A quality detection method for a traditional Chinese medicine compound preparation for treating dyspepsia

Abstract

The present invention is a quality detection method for a Chinese medicine compound preparation for treating dyspepsia. A quality detection method for a Chinese medicine compound preparation for treating dyspepsia comprises the following steps: (1) preparing a test solution: completely dissolving the Chinese medicine compound preparation for treating dyspepsia in a methanol solution, ultrasonically treating for 20-60 minutes, and filtering to obtain a test solution; (2) preparing a reference solution: dissolving chlorogenic acid, rosmarinic acid, limonene glycoside, artemisinic acid and vitexin in methanol, filtering through micropores to obtain a reference solution; (3) detecting: 5-20 μl of the reference solution and the test solution are respectively drawn, injected into a high performance liquid chromatograph, and the chromatogram within 0-95 minutes is recorded. The quality detection method for a Chinese medicine compound preparation for treating dyspepsia described in the present invention can more objectively, comprehensively and accurately control the quality of the Chinese medicine compound preparation.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a quality detection method for a traditional Chinese medicine compound preparation for treating dyspepsia. Background Art

[0002] Functional dyspepsia is the most common functional gastrointestinal disease in clinical practice. Clinically, it manifests as a syndrome of upper abdominal pain or heartburn, upper abdominal fullness and early satiety after meals, which may be accompanied by a clinical syndrome of loss of appetite, nausea or vomiting. With the development of the times, more and more people choose Chinese medicine for treatment. Chinese medicine defines diseases based on symptoms and treats them based on syndrome differentiation. Since ancient times, there has been no such disease name as functional dyspepsia. For diseases caused by food digestion and absorption disorders, Chinese medicine has diseases such as "fullness", "epigastric pain", "noisy", "belching", "hiccups", and "vomiting".

[0003] Artemisia rupestris L. is the whole herb of Asteraceae plant Artemisia rupestris L., which is included in the Uyghur volume of "Chinese Materia Medica". It has the functions of clearing away heat and relieving fever, relieving inflammation and pain, cooling blood and detoxifying, strengthening the stomach and digestion, and promoting blood circulation and removing blood stasis. The chemical composition of Xinjiang Artemisia rupestris L. was studied, and keto acids, flavonoids, volatile oils, alkaloids, amino acids, lactones and other components were isolated from it. The pharmacological activity test of the effective part of Artemisia rupestris L. showed that it has the functions of protecting liver and reducing enzymes, aiding digestion, anti-allergy, anti-inflammatory, anti-oxidation, anti-virus, anti-esophageal reflux, immunomodulation, and inducing cancer cell apoptosis. Pinne is a plant of the Lamiaceae family. Its Uyghur name is "Pinne". It is a second-level dry heat herb with the functions of dispersing qi, sweating, anti-inflammatory, enhancing metabolism, calming the nerves, and expelling non-mucus from the body. It can dispel wind and heat, relieve the symptoms and harmonize the middle, regulate qi and relieve pain. It is used for colds, coughs, headaches, sore throats, stomachaches, abdominal distension, vomiting and diarrhea, dysmenorrhea, limb numbness, and swelling and pain from falls. Haoping Granule is a preparation of Xinjiang Uyghur Medical Hospital. It is composed of two medicinal materials, Artemisia striata and Pinne. It has the effects of relieving the symptoms and harmonizing the middle, regulating qi and relieving pain, and strengthening the stomach and digestion. Artemisia striata is often used to treat food stagnation, stomachache, abdominal distension, vomiting and diarrhea. It has rich experience in medication and has shown good therapeutic effects in the treatment of functional dyspepsia.

[0004] At present, the quality inspection methods for Chinese herbal compound preparations have the defect that they can only partially inspect a single or several components and cannot comprehensively characterize the Chinese herbal ingredients.

[0005] In view of this, the present invention proposes a quality detection method for a Chinese herbal compound preparation for treating dyspepsia, which is convenient, easy to implement and contains relatively comprehensive information. Summary of the invention

[0006] The purpose of the present invention is to provide a quality detection method for a Chinese medicine compound preparation for treating dyspepsia, which can fully reflect the components of the Chinese medicine compound preparation.

