Specific chromatogram establishment method of concha haliotidis compound traditional Chinese medicine preparation

Abstract

The invention provides a specific chromatogram establishing method of an abalone shell compound traditional Chinese medicine preparation, and relates to the technical field of medicine analysis. The method comprises the following steps: mixing a concha haliotidis compound traditional Chinese medicine preparation with a methanol aqueous solution (with the volume fraction of 75%), and carrying out ultrasonic extraction to obtain a test solution; performing high performance liquid chromatography detection on the test sample solution to obtain a liquid chromatogram of a test sample, comparing the liquid chromatogram of the test sample with a preset reference spectrum, and selecting a common peak from the liquid chromatogram of the test sample to obtain a characteristic spectrum of the concha haliotidis compound traditional Chinese medicine preparation; the reference spectrum is a liquid chromatogram obtained by a reference substance solution under the same high performance liquid chromatography detection conditions. The characteristic chromatogram obtained by the establishment method provided by the invention can comprehensively reflect the components of the concha haliotidis compound traditional Chinese medicine preparation, and the quality control of the concha haliotidis-containing traditional Chinese medicine preparation is realized; and the establishment method provided by the invention has the characteristics of high accuracy, high stability and good repeatability, and can realize full-appearance monitoring of the quality of the concha haliotidis preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for establishing a characteristic spectrum of a compound Chinese medicine preparation of Semen Cassiae. Background Art

[0002] The first batch of ancient classic prescriptions published by the State Food and Drug Administration includes Shijueming Powder. Its source is: "Puji Prescriptions" (Ming Zhu Su) "Shijueming Powder, treats wind poison gas attacking the head, causing dark eyes and poor vision." The prescription is: Shijueming, Qianghuo (remove the reed head), Caocassia, chrysanthemum, each 1 liang, and licorice (roasted, chopped) half a liang. Preparation and usage are as follows: The above is a powder, take 2 qian each time, with a cup of water. Decoction for 6 minutes, mix and dissolve, take warm after meals and before going to bed. Boil and disperse. This prescription is widely used in ophthalmology, but it is always related to the liver and gallbladder meridians, and is mostly used for liver heat and cataract eye diseases. Modern ophthalmology clinics also follow this purpose.

[0003] After that, according to the improvement of Shijueming powder, Nepeta replaces Qianghuo, and the prescription is Shijueming, Cassia seed, chrysanthemum, Nepeta, and licorice, and Shijueming Chinese medicine preparation is made. The formula first selects Cassia seed and Shijueming as the main medicine. Both Cassia seed and Shijueming have the effect of improving eyesight and are "ophthalmic holy medicines", so they are called Cassia. Cassia seed is also called grass Cassia, which is sweet, bitter, salty, and slightly cold in nature. It belongs to the liver and large intestine meridians. It has the functions of clearing heat and improving eyesight (removing heat evil in the body and promoting vision recovery), moistening the intestines and relieving constipation, and is used for red and astringent eyes, shameful eyes and tears, headache and dizziness, dark eyes, and constipation. Modern studies have shown that it has the effects of lowering blood pressure, lowering blood lipids, and inhibiting bacteria. Shijueming is salty and cold in nature, belongs to the liver meridian, and has the effects of calming the liver and suppressing yang (suppressing the excessive yang rising in the liver), clearing the liver and improving eyesight (clearing liver fire to promote vision recovery). It is often used for headache and dizziness, red eyes, blurred vision, and amblyopia. Current studies have shown that it has the effects of lowering blood pressure, anti-oxidation, and neutralizing gastric acid. The two are combined as the main medicine to synergistically treat headaches, dizziness, red and swollen eyes, dryness, blurred vision and other symptoms. Schizonepeta is slightly warm in nature and has the effect of relieving the exterior and dispersing wind with pungent and warm properties. It is used as a minister to assist the main medicines, Cassiae and Cassiae seeds, in their detoxification effects. Chrysanthemum is sweet and bitter, slightly cold, and enters the lung and liver meridians. It has the effects of dispersing wind-heat, calming the liver and improving eyesight, and clearing heat and detoxifying. It is suitable for wind-heat in the liver meridian, inflammation of liver fire, and red and swollen eyes. The liver opens up the eyes, and the eyes are red and swollen when the liver fire is strong. This prescription is combined with chrysanthemum as an adjuvant to enhance the effects of clearing heat and detoxifying, calming the liver and improving eyesight. It cooperates with licorice to introduce the drug into the liver meridian, and the combination of various drugs directly reaches the root of nourishing the liver and improving eyesight. Licorice, sweet and flat, has the effects of tonifying the spleen and replenishing qi, moistening the lungs and relieving cough, detoxifying, and harmonizing various drugs. Roasting with honey can warm and nourish the body, while using it raw can detoxify. Using it in compound prescriptions can alleviate the potency of other drugs and enable drugs of different properties to achieve a coordinated effect.

