent is 2.5:20 - 30, g / mL; A2) In step 1), the ultrasonic extraction time is 25 - 35 min; A3) In step 1), the ultrasonic extraction power is 150 - 250 W, and the ultrasonic extraction frequency is 30 - 50 kHz; A4) In step 1), after ultrasonic extraction, it is necessary to weigh precisely again and make up the weight loss with the second solvent; the second solvent is an aqueous solution containing 65 - 75% methanol by volume percentage; A5) In step 1), the centrifugation conditions are: the centrifugation time is 5 - 15 min; the centrifugation rate is 5000 - 7000 rpm; A6) In step 1), the supernatant can directly take the supernatant after centrifugation or be obtained by filtration.

3. The detection method for the fingerprint spectrum of Jingyin Granules according to claim 1, characterized in that, in step 3), the determination conditions of the high performance liquid chromatography method include any one or more of the following: B1) The column temperature is 30 - 40 °C; B2) The injection volume is 5 - 15 μL; B3) The flow rate is 0.5 - 2.0 mL / min; B4) The detection wavelength is 205 - 215 nm.

4. A method for constructing the standard fingerprint of Jingyin Granules, comprising: Detecting multiple batches of Jingyin Granule samples respectively by using the detection method of the fingerprint of Jingyin Granules described in any one of claims 1-3, obtaining a common mode control map of the fingerprints of multiple batches of Jingyin Granule samples, taking the chromatographic peaks existing in all the maps as common characteristic peaks, determining the relative retention time of the common characteristic peaks, the ratio of the area of each common characteristic peak to the total peak area, and attributing and positioning the index components in the fingerprint of Jingyin Granules according to the relative retention time, so as to establish the standard fingerprint of Jingyin Granules.

5. The method for constructing the standard fingerprint of Jingyin Granules according to claim 4, characterized in that The relative retention time of the standard fingerprint of Jingyin Granules is the ratio of the retention time of each common characteristic peak to the retention time of the reference peak, including 16 common characteristic peaks. Taking the 2nd peak as the reference peak S peak, the retention time is 1.000, and the relative retention times of the other 15 common characteristic peaks are in turn: the 1st peak is 0.625, the 3rd peak is 1.080, the 4th peak is 1.517, the 5th peak is 1.609, the 6th peak is 1.739, the 7th peak is 3.268, the 8th peak is 3.561, the 9th peak is 3.808, the 10th peak is 4.008, the 11th peak is 4.543, the 12th peak is 4.624, the 13th peak is 4.662, the 14th peak is 4.767, the 15th peak is 5.216, the 16th peak is 6.105; the relative deviation of the relative retention times of the 15 common characteristic peaks except the 2nd peak is ≤±0.24%.

6. The method for constructing the standard fingerprint of Jingyin Granules according to claim 4, characterized in that In the standard fingerprint of Jingyin Granules, 13 fingerprint peaks are determined: the 1st peak is protocatechuic acid, the 2nd peak is chlorogenic acid, the 5th peak is caffeic acid, the 6th peak is cryptochlorogenic acid, the 7th peak is prim-O-glucosylcimifugin, the 8th peak is liquiritin, the 9th peak is rutin, the 10th peak is luteoloside, the 11th peak is isochlorogenic acid B, the 13th peak is isochlorogenic acid A, the 14th peak is 5-O-methylvisammioside, the 15th peak is isochlorogenic acid C, the 16th peak is arctiin.

7. A quality detection method for the fingerprint of Jingyin Granules, comprising: Obtaining the fingerprint of Jingyin Granules by using the detection method of the fingerprint of Jingyin Granules described in any one of claims 1-3, and comparing the similarity with the standard fingerprint of Jingyin Granules obtained by using the method for constructing the standard fingerprint of Jingyin Granules described in any one of claims 4-6.

8. The quality detection method for the fingerprint of Jingyin Granules according to claim 7, characterized in that The similarity ≥0.

97.

9. A detection method for the content of 5 components in Jingyin Granules, comprising the following steps: A) Preparation of the test solution: The same as step 1) of the detection method of the fingerprint of Jingyin Granules described in any one of claims 1-3; B) Preparation of the reference solution: Providing a reference solution, which includes protocatechuic acid, caffeic acid, 5-O-methylvisammioside, arctiin, chlorogenic acid and a first solvent; C) Determination: Using high performance liquid chromatography under the same chromatographic conditions as those in the detection method of the fingerprint spectrum of Jingyin Granules described in any one of claims 1 - 3, respectively determine the test solution in step A) and the reference solution in step B), and calculate the contents of 5 components in the test solution by the external standard method.

10. A method for attributing and locating characteristic peaks of the fingerprint spectrum of Jingyin Granules, comprising the following steps: a) Preparation of the sample solution of single herbs: Take any one or more of the 9 herb samples including Schizonepeta tenuifolia Briq., Lonicera japonica Thunb., Ilex chinensis Sims., Houttuynia cordata Thunb., Isatis indigotica Fort., Taraxacum mongolicum Hand.-Mazz., Arctium lappa L., Saposhnikovia divaricata (Turcz.) Schischk., and Glycyrrhiza uralensis Fisch. in Jingyin Granules, and prepare them according to step 1) of the detection method of the fingerprint spectrum of Jingyin Granules described in any one of claims 1 - 3 to obtain at least one sample solution of single herbs respectively; b) Determination: Using high performance liquid chromatography under the same chromatographic conditions as those in step 3) of the detection method of the fingerprint spectrum of Jingyin Granules described in any one of claims 1 - 3 to determine the sample solution of single herbs and obtain the fingerprint spectrum of the sample solution of single herbs; c) Obtaining the reference fingerprint spectrum: Using the same steps as those in the detection method of the fingerprint spectrum of Jingyin Granules described in any one of claims 1 - 3 to obtain the reference fingerprint spectrum of Jingyin Granules; d) Quality inspection: Compare the fingerprint spectrum of the sample solution of single herbs with the reference fingerprint spectrum of Jingyin Granules, and identify the corresponding characteristic peaks of the sample solution of single herbs in the reference fingerprint spectrum of Jingyin Granules through the relative retention time, so as to attribute and locate the characteristic peaks in the fingerprint spectrum.

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