[0007] In order to achieve the above purpose, the technical solution adopted is:

[0008] A quality detection method for a Chinese medicinal compound preparation for treating dyspepsia comprises the following steps:

[0009] (1) Preparation of test solution:

[0010] The Chinese herbal compound preparation for treating dyspepsia is completely dissolved in a methanol solution, ultrasonically treated for 20-60 minutes, and then filtered to obtain a test solution;

[0011] (2) Preparation of reference solution:

[0012] After dissolving chlorogenic acid, rosmarinic acid, montanol, artemisinic acid and vitexin in methanol, the solution was filtered through a micropore to obtain a reference solution;

[0013] (3) Detection:

[0014] 5-20 μl of the reference solution and the test solution are respectively taken and injected into a high performance liquid chromatograph, and a chromatogram within 0-95 min is recorded; wherein the filler is octadecylsilane bonded silica gel; the mobile phase A is a formic acid solution with a volume fraction of 0.1-0.4%, and the mobile phase B is acetonitrile; the elution method is gradient elution; and the detector is a DAD detector.

[0015] Furthermore, the Chinese herbal compound preparation for treating indigestion contains Artemisia annua and Pinellia ternata medicinal materials.

[0016] Furthermore, in the step (1), the mass volume ratio of the Chinese herbal compound preparation for treating indigestion to the methanol solution is 0.2-1 g: 15-40 ml;

[0017] The volume fraction of the methanol solution is 20-70%;

[0018] The power of ultrasonic treatment is 140-240W, the frequency is 30-100kHz;

[0019] The pore size of the microporous filter membrane is 0.2-0.5μm.

[0020] Furthermore, in the step (1), the mass volume ratio of the Chinese herbal compound preparation for treating indigestion to the methanol solution is 0.2 g:20 ml;

[0021] The volume fraction of methanol solution is 40%;

[0022] The power of ultrasonic treatment was 200 W and the frequency was 60 kHz.

[0023] Furthermore, in the step (2), the concentrations of the solutes in the reference solution are: 5-15 μg / ml chlorogenic acid, 8-25 μg / ml rosmarinic acid, 8-20 μg / ml montana glycosides, 45-65 μg / ml artemisinic acid, and 2-12 μg / ml of vitexin.

[0024] Furthermore, in the step (3), the column temperature is 20-35°C;

[0025] Flow rate is 0.5-1.5ml / min;

[0026] The detection wavelength is 200-254nm.

[0027] Furthermore, in the step (3), the column temperature is 30°C;

[0028] Mobile phase A was a 0.1% volume fraction of formic acid in water;

[0029] Flow rate: 1.0 ml / min;

[0030] The detection wavelength is 240nm.

[0031] Furthermore, in the step (3), the relative retention time of the reference peak is taken as 1, and the relative retention time of each characteristic peak is calculated to generate a fingerprint spectrum or a characteristic spectrum.

[0032] Furthermore, in the step (3), the reference peak is artemisinic acid, and the theoretical plate number is not less than 10,000.

[0033] Furthermore, in step (3), the volume fractions of mobile phases A and B in gradient elution are changed as follows:

[0034] 0-30min, phase A 98%→70%, phase B 2%→30%;

[0035] 30-40min, phase A 70%, phase B 30%;

[0036] 40-78min, phase A 70%→60%, phase B 30%→40%;

[0037] 78-85min, phase A 60%→48%, phase B 40%→52%;

[0038] 85-90min, phase A 48%→98%, phase B 52%→2%;

[0039] 90-95min, phase A 98%, phase B 2%.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] In recent years, multi-index component characteristic spectrum technology has begun to be widely used in the field of quality control of traditional Chinese medicine, providing an effective method for controlling the quality of traditional Chinese medicine compound and its preparations. The characteristic spectrum of traditional Chinese medicine has the characteristics of integrity, highlighting the complete appearance of traditional Chinese medicine, and providing a broader perspective for the quality control research of traditional Chinese medicine compound. High performance liquid chromatography is widely used in the detection of chemical components of traditional Chinese medicine with its advantages of high pressure, high sensitivity, high efficiency and automation, making the components of traditional Chinese medicine preparations more controllable. This technology combines extraction and separation technology, spectroscopy method and high performance liquid chromatography technology to clarify the main effective ingredients of traditional Chinese medicine, determine the index components that are focused on monitoring the characteristic spectrum, and not only show the overall characteristics of traditional Chinese medicine from a macroscopic perspective, but also monitor its multiple index components from a microscopic perspective, making the quality standards of traditional Chinese medicine preparations more perfect. It is necessary to establish a detection method for the characteristic spectrum of traditional Chinese medicine compound components on the basis of existing technologies in order to more comprehensively and effectively control the quality of the traditional Chinese medicine compound preparation, improve its quality control level, and ensure the safety and efficacy of clinical medication.