[0004] Due to the complex situation of multiple sources and production areas of Chinese medicinal materials, the quality of Chinese medicine products varies greatly, especially the amount of effective ingredients varies significantly. The characteristic spectrum of Chinese medicine is an effective method used to control the quality of Chinese medicine compound and its preparations in recent years. The characteristic spectrum technology of Chinese medicine provides a broader perspective for the quality control research of Chinese medicine compound. The characteristic spectrum of Chinese medicine has the characteristics of integrity, highlighting the complete appearance of Chinese medicine compound. At the same time, the characteristic spectrum of similar medicinal materials has the characteristics of similarity. Relying on this spectrum, the comprehensive evaluation of the intrinsic chemical components of Chinese medicine and the comprehensive control of the overall quality are realized, making the composition of Chinese medicine preparations more controllable.

[0005] At present, the above-mentioned improved traditional Chinese medicine preparations containing Semen Cassiae have not been included in any standard, lack of quality control, and cannot guarantee the clinical efficacy of this variety. It is of great significance to study and establish a map that can reflect the multi-component information of Semen Cassiae traditional Chinese medicine preparations and reflect their quality characteristics to ensure the quality of Semen Cassiae traditional Chinese medicine preparations. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a method for establishing a characteristic spectrum of a compound Chinese medicine preparation containing Semen Cassiae. The characteristic spectrum obtained by the establishment method provided by the present invention can comprehensively reflect the components of the compound Chinese medicine preparation containing Semen Cassiae, and realize the quality control of the Chinese medicine preparation containing Semen Cassiae.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for establishing a characteristic spectrum of a compound Chinese medicine preparation of Semen Cassiae, comprising the following steps:

[0009] The compound Chinese medicine preparation of Shijueming is mixed with methanol aqueous solution for ultrasonic extraction to obtain a test solution; the volume fraction of the methanol aqueous solution is 75%; the raw materials for preparing the compound Chinese medicine preparation of Shijueming include Shijueming, Cassia seed, Nepeta tenuifolia, chrysanthemum and liquorice;

[0010] The test solution is subjected to high performance liquid chromatography to obtain a liquid chromatogram of the test sample, the liquid chromatogram of the test sample is compared with a predetermined reference spectrum, and common peaks are selected from the liquid chromatogram of the test sample to obtain a characteristic spectrum of the Shijueming compound Chinese medicine preparation; the reference spectrum is a liquid chromatogram obtained under the same high performance liquid chromatography detection conditions of a reference solution, and the reference solution contains reference substances of caffeic acid, chlorogenic acid, liquiritin, luteolin, resveratrol, hesperidin and citrus cassia bark;

[0011] The conditions for the high performance liquid chromatography detection include:

[0012] The chromatographic column is Thermo Hypersil GOLD C 18 ;

[0013] The column temperature was 30°C;

[0014] The detection wavelength is 285nm;

[0015] The injection volume was 10 μL;

[0016] The mobile phase includes mobile phase A, mobile phase B and mobile phase C, wherein the mobile phase A is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution with a volume fraction of 0.1%, and the mobile phase C is methanol;

[0017] The flow rate of the mobile phase is 0.3 mL / min;

[0018] The elution program is a gradient elution program;

[0019] The gradient elution procedure is:

[0020] 0-9min: the volume percentage of mobile phase A increases from 1% to 6% at a constant rate, the volume percentage of mobile phase B decreases from 97% to 92% at a constant rate, and the volume percentage of mobile phase C remains at 2%;

[0021] 9-21min: the volume percentage of mobile phase A is maintained at 6%, the volume percentage of mobile phase B is maintained at 92%, and the volume percentage of mobile phase C is maintained at 2%;

[0022] 21-26 min: the volume percentage of mobile phase A increased from 6% to 11% at a constant rate, the volume percentage of mobile phase B decreased from 92% to 87% at a constant rate, and the volume percentage of mobile phase C remained at 2%;

[0023] 26-33 min: the volume percentage of mobile phase A was maintained at 11%, the volume percentage of mobile phase B was maintained at 87%, and the volume percentage of mobile phase C was maintained at 2%;

[0024] 33-36min: the volume percentage of mobile phase A increased from 11% to 13% at a constant rate, the volume percentage of mobile phase B decreased from 87% to 85% at a constant rate, and the volume percentage of mobile phase C was maintained at 2%;

[0025] 36-42min: the volume percentage of mobile phase A increased from 13% to 19% at a constant rate, the volume percentage of mobile phase B decreased from 85% to 79% at a constant rate, and the volume percentage of mobile phase C was maintained at 2%;

[0026] 42-46 min: the volume percentage of mobile phase A was maintained at 19%, the volume percentage of mobile phase B was maintained at 79%, and the volume percentage of mobile phase C was maintained at 2%;

[0027] 46-49min: the volume percentage of mobile phase A increased from 19% to 44% at a constant rate, the volume percentage of mobile phase B decreased from 79% to 54% at a constant rate, and the volume percentage of mobile phase C remained at 2%;

[0028] 49-55min: the volume percentage of mobile phase A is maintained at 44%, the volume percentage of mobile phase B is maintained at 54%, and the volume percentage of mobile phase C is maintained at 2%;

[0029] 55-55.5min: the volume percentage of mobile phase A was uniformly decreased from 44% to 1%, the volume percentage of mobile phase B was uniformly increased from 54% to 97%, and the volume percentage of mobile phase C was maintained at 2%;

[0030] 55.5-60 min: the volume percentage of mobile phase A was maintained at 1%, the volume percentage of mobile phase B was maintained at 97%, and the volume percentage of mobile phase C was maintained at 2%.