[0042] The quality detection method provided by the present invention has the characteristics of high accuracy, high stability and good repeatability through the establishment of the fingerprint spectrum of the Chinese medicine compound preparation. The present invention detects 10 different batches of Chinese medicine compound preparations, and the results show that the RSD of the relative retention time of the chromatographic peaks at the same position is within 10%, indicating that the fingerprint spectrum provided by the present invention has good reproducibility, and the method for establishing the fingerprint spectrum of the Chinese medicine compound preparation provided by the present invention has high reliability; and the fingerprint spectrum of the Chinese medicine compound preparation established by the above method of the present invention has 5 characteristic peaks, and the fingerprint spectrum can effectively characterize the quality of the Chinese medicine compound preparation, which is conducive to comprehensive monitoring of the quality of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is the atlas of mixed reference substances for compound preparations of traditional Chinese medicine;

[0044] Figure 2 Characteristic spectra of 10 batches of Chinese herbal compound preparations;

[0045] Figure 3 It is a spectrum of the medicinal flavor attribution of chromatographic peaks in Chinese herbal compound preparations. DETAILED DESCRIPTION

[0046] In order to further explain the quality detection method of a Chinese medicine compound preparation for treating dyspepsia of the present invention and achieve the expected purpose of the invention, the quality detection method of a Chinese medicine compound preparation for treating dyspepsia proposed according to the present invention, its specific implementation method, structure, characteristics and efficacy are described in detail as follows in combination with the preferred embodiments. In the following description, different "one embodiment" or "embodiment" does not necessarily refer to the same embodiment. In addition, specific features, structures or characteristics in one or more embodiments can be combined in any suitable form.

[0047] The quality detection method of a Chinese medicine compound preparation for treating dyspepsia of the present invention will be further described in detail below in conjunction with specific embodiments:

[0048] The present invention discloses a quality detection method for a Chinese medicine compound preparation for treating indigestion, which is a detection method for the characteristic spectrum of the Chinese medicine compound preparation. The method includes the preparation of a test solution; the preparation of a reference solution; and determination by liquid chromatography, taking the reference peak retention time as 1, calculating the relative retention time of the common peaks, and obtaining the characteristic spectrum of the Chinese medicine compound preparation. The method establishes the characteristic peaks of five components in the Chinese medicine compound preparation, selects artemisinic acid as the reference peak, and determines the relative retention time of each characteristic peak. The characteristic spectrum detection method for the components in the Chinese medicine compound preparation provided by the present invention has high sensitivity and precision, good stability and repeatability. Compared with the existing quality standards, this method can more objectively, comprehensively and accurately control the quality of the components in the Chinese medicine compound preparation, which is of great significance for ensuring clinical efficacy. The technical scheme of the present invention is:

[0049] (1) Optimization of chromatographic conditions: mobile phase A is formic acid solution, mobile phase B is acetonitrile; gradient elution, flow rate: 0.5-1.5 mL / min; injection volume 5-20 ul.

[0050] (2) Sample preparation

[0051] ①Prepare negative sample solution;

[0052] ②Prepare reference solution;

[0053] ③Prepare the test solution;

[0054] ④Precision detection test;

[0055] ⑤Repeatability test;

[0056] ⑥ Check the stability of the test solution.

[0057] (3) Determination

[0058] The test solution, negative solution and reference solution obtained in step (2) are injected into a high performance liquid chromatograph for determination according to the chromatographic conditions of step (1) to obtain a characteristic spectrum.

[0059] Preferably, the Chinese herbal compound preparation includes Artemisia annua and Pinellia ternata medicinal materials.

[0060] Further preferably, the amount of the Artemisia argyi used is 50-400 parts by weight, and the amount of the Pinnatifida used is 10-300 parts by weight.

[0061] Preferably, in the step (1), the chromatographic conditions are: a chromatographic column with octadecylsilane bonded silica gel as a filler, with a specification of 250×4.6 mm, 5 um; mobile phase A is a formic acid solution with a volume fraction of 0.1-0.4%, and mobile phase B is acetonitrile; gradient elution is detailed in Table 1; column temperature: 20-35°C.

[0062] Table 1 Gradient elution schedule

[0063]

[0064]

[0065] Further preferably, the column temperature is 30° C.; the mobile phase A is a 0.1% by volume formic acid aqueous solution; the flow rate is 1.0 ml / min; and the detection wavelength is 240 nm.

[0066] Preferably, in the step (2), the preparation method of the reference solution is: take appropriate amounts of chlorogenic acid reference, rosmarinic acid reference, limonene glycoside reference, artemisinic acid reference and vitexin reference, accurately weigh them, and add methanol to prepare mixed reference solutions, wherein the concentrations of the solutes in the mixed reference solutions are 5-15 μg / ml of chlorogenic acid, 10-25 μg / ml of rosmarinic acid, 10-20 μg / ml of limonene glycoside, 50-65 μg / ml of artemisinic acid, and 2-12 μg / ml of vitexin, respectively.

[0067] Preferably, in the step (2), the preparation method of the test solution is: take a sample, grind it, take about 0.2-1g, accurately weigh it, put it in a stoppered conical flask, accurately add 15-40ml of 20-70% methanol by volume fraction, weigh it, ultrasonically treat it (power 140-240W, frequency 30-100kHz) for 20-60 minutes, cool it, weigh it again, make up the lost weight with 20-70% methanol, shake it well, centrifuge it, take the supernatant, filter it (the pore size of the microporous filter membrane is 0.2-0.5μm), and take the filtrate to obtain it.

[0068] Further preferably, the mass volume ratio of the Chinese herbal compound preparation for treating dyspepsia to the methanol solution is 0.2 g:20 ml; the volume fraction of the methanol solution is 40%; the power of the ultrasonic treatment is 200 W, and the frequency is 60 kHz.

[0069] Preferably, in the step (2), the method for preparing the negative sample solution is: preparing negative samples lacking Artemisia uniflora and lacking Pinna respectively, and preparing the negative sample solution according to the preparation method of the test solution in step (2).

[0070] The precision test detection method is as follows: take the test solution prepared in step (2), inject it under the chromatographic conditions selected in step (1), and record the characteristic spectrum; use peak 4, artemisinic acid, as the reference peak S1, calculate the relative retention time of peaks 1-5, and use the relative retention time of each peak as a reference indicator.

[0071] The repeatability test detection method is as follows: take the test solution in step (2), repeat the injection 6 times according to the chromatographic conditions selected in step (1), and record the fingerprint spectrum; take peak 4, artemisinic acid, as the reference peak S1, calculate the relative retention time of peaks 1-5, and use the relative retention time of each peak as a reference indicator to calculate the RSD of the relative retention time.

[0072] The stability test method of the test solution is as follows: take the test solution prepared in step (2) and place it at room temperature for 0h, 3h, 6h, 9h, 12h, 24h, and 48h, then inject it under the chromatographic conditions selected in step (1) and record the characteristic spectrum; take peak 4, artemisinic acid, as the reference peak S1, calculate the relative retention time of peaks 1-5, and use the relative retention time of each peak as a reference index to calculate the RSD of the relative retention time.

[0073] Preferably, the reference peak is artemisinic acid, and the theoretical plate number, calculated based on artemisinic acid, is not less than 10,000.

[0074] The reference solution is tested under the same HPLC conditions as the test solution to obtain a chromatogram of the mixed reference solution; the chromatogram of the mixed reference solution is compared with the obtained fingerprint of the Chinese medicine compound preparation to identify the chromatographic peaks in the fingerprint of the Chinese medicine compound preparation. The characteristic spectrum presents 5 characteristic peaks, peak 1: chlorogenic acid; peak 2: rosmarinic acid; peak 3: montanol; peak 4 (S): artemisia ketone acid; peak 5: vitexin; peaks 4 and 5 are from Artemisia striata, and peaks 1 and 2 are from Pinna.

[0075] Further preferably, the characteristic spectrum has a relative retention time of 1 for the chromatographic peak of artemisinic acid, peak 4, and each characteristic peak is:

[0076] Peak 1: relative retention time 0.25-0.31,

[0077] Peak 2: relative retention time 0.61-0.75,

[0078] Peak 3: relative retention time 0.74-0.9,

[0079] Peak 4: relative retention time 1.000,

[0080] Peak 5: relative retention time 1.09-1.33.

[0081] Further preferably, the characteristic spectrum has a relative retention time of 1 for the chromatographic peak of artemisinic acid, peak 4, and each characteristic peak is:

[0082] Peak 1: relative retention time 0.29,

[0083] Peak 2: relative retention time 0.69,

[0084] Peak 3: relative retention time 0.83,

[0085] Peak 4: relative retention time 1.00,

[0086] Peak 5: relative retention time 1.22

[0087] The relative deviation of the retention time of each characteristic peak was ±10%.