[0031] Preferably, the dosage ratio of the compound Chinese medicine preparation of Semen Cassiae to the methanol aqueous solution is 0.5 g: 15-20 mL.

[0032] Preferably, the frequency of the ultrasonic extraction is 40kHz and the power is 300W.

[0033] Preferably, the ultrasonic extraction time is 90 min.

[0034] Preferably, the concentration of each reference substance in the reference substance solution is 10 μg / mL.

[0035] Preferably, the chromatographic column has a length of 100 mm and an inner diameter of 2.1 mm.

[0036] Preferably, the concentration of the test solution is 0.02 g / mL.

[0037] Preferably, the amount of Semen Cassiae is 250 parts by mass, the amount of Cassia seed is 250 parts by mass, the amount of Schizonepeta tenuifolia is 250 parts by mass, the amount of chrysanthemum is 250 parts by mass, and the amount of liquorice is 125 parts by mass.

[0038] Preferably, the preparation method of the compound Chinese medicine preparation of Semen Cassiae comprises the following steps:

[0039] Add water to Cassiae Semen, Cassia Seed, Nepeta Tenuifolia, Chrysanthemum and Licorice and boil twice, filter the decoctions obtained from the two decoctions respectively, and combine the filtrates;

[0040] The combined filtrate is concentrated and then maltodextrin is added, followed by granulation and drying to obtain the compound Chinese medicine preparation of Semen Cassiae.

[0041] Preferably, the amount of water added for the first decoction is 12 times the mass of the raw material, the amount of water added for the second decoction is 10 times the mass of the raw material, the time for the first decoction is 2 hours, and the time for the second decoction is 1.5 hours.

[0042] The invention provides a method for establishing a characteristic spectrum of a compound Chinese medicine preparation of Cassia Cassiae, comprising the following steps: mixing the compound Chinese medicine preparation of Cassia Cassiae with a methanol aqueous solution for ultrasonic extraction to obtain a test solution; the volume fraction of the methanol aqueous solution is 75%; the raw materials for preparing the compound Chinese medicine preparation of Cassia Cassiae include Cassia Cassiae, Cassia Seed, Schizonepeta Tenuifolia, Chrysanthemum and Licorice; performing high performance liquid chromatography detection on the test solution to obtain a liquid chromatogram of the test sample, comparing the liquid chromatogram of the test sample with a predetermined reference spectrum, selecting common peaks from the liquid chromatogram of the test sample, and obtaining a characteristic spectrum of the compound Chinese medicine preparation of Cassia Cassiae; the reference spectrum is a liquid chromatogram obtained by a reference solution under the same high performance liquid chromatography detection conditions, and the reference solution contains reference substances of caffeic acid, chlorogenic acid, liquiritin, luteolin, resveratrol, hesperidin and citrus cassiae. According to the prescription composition of the Shijueming Chinese medicine preparation, the present invention studies and analyzes the chemical components of each Chinese medicine, combines the anti-oxidation and anti-inflammatory pharmacological effects of each chemical component, and can detect corresponding reference substances in corresponding medicinal materials and finished granules. In order to establish more reference substances for confirmation, the test solution and the reference solution containing caffeic acid, chlorogenic acid, liquiritin, luteolin, resveratrol, hesperidin and tangerine cassia bark are respectively subjected to high-performance liquid chromatography detection, and the liquid chromatogram of the reference substance is used as a reference spectrum, and the common peaks are selected from the liquid chromatogram of the test sample to construct the characteristic spectrum of the Shijueming compound Chinese medicine preparation. The present invention ensures the efficient preparation of the test solution by optimizing the extraction conditions of the test sample; and establishes the best chromatographic parameters that can best reflect the component characteristics of the Shijueming compound Chinese medicine preparation by investigating the chromatographic conditions of the high-performance liquid chromatography detection. The characteristic spectrum obtained by the establishment method provided by the present invention can comprehensively reflect the ingredients of the compound Chinese medicine preparation containing Shijueming, realize the quality control of the Chinese medicine preparation containing Shijueming, ensure the authenticity, consistency and stability of the product, and ensure the clinical efficacy of the drug. The establishment method of the characteristic spectrum of the compound Chinese medicine preparation of Shijueming provided by the present invention has the characteristics of high accuracy, high stability and good repeatability. The embodiment of the present invention tested 6 different batches of the compound Chinese medicine preparation of Shijueming, and the results showed that the RSD of the relative retention time of the chromatographic peaks at the same position was within 5%, and the establishment method had good reproducibility and high reliability; and the obtained characteristic spectrum had 7 common peaks that achieved effective separation, which could effectively characterize the quality level of the Shijueming preparation, and was conducive to the comprehensive monitoring of the quality of the Shijueming preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1is the HPLC chromatogram of the control solution test, where 1 is caffeic acid, 2 is chlorogenic acid, 3 is liquiritin, 4 is luteolin, 5 is resveratrol, 6 is hesperidin, and 7 is aurantin;