[0088] In the embodiments of the present invention, the instruments and reagents used are as follows:

[0089] Instruments and equipment LC-20AT high performance liquid chromatograph, equipped with quaternary pump and DAD detector (Shimadzu Corporation, Japan); ME-204T electronic balance (Mettler-Toledo Instrument (Shanghai) Instrument Co., Ltd.); ME-55 electronic balance (Mettler-Toledo Instrument (Shanghai) Instrument Co., Ltd.); KQ-300 dual-frequency ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); TD52 desktop low-speed centrifuge (Hunan Kaida Industrial Co., Ltd.)

[0090] Reference substances, test substances and reagents:

[0091] Reference substances: artemisinic acid (batch number: GSB11-2530-2020, mass fraction 99.96%, Xinjiang Institute of Physical and Chemical Technology, Chinese Academy of Sciences), chlorogenic acid (batch number: 110753-202119, mass fraction 96.3%), rosmarinic acid (batch number: 111871-202007, mass fraction 98.1%), montana glycoside (batch number: 111528-202213, mass fraction 94.6%), and vitexin (batch number: 111554-201705, mass fraction 98.3%), China Food and Drug Inspection Institutes.

[0092] Test sample: The batch numbers of the Chinese medicine compound preparations used in the present invention are: 20210813, 20211028, 20211029, 20210908-1, 20210908-2, 20220314, 20220316, 20220161, 20220162, 20220237, and negative samples are all homemade. The Chinese medicine compound preparation includes Artemisia striata and Pinna, the amount of Artemisia striata is 50-400 parts by weight, and the amount of Pinna is 10-300 parts by weight.

[0093] The preparation methods of the test sample solutions of the above batches are the same, which are:

[0094] Take the above samples respectively, grind them into powder, take about 0.2g of the Chinese medicine compound preparation, weigh it accurately, put it in a stoppered conical flask, add 20ml of 40% methanol accurately, weigh it, and treat it ultrasonically (power 200W, frequency 60kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 40% methanol, shake it well, centrifuge it, take the supernatant, filter it, and take the filtrate to get the test solution.

[0095] Reagents: acetonitrile, chromatographic grade, Fisher Company, USA; water is pure water; other reagents are of analytical grade.

[0096] Example 1: Precision Experiment

[0097] Take the above samples respectively, grind them into powder, take about 0.2g of the Chinese medicine compound preparation, weigh it accurately, put it in a stoppered conical flask, add 20ml of 40% methanol accurately, weigh it, treat it ultrasonically (power 200W, frequency 60kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 40% methanol, shake it well, centrifuge it, take the supernatant, filter it, take the filtrate to get the test solution.

[0098] The following HPLC conditions were used for testing:

[0099] Chromatographic column: Diamonsil C18 column;

[0100] Detection wavelength: 240nm;

[0101] Mobile phase: Mobile phase A is 0.1% by volume formic acid aqueous solution; Mobile phase B is acetonitrile;

[0102] The elution method is gradient elution, and the elution program of the gradient elution is shown in Table 1;

[0103] Flow rate: 1.0 mL / min;

[0104] Column temperature: 30°C;

[0105] Column specifications: 250mm×4.6mm, 5μm;

[0106] Injection volume: 10 μL;

[0107] According to the above chromatographic conditions, 6 injections were performed continuously, and the artemisia ketoic acid peak was used as the reference peak. The relative retention time of each common peak and the reference peak was calculated, and the RSD value was calculated. The results are shown in Table 2. As can be seen from Table 2, the relative retention time RSD% of each common peak is less than 5%, indicating that the detection method provided by the present invention has good precision.

[0108] Table 2 Precision experiment (relative retention time of common peaks)

[0109]

[0110] Example 2: Repeatability Experiment

[0111] Take the Chinese medicine compound preparation (batch 20220162), weigh 6 portions in parallel, prepare 6 test solutions according to the preparation method of the test solution in Example 1, inject them respectively according to the chromatographic conditions in Example 1, take the artemisia ketoacid peak as the reference peak, calculate the relative retention time of each common peak and the reference peak, and calculate the RSD value. The results are shown in Table 3:

[0112] Table 3 Repeatability experiment (relative retention time of common peaks)

[0113]

[0114] Example 3: Stability test

[0115] Take the Chinese medicine compound preparation (batch 20220162), prepare the test solution according to the preparation method of the test solution in Example 1, and inject the sample at 0h, 3h, 6h, 9h, 12h, 24h, and 48h according to the chromatographic conditions in Example 1, and use the artemisia ketoacid peak as the reference peak to calculate the relative retention time of each common peak, and calculate the RSD value. The results are shown in Table 4:

[0116] Table 4 Stability experiment (relative retention time of common peaks)

[0117]

[0118] As shown in Table 4, the relative retention time RSD% of each common peak is less than 5%, indicating that the test solution is basically stable within 48 hours.

[0119] Example 4: Establishment of fingerprint of Chinese medicine compound preparation

[0120] (1) Preparation of test solution

[0121] Take the above 10 batches of Chinese medicine compound preparations, with batch numbers 20210813, 20211028, 20211029, 20210908-1, 20210908-2, 20220314, 20220316, 20220161, 20220162, 20220237, and number them S1, S2, S3, S4, S5, S6, S7, S8 respectively. , S9, S10, respectively take the above samples, grind them into powder, take about 0.2g, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of 40% methanol, weigh it, treat it with ultrasound (power 200W, frequency 60kHz) for 30 minutes, cool it, filter it (the pore size of the microporous filter membrane is 0.45μm), weigh it again, make up the lost weight with 40% methanol, and get 10 batches of test solutions of Chinese herbal compound preparations.

[0122] (2) Preparation of mixed reference solution

[0123] Take appropriate amounts of chlorogenic acid reference substance, rosmarinic acid reference substance, limonene glycoside reference substance, artemisinic acid reference substance and vitexin reference substance, accurately weigh them, add methanol to prepare a mixed reference solution, and obtain a reference solution. The mass concentrations of chlorogenic acid, rosmarinic acid, limonene glycoside, artemisinic acid and vitexin in the reference solution are 10μg / mL, 15μg / mL, 10μg / mL, 50μg / mL and 5μg / mL, respectively.

[0124] (3) HPLC conditions

[0125] Chromatographic column: Diamonsil C18 column;

[0126] Detection wavelength: 240nm;

[0127] Mobile phase: Mobile phase A is 0.1% by volume formic acid aqueous solution; Mobile phase B is acetonitrile;

[0128] The elution method is gradient elution, and the elution program of the gradient elution is shown in Table 1;

[0129] Flow rate: 1.0 mL / min;

[0130] Column temperature: 30°C;

[0131] Column specifications: 250mm×4.6mm, 5μm;

[0132] Injection volume: 10 μL;

[0133] (4) Accurately pipette 10 μL of the test solution and the reference solution, respectively, and perform the test using the above-mentioned HPLC conditions to obtain the liquid chromatograms of the test solution and the liquid chromatograms of the reference solution, respectively.

[0134] The reference peak is artemisinic acid, and the theoretical plate number is not less than 10,000.

[0135] Liquid chromatography of the reference solution Figure 1 As shown, Figure 1 Peak 1 is the chlorogenic acid reference substance, Peak 2 is the rosmarinic acid reference substance, Peak 3 is montmorillonate, Peak 4 is the artemisinic acid reference substance, and Peak 5 is the vitexin reference substance.

[0136] The liquid chromatography of the test solution is analyzed by liquid chromatography software, and the S1 test sample is used as the reference spectrum. The chromatogram is superimposed, i.e., the fingerprint spectrum of the Chinese herbal compound preparation, such as Figure 2 As shown, the relative retention time of each common peak and the reference peak was calculated, and the RSD value was calculated. The results are shown in Table 5:

[0137] As shown in Table 5, for the characteristic spectra of different batches of Chinese medicine compound preparations, the relative retention time RSD% of each common peak is less than 5%, indicating that the similarity between different batches is high and the testing method provided by the present invention has good stability.

[0138] Table 5 Relative retention time of chromatographic peaks of 10 batches of samples

[0139]

[0140]

[0141] Example 5: Chromatographic peak attribution

[0142] According to the method of Example 1, the positive control chromatograms and negative control chromatograms of Artemisia annua and Pinnatifida in the Chinese medicine compound preparation were respectively determined, and the ultraviolet absorption of the chromatographic peaks in the fingerprint of the Chinese medicine compound preparation and the positive control chromatograms and negative control chromatograms of each medicinal material were analyzed by DAD detector, and the retention time of the chromatographic peaks was compared. Finally, the attribution peaks of the common peaks in the fingerprint of the Chinese medicine compound preparation were confirmed, as shown in FIG. Figure 3 , from top to bottom, they are: mixed standard solution, preparation sample, Pinna medicinal material negative sample, Artemisia striata medicinal material negative sample. Peaks 1 and 2 are Pinna medicinal material components, peaks 4 and 5 are characteristic peaks of Artemisia striata medicinal material, and peak 3 is a common peak of the two medicinal materials.