[0044] Figure 2 The HPLC chromatogram of the test sample extracted by heating with water solvent;

[0045] Figure 3 This is the HPLC chromatogram of the test sample extracted by heating with 25% methanol solvent;

[0046] Figure 4 This is the HPLC chromatogram of the test sample extracted by heating with 50% methanol solvent;

[0047] Figure 5 This is the HPLC chromatogram of the test sample extracted by heating with 75% methanol solvent;

[0048] Figure 6 This is the HPLC chromatogram of the test sample extracted by ultrasonic extraction with water solvent;

[0049] Figure 7 This is the HPLC chromatogram of the test sample extracted by ultrasonication with 25% methanol solvent;

[0050] Figure 8 This is the HPLC chromatogram of the test sample extracted by ultrasonication with 50% methanol solvent;

[0051] Fig. 9 This is the HPLC chromatogram of the test sample extracted with 75% methanol solvent for 90 minutes by ultrasonic;

[0052] Fig.10 This is the HPLC chromatogram of the test sample extracted with 75% methanol solvent for 30 minutes by ultrasonic;

[0053] Fig.11 The HPLC chromatogram of the test sample extracted by ultrasonication with 75% methanol solvent for 60 minutes;

[0054] Fig.12 The HPLC chromatogram of the test sample extracted with 75% methanol solvent for 120 minutes by ultrasonication;

[0055] Fig.13 This is the HPLC chromatogram of the test sample at a column temperature of 40°C;

[0056] Fig.14 The HPLC chromatogram of the test sample with the mobile phase system being acetonitrile-water;

[0057] Fig.15 It is the HPLC chromatogram of the test product under the condition of 68 minutes mobile phase gradient elution program;

[0058] Fig.16This is the HPLC chromatogram of the test product under the conditions of 67-minute mobile phase gradient elution program;

[0059] Fig.17 It is the HPLC chromatogram of the test product at a wavelength of 235 nm;

[0060] Fig.18 It is the HPLC chromatogram of the test product at a wavelength of 255 nm;

[0061] Fig.19 It is the HPLC chromatogram of the test product at a wavelength of 275 nm;

[0062] Fig. 20 It is the HPLC chromatogram of the test product at a wavelength of 305 nm;

[0063] Fig.21 It is the HPLC chromatogram of the test product at a wavelength of 325 nm;

[0064] Fig. 22 This is the overlapping chromatogram of 6 consecutive injections of the test solution for precision test;

[0065] Fig.23 Overlapping HPLC chromatograms of the test sample solutions for solution stability test;

[0066] Fig.24 Overlapping HPLC chromatograms of the test solution for repeatability test;

[0067] Fig.25 The HPLC overlapping chromatograms of 6 batches of test sample solutions show that peak 1 is caffeic acid, peak 2 is chlorogenic acid, peak 3 is liquiritin, peak 4 is luteolin, peak 5 is resveratrol, peak 6 is hesperidin, and peak 7 is aurantin.

[0068] Fig.26 It is the HPLC overlapping chromatogram of 4 kinds of Chinese medicinal materials, mixed reference substance solution and test substance solution;

[0069] Fig. 27 This is the HPLC chromatogram of Cassia seed positive control;

[0070] Fig.28 This is the HPLC chromatogram of Nepeta tenuifolia positive control;

[0071] Fig.29 This is the HPLC chromatogram of the chrysanthemum positive control;

[0072] Fig.30 This is the HPLC chromatogram of the positive control of licorice. DETAILED DESCRIPTION

[0073] The present invention provides a method for establishing a characteristic spectrum of a compound Chinese medicine preparation of Semen Cassiae, comprising the following steps:

[0074] The compound Chinese medicine preparation of Shijueming is mixed with methanol aqueous solution for ultrasonic extraction to obtain a test solution; the volume fraction of the methanol aqueous solution is 75%; the raw materials for preparing the compound Chinese medicine preparation of Shijueming include Shijueming, Cassia seed, Nepeta tenuifolia, chrysanthemum and liquorice;

[0075] The test solution is subjected to high performance liquid chromatography to obtain a liquid chromatogram of the test sample, the liquid chromatogram of the test sample is compared with a predetermined reference spectrum, common peaks are selected from the liquid chromatogram of the test solution, and a characteristic spectrum of the Shijueming compound Chinese medicine preparation is constructed; the reference spectrum is a liquid chromatogram obtained under the same high performance liquid chromatography detection conditions of a reference solution, and the reference solution contains reference substances of caffeic acid, chlorogenic acid, liquiritin, luteolin, resveratrol, hesperidin and citrus juncea.

[0076] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0077] The present invention mixes a compound Chinese medicinal preparation of Semen Cassiae (i.e. a Chinese medicinal preparation containing Semen Cassiae, Semen Cassiae Chinese medicinal granules in the embodiment) with a methanol aqueous solution and performs ultrasonic extraction to obtain a test solution.

[0078] In the present invention, the raw materials for preparing the compound Chinese medicine preparation of Shijueming include Shijueming, Cassia seed, Nepeta, Chrysanthemum and Licorice, wherein the Shijueming is preferably 250 parts by mass, the Cassia seed is preferably 250 parts by mass, the Nepeta is preferably 250 parts by mass, the Chrysanthemum is preferably 250 parts by mass, and the Licorice is preferably 125 parts by mass. In the present invention, the preparation method of the compound Chinese medicine preparation of Shijueming preferably includes the following steps:

[0079] Add water to Cassiae Semen, Cassia Seed, Nepeta Tenuifolia, Chrysanthemum and Licorice and boil twice, filter the decoctions obtained from the two decoctions respectively, and combine the filtrates;

[0080] The combined filtrate is concentrated and then maltodextrin is added, followed by granulation and drying to obtain the compound Chinese medicine preparation of Semen Cassiae.

[0081] In the present invention, the amount of water added for the first decoction is preferably 12 times the mass of the raw material, and the amount of water added for the second decoction is preferably 10 times the mass of the raw material. The time for the first decoction is preferably 2 hours, and the time for the second decoction is preferably 1.5 hours.

[0082] In the present invention, the volume fraction of the methanol aqueous solution is preferably 75%, and the dosage ratio of the Shijueming compound Chinese medicine preparation to the methanol aqueous solution is preferably 0.5g:15-20mL. In the embodiment of the present invention, water, a methanol aqueous solution with a volume fraction of 75%, a methanol aqueous solution with a volume fraction of 50%, and a methanol aqueous solution with a volume fraction of 25% are respectively used as extraction solvents, wherein the methanol aqueous solution with a volume fraction of 75% has the largest number of chromatographic peaks in the chromatogram of the sample extracted, the separation of each chromatographic peak is good, and the extraction is complete; while the chromatographic peaks in the chromatogram of the sample extracted with water have no obvious chromatographic peaks, and the extraction is incomplete. Therefore, the present invention selects a methanol aqueous solution with a volume fraction of 75% as the extraction solvent.

[0083] In the present invention, the frequency of the ultrasonic extraction is preferably 40kHz, and the power is preferably 300W; the time of the ultrasonic extraction is preferably 90min; the ultrasonic extraction is carried out at room temperature, that is, no additional heating is required. The embodiments of the present invention respectively extract the test sample by ultrasonic and water bath heating, wherein water, methanol aqueous solution with a volume fraction of 75%, methanol aqueous solution with a volume fraction of 50%, and methanol aqueous solution with a volume fraction of 25% are respectively used as extraction solvents to extract the test sample chromatogram by water bath heating, and the peak area of ​​the chromatographic peak is obviously smaller than the peak area of ​​the chromatographic peak in the chromatogram of the test sample extracted by ultrasonic method; therefore, the present invention sets the extraction method as ultrasonic extraction. In addition, in the embodiment of the present invention, methanol aqueous solution with a volume fraction of 75% is used as the extraction solvent for ultrasonic extraction for 90min, 30min, 60min and 120min respectively, wherein the peak area of ​​the chromatographic peak in the chromatogram of the test sample for ultrasonic extraction for 30min and 60min is obviously smaller, the chromatographic peaks in the chromatogram of the test sample for ultrasonic extraction time of 120min interfere with the determination, and the separation degree between the chromatographic peaks in the chromatogram of the test sample for ultrasonic extraction time of 90min is better, and does not interfere with the detection; therefore, the present invention sets the ultrasonic extraction time to 90min.

[0084] In the embodiment of the present invention, 0.5 g of the compound Chinese medicine preparation granules of Shijueming are accurately weighed and placed in a 25 mL volumetric flask, 15 mL of a 75% methanol aqueous solution by volume is added, ultrasonicated for 90 min, taken out and allowed to stand to room temperature, diluted to the scale with a 75% methanol aqueous solution by volume, shaken, filtered, and the filtrate is taken to obtain the test solution.

[0085] After obtaining the test sample solution, the present invention performs high performance liquid chromatography on the test sample solution to obtain a liquid chromatogram of the test sample.

[0086] In the present invention, the conditions for the HPLC detection include:

[0087] The chromatographic column is Thermo Hypersil GOLD C 18, the column length is preferably 100 mm, the inner diameter is preferably 2.1 mm, the filler is octadecylsilane bonded silica gel, and the particle size of the filler is preferably 1.9 μm;

[0088] The column temperature was 30°C;

[0089] The detection wavelength is 285 nm (VWD detector);

[0090] The injection volume was 10 μL;

[0091] The concentration of the test solution (i.e., injection concentration) is preferably 0.02 g / mL;

[0092] The mobile phase includes mobile phase A, mobile phase B and mobile phase C, wherein the mobile phase A is acetonitrile, the mobile phase B is a 0.1% by volume phosphoric acid aqueous solution (i.e., a 0.1% phosphoric acid solution, prepared by diluting commercially available 85% phosphoric acid with water, wherein the volume fraction of 85% phosphoric acid is 0.1%), and the mobile phase C is methanol;

[0093] The flow rate of the mobile phase is 0.3 mL / min;

[0094] The elution program is a gradient elution program;

[0095] The gradient elution program is (listed in Table 1):

[0096] 0-9min: the volume percentage of mobile phase A increases from 1% to 6% at a constant rate, the volume percentage of mobile phase B decreases from 97% to 92% at a constant rate, and the volume percentage of mobile phase C remains at 2%;

[0097] 9-21min: the volume percentage of mobile phase A is maintained at 6%, the volume percentage of mobile phase B is maintained at 92%, and the volume percentage of mobile phase C is maintained at 2%;

[0098] 21-26 min: the volume percentage of mobile phase A increased from 6% to 11% at a constant rate, the volume percentage of mobile phase B decreased from 92% to 87% at a constant rate, and the volume percentage of mobile phase C remained at 2%;

[0099] 26-33 min: the volume percentage of mobile phase A was maintained at 11%, the volume percentage of mobile phase B was maintained at 87%, and the volume percentage of mobile phase C was maintained at 2%;

[0100] 33-36min: the volume percentage of mobile phase A increased from 11% to 13% at a constant rate, the volume percentage of mobile phase B decreased from 87% to 85% at a constant rate, and the volume percentage of mobile phase C was maintained at 2%;

[0101] 36-42min: the volume percentage of mobile phase A increased from 13% to 19% at a constant rate, the volume percentage of mobile phase B decreased from 85% to 79% at a constant rate, and the volume percentage of mobile phase C was maintained at 2%;

[0102] 42-46 min: the volume percentage of mobile phase A was maintained at 19%, the volume percentage of mobile phase B was maintained at 79%, and the volume percentage of mobile phase C was maintained at 2%;

[0103] 46-49min: the volume percentage of mobile phase A increased from 19% to 44% at a constant rate, the volume percentage of mobile phase B decreased from 79% to 54% at a constant rate, and the volume percentage of mobile phase C remained at 2%;

[0104] 49-55min: the volume percentage of mobile phase A is maintained at 44%, the volume percentage of mobile phase B is maintained at 54%, and the volume percentage of mobile phase C is maintained at 2%;

[0105] 55-55.5min: the volume percentage of mobile phase A was uniformly decreased from 44% to 1%, the volume percentage of mobile phase B was uniformly increased from 54% to 97%, and the volume percentage of mobile phase C was maintained at 2%;

[0106] 55.5-60 min: the volume percentage of mobile phase A was maintained at 1%, the volume percentage of mobile phase B was maintained at 97%, and the volume percentage of mobile phase C was maintained at 2%.

[0107] HPLC characteristic spectrum is the most widely used chromatographic analysis method at present. It is widely used in the detection of chemical components of traditional Chinese medicine with the advantages of high pressure, high sensitivity, high efficiency and automation, making the components of traditional Chinese medicine preparations more controllable. The present invention uses high performance liquid chromatography under specific parameter conditions for detection, and the obtained spectrum can fully reflect the components of traditional Chinese medicine compound preparations, realize the quality control of traditional Chinese medicine preparations, and ensure the clinical efficacy of drugs.

[0108] In the embodiments of the present invention, the chromatograms of the test sample when the column temperature is 30°C and 40°C are respectively examined. As a result, when the column temperature is 40°C, the retention time of the chromatographic peak in the chromatogram of the test sample is advanced, and the chromatographic peak separation effect is not as good as the chromatographic peak in the chromatogram of the test sample when the column temperature is 30°C. Therefore, the column temperature of the present invention is selected to be 30°C.

[0109] The embodiment of the present invention investigates the high performance liquid chromatograms of the test sample under different mobile phase systems, and compares the high performance liquid chromatogram of the test sample under the conditions of using an acetonitrile-water mobile phase system, that is, acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with the high performance liquid chromatogram of the test sample under the conditions of using an acetonitrile-water-methanol mobile phase system (i.e., acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and methanol as mobile phase C). The separation degree of each chromatographic peak of the chromatogram of the test sample under the condition that the mobile phase system is acetonitrile-water cannot reach the baseline separation, while the separation degree of each chromatographic peak of the chromatogram of the test sample under the condition that the mobile phase system is acetonitrile-water-methanol basically meets the requirements and can achieve baseline separation. Therefore, the mobile phase system selected by the present invention is acetonitrile-water-methanol, that is, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and methanol as mobile phase C.

[0110] The embodiment of the present invention examines the chromatograms of the test sample at different detection wavelengths, and detects the test solution at wavelengths of 235nm, 255nm, 275nm, 285nm, 305nm, and 325nm, respectively. The results show that the number of chromatographic peaks and the peak area detected at 285nm are more reasonable, so the present invention selects 285nm as the detection wavelength.

[0111] After obtaining the liquid chromatogram of the test sample, the present invention compares the liquid chromatogram of the test sample with a predetermined reference spectrum, selects common peaks from the liquid chromatogram of the test sample, and obtains a characteristic spectrum of the Shijueming compound Chinese medicine preparation; the reference spectrum is a liquid chromatogram obtained under the same high performance liquid chromatography detection conditions of a reference solution, and the reference solution contains reference substances of caffeic acid, chlorogenic acid, liquiritin, luteolin, resveratrol, hesperidin and citrus juncea.

[0112] The present invention preferably mixes the reference substances of caffeic acid, chlorogenic acid, liquiritin, luteolin, resveratrol, hesperidin and aurantium with a solvent to obtain a reference substance solution (i.e., a mixed standard solution). In the present invention, the solvent is preferably a methanol aqueous solution, and the volume fraction of the methanol aqueous solution is preferably 50%; the concentration of each reference substance in the reference substance solution is preferably 10 μg / mL.

[0113] The present invention selects the above 7 reference substances, and can establish more reference substances for confirmation; the corresponding reference substances can also be detected in the corresponding medicinal materials and finished granules, and the selected reference substances all have pharmacological effects such as anti-inflammatory and antioxidant effects.

[0114] The present invention adopts the same high performance liquid chromatography conditions as those of the test solution of the compound Chinese medicine preparation of Semen Cassiae to test the reference solution, and compares the chromatogram of the reference solution with the characteristic spectrum of the Chinese medicine preparation test solution to identify the chromatographic peaks in the characteristic spectrum of the Chinese medicine preparation.

[0115] In the present invention, the characteristic spectrum of the Shijueming compound Chinese medicine preparation, with the chromatographic peak of luteolin as a reference, has 7 common peaks, which are:

[0116] Peak 1, relative retention time 0.32;

[0117] peak 2, relative retention time 0.33;

[0118] peak 3, relative retention time 0.87;

[0119] Peak 4, relative retention time 1.00;

[0120] peak 5, relative retention time 1.16;

[0121] peak 6, relative retention time 1.23;

[0122] peak 7, relative retention time 1.53;

[0123] Among them, peak 1 is caffeic acid; peak 2 is chlorogenic acid; peak 3 is liquiritin; peak 4 is oleoside; peak 5 is resveratrol; peak 6 is hesperidin; peak 7 is cassia bark extract, among which peaks 1 and 6 belong to Schizonepeta tenuifolia, peaks 2, 4 and 5 belong to Chrysanthemum, peak 3 belongs to Licorice, and peak 7 belongs to Cassia seed.

[0124] The present invention provides a method for establishing a characteristic spectrum of a sesquiterpenoids Chinese medicine preparation, wherein a 75% methanol aqueous solution is used to ultrasonically extract the sesquiterpenoids Chinese medicine compound preparation, and then a high performance liquid chromatography method is used to test the test solution, and the parameters of each stage are specially controlled. According to "High Performance Liquid Chromatography (General Rule 0512)", a characteristic spectrum of a Chinese medicine preparation is generated from the obtained chromatographic data, and the method is simple to operate, and the method specificity, solution stability, method precision and repeatability meet the requirements. The present invention establishes a scientific, reasonable and feasible characteristic spectrum, which can ensure that the quality of the sesquiterpenoids Chinese medicine preparation is controllable.

[0125] In order to further illustrate the present invention, the method for establishing the characteristic spectrum of the compound Chinese medicine preparation of Semen Cassiae provided by the present invention is described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present invention.

[0126] The instruments, equipment, and reagents used in the examples are as follows:

[0127] Instruments and equipment:

[0128] WatersAcquity HPLC, VWD detector, Waters Audit Trail software;

[0129] KQ5200DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0130] XPE105 analytical balance (METTLER);

[0131] BSA124S electronic balance (sartorius);

[0132] JC-GGC-12W water bath vibrator (Qingdao Juchuang Environmental Protection Group Co., Ltd.);

[0133] Reference substances, test substances and decoction pieces:

[0134] Caffeic acid (China Food and Drug Inspection Institute, batch number: 110885-201703);

[0135] Chlorogenic acid (China Food and Drug Inspection Institute, batch number: 110753-202119);

[0136] Liquorice glycoside (China Food and Drug Inspection Institute, batch number: 111610-202209);

[0137] Luteolin (China Food and Drug Inspection Institute, batch number: 111720-202312);

[0138] Resveratrol (China Food and Drug Administration, batch number: 111535-201703);

[0139] Hesperidin (China Food and Drug Inspection Institute, batch number: 110721-202220);

[0140] Cassia tangerina (China Food and Drug Inspection Institute, batch number: 111900-202006);

[0141] The commissioned production of Shijueming Chinese medicinal granules for research use (batch numbers: 20230301, 20230302, 20230303, 20231101, 20231102, 20231103, Shenyang Feilong Pharmaceutical Co., Ltd.), the preparation method of the above-mentioned Shijueming Chinese medicinal granules is as follows: raw materials: 625g of Shijueming, 625g of Cassia seed, 625g of Schizonepeta, 625g of Chrysanthemum, and 312.5g of Licorice; preparation method: weigh Shijueming, Cassia seed, Schizonepeta, Chrysanthemum, and Licorice slices respectively, add water and decoct twice, add 12 times of water for the first time and decoct for 2 hours, and add 10 times of water for the second time and decoct for 1.5 hours, filter, combine the filtrate, and concentrate under reduced pressure to a clear paste with a relative density of 1.05-1.12 (60°C), add appropriate amount of dextrin, granulate, dry, and granulate to make 1000g, and package to obtain.

[0142] Medicinal materials such as Semen Cassiae, Semen Cassiae, Chrysanthemum, Schizonepeta and Licorice were purchased from Anguo Hongfa Chinese Medicinal Materials Pieces Co., Ltd.

[0143] Example 1

[0144] A method for establishing a characteristic spectrum of a Chinese medicine preparation containing Semen Cassiae, comprising the following steps:

[0145] Step A, preparation of reference solution:

[0146] Take appropriate amount of caffeic acid, chlorogenic acid, liquiritin, luteolin, resveratrol, hesperidin, and aurantium dulcis reference substances respectively, add 50% methanol aqueous solution to dilute to prepare mixed standard solution, i.e., reference substance solution, wherein the concentration of each reference substance is 10 μg / mL;

[0147] Step B, preparation of test solution:

[0148] Take 0.5 g of Shijueming Chinese medicine granules (batch number: 20231101, Shenyang Feilong Pharmaceutical Co., Ltd.), accurately weigh, put into a 25 mL volumetric flask, add 15 mL of 75% methanol aqueous solution, and dissolve it by ultrasound (frequency is 40 kHz, power is 300 W) for 90 min. Then take it out and let it stand to room temperature, dilute it to the scale with 75% methanol aqueous solution, shake well, filter, and take the filtrate to obtain the test solution.

[0149] Step C, detection:

[0150] Using high performance liquid chromatography, 10 μL of the reference solution and the test solution were injected into the high performance liquid chromatography instrument. The chromatographic conditions were as follows: Thermo Hypersil GOLD C 18 The column is 100 mm long and 2.1 mm in inner diameter. Octadecylsilane bonded silica gel is used as filler and the filler particle size is 1.9 μm. Acetonitrile is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, methanol is used as mobile phase C, the detection wavelength is 285 nm, and the flow rate is 0.3 mL / min. The column temperature is 30° C. The gradient elution program is shown in Table 1, where % is the volume percentage.

[0151] Table 1 Gradient elution program

[0152] Time (min) Mobile phase A% Mobile phase B% Mobile phase C% 0 1 97 2 9 6 92 2 21 6 92 2 26 11 87 2 33 11 87 2 36 13 85 2 42 19 79 2 46 19 79 2 49 44 54 2 55 44 54 2 55.5 1 97 2 60 1 97 2

[0153] Figure 1 is the HPLC chromatogram of the reference solution. Figure 1 Peak 1 is caffeic acid, peak 2 is chlorogenic acid, peak 3 is liquiritin, peak 4 is luteolin, peak 5 is resveratrol, peak 6 is hesperidin, and peak 7 is aurantium dulcisin.

[0154] Comparative Example 1

[0155] Water, 50% by volume methanol aqueous solution, and 25% by volume methanol aqueous solution were respectively used as extraction solvents and ultrasonicated for 90 minutes. The rest was the same as in Example 1.

[0156] Figure 6 This is the HPLC chromatogram of the test sample extracted with water solvent for 90 minutes by ultrasonic; Figure 7 The HPLC chromatogram of the test sample extracted with a 25% methanol-water solution (i.e., 25% methanol) by ultrasonic for 90 minutes;

[0157] Figure 8 The HPLC chromatogram of the test sample extracted with a 50% methanol-water solution (i.e., 50% methanol) by ultrasonic for 90 minutes; Fig. 9 It is the HPLC chromatogram of the test sample extracted by ultrasonication for 90 minutes with a 75% volume fraction methanol aqueous solution (i.e., 75% methanol).

[0158] Depend on Figure 6 to Figure 9 It can be seen that water, 75% methanol, 50% methanol and 25% methanol were used as extraction solvents respectively. Among them, the chromatogram of the test sample extracted with 75% methanol had the largest number of chromatographic peaks, the separation of each chromatographic peak was good, and the extraction was complete; no obvious chromatographic peaks were seen in the chromatogram of the test sample extracted with water, and the extraction was incomplete; therefore, 75% methanol was selected as the extraction solvent.

[0159] Comparative Example 2

[0160] Water, 50% by volume methanol aqueous solution, 25% by volume methanol aqueous solution and 75% by volume methanol aqueous solution were used as extraction solvents respectively, and the mixture was heated in a water bath and refluxed for 90 minutes. The rest was the same as in Example 1.

[0161] Figure 2 The HPLC chromatogram of the test sample extracted by heating with water solvent; Figure 3 This is the HPLC chromatogram of the test sample extracted by heating with 25% methanol solvent; Figure 4 This is the HPLC chromatogram of the test sample extracted by heating with 50% methanol solvent; Figure 5 The HPLC chromatogram of the sample extracted by heating with 75% methanol solvent. It can be seen that the peak areas of the chromatograms of the sample extracted by water bath heating with water, 75% methanol, 50% methanol