[0143] Example 6: Establishment of fingerprint of Chinese medicine compound preparation

[0144] (1) Preparation of test solution

[0145] Take the above 10 batches of Chinese medicine compound preparations, with batch numbers of 20210813, 20211028, 20211029, 20210908-1, 20210908-2, 20220314, 20220316, 20220161, 20220162, and 20220237, and number them as S1, S2, S3, S4, S5, S6, S7, and S8, respectively. 8. S9 and S10, respectively take the above samples, grind them into powder, take about 1.0 g, weigh it accurately, put it in a stoppered conical flask, accurately add 40 ml of 20% methanol, weigh it, treat it with ultrasound (power 240 W, frequency 30 kHz) for 60 minutes, cool it, filter it (the pore size of the microporous filter membrane is 0.2 μm), weigh it again, make up the lost weight with 40% methanol, and obtain 10 batches of test solutions of Chinese herbal compound preparations.

[0146] (2) Preparation of mixed reference solution

[0147] Take appropriate amount of chlorogenic acid reference substance, rosmarinic acid reference substance, limonene glycoside reference substance, artemisinic acid reference substance and vitexin reference substance, accurately weigh, add methanol to make a mixed reference solution, and obtain a reference solution. The mass concentrations of chlorogenic acid, rosmarinic acid, limonene glycoside, artemisinic acid and vitexin in the reference solution are 15μg / mL, 25μg / mL, 20μg / mL, 65μg / mL and 12μg / mL, respectively.

[0148] (3) HPLC conditions

[0149] Chromatographic column: Diamonsil C18 column;

[0150] Detection wavelength: 254nm;

[0151] Mobile phase: Mobile phase A is 0.4% by volume formic acid aqueous solution; Mobile phase B is acetonitrile;

[0152] The elution method is gradient elution, and the elution program of the gradient elution is shown in Table 1;

[0153] Flow rate: 1.5 mL / min;

[0154] Column temperature: 35°C;

[0155] Column specifications: 250mm×4.6mm, 5μm;

[0156] Injection volume: 5 μL;

[0157] (4) Accurately pipette 5 μL of the test solution and the reference solution, respectively, and test using the above-mentioned HPLC conditions to obtain the liquid chromatograms of the test solution and the liquid chromatograms of the reference solution, respectively. The reference peak is artemisinic acid, and the theoretical plate number is not less than 10,000.

[0158] Example 7: Establishment of fingerprint of Chinese medicine compound preparation

[0159] (1) Preparation of test solution

[0160] Take the above 10 batches of Chinese medicine compound preparations, with batch numbers 20210813, 20211028, 20211029, 20210908-1, 20210908-2, 20220314, 20220316, 20220161, 20220162, 20220237, and number them S1, S2, S3, S4, S5, S6, S7, S8 respectively. , S9, S10, respectively take the above samples, grind them into powder, take about 0.3 g, weigh it accurately, put it in a stoppered conical flask, accurately add 15 ml of 70% methanol, weigh it, treat it with ultrasound (power 140 W, frequency 100 kHz) for 20 minutes, cool it, filter it (the pore size of the microporous filter membrane is 0.3 μm), weigh it again, make up the lost weight with 40% methanol, and get 10 batches of test solutions of Chinese herbal compound preparations.

[0161] (2) Preparation of mixed reference solution

[0162] Take appropriate amounts of chlorogenic acid reference substance, rosmarinic acid reference substance, limonene glycoside reference substance, artemisinic acid reference substance and vitexin reference substance, accurately weigh them, add methanol to prepare a mixed reference solution, and obtain a reference solution. The mass concentrations of chlorogenic acid, rosmarinic acid, limonene glycoside, artemisinic acid and vitexin in the reference solution are 5 μg / mL, 8 μg / mL, 8 μg / mL, 45 μg / mL and 2 μg / mL, respectively.

[0163] (3) HPLC conditions

[0164] Chromatographic column: Diamonsil C18 column;

[0165] Detection wavelength: 200nm;

[0166] Mobile phase: Mobile phase A is 0.2% by volume formic acid aqueous solution; Mobile phase B is acetonitrile;

[0167] The elution method is gradient elution, and the elution program of the gradient elution is shown in Table 1;

[0168] Flow rate: 0.5 mL / min;

[0169] Column temperature: 20°C;

[0170] Column specifications: 250mm×4.6mm, 5μm;

[0171] Injection volume: 20 μL;

[0172] (4) Accurately pipette 20 μL of the test solution and the reference solution, respectively, and test using the above-mentioned HPLC conditions to obtain the liquid chromatograms of the test solution and the liquid chromatograms of the reference solution, respectively. The reference peak is artemisinic acid, and the theoretical plate number is not less than 10,000.

[0173] The above is only a preferred embodiment of the embodiment of the present invention, and does not impose any form of limitation on the embodiment of the present invention. Any simple modification, equivalent changes and modifications made to the above embodiment based on the technical essence of the embodiment of the present invention are still within the scope of the technical solution of the embodiment of the present invention.

Claims

1. A quality detection method for a Chinese medicinal compound preparation for treating dyspepsia, characterized in that: The following steps are involved: (1) Preparation of test solution: The Chinese herbal compound preparation for treating dyspepsia is completely dissolved in a methanol solution, ultrasonically treated for 20-60 minutes, and then filtered to obtain a test solution; The Chinese herbal compound preparation for treating indigestion is Artemisia selengensis and Pinnatulina; (2) Preparation of reference solution: After dissolving chlorogenic acid, rosmarinic acid, montanol, artemisinic acid pair and vitexin in methanol, the solution was filtered through micropore to obtain a reference solution; (3) Testing: 5-20 μl of the reference solution and the test solution were respectively taken and injected into a high performance liquid chromatograph to record the chromatogram within 0-95 min; wherein the filler was octadecylsilane bonded silica gel, the mobile phase A was a formic acid solution with a volume fraction of 0.1-0.4%, and the mobile phase B was acetonitrile; the elution method was gradient elution; and the detector was a DAD detector; The volume fraction of mobile phases A and B in the gradient elution is changed as follows: 0-30min, phase A 98%→70%, phase B 2%→30%; 30-40min, phase A 70%, phase B 30%; 40-78min, phase A 70%→60%, phase B 30%→40%; 78-85min, phase A 60%→48%, phase B 40%→52%; 85-90min, phase A 48%→98%, phase B 52%→2%; 90-95min, phase A 98%, phase B 2%.

2. The quality inspection method according to claim 1, characterized in that: In the step (1), the mass volume ratio of the Chinese herbal compound preparation for treating indigestion to the methanol solution is 0.2-1 g: 15-40 ml; The volume fraction of methanol solution is 20-70%; The power of ultrasonic treatment is 140-240W, the frequency is 30-100kHz; The pore size of the microporous filter membrane is 0.2-0.5μm.

3. The quality inspection method according to claim 2, characterized in that: In the step (1), the mass volume ratio of the Chinese herbal compound preparation for treating indigestion to the methanol solution is 0.2 g:20 ml; The volume fraction of methanol solution is 40%; The power of ultrasonic treatment was 200 W and the frequency was 60 kHz.

4. The quality inspection method according to claim 1, characterized in that: In the step (2), the concentration of the solute in the reference solution is: chlorogenic acid 5-15 μg / ml, rosmarinic acid 8-25 μg / ml, montana glycoside 8-20 μg / ml, artemisinic acid 45-65 μg / ml, and vitexin 2-12 μg / ml.

5. The quality inspection method according to claim 1, characterized in that: In the step (3), the column temperature is 20-35°C; Flow rate is 0.5-1.5ml / min; The detection wavelength is 200-254nm.

6. The quality inspection method according to claim 5, characterized in that: In the step (3), the column temperature is 30°C; Mobile phase A was a 0.1% volume fraction of formic acid in water; Flow rate: 1.0 ml / min; The detection wavelength is 240nm.

7. The quality inspection method according to claim 1, characterized in that: In the step (3), the relative retention time of the reference peak is taken as 1, and the relative retention time of each characteristic peak is calculated to generate a fingerprint spectrum or a characteristic spectrum.

8. The quality inspection method according to claim 7, characterized in that: In the step (3), the reference peak is artemisinic acid, and the theoretical plate number is not less than 10,000.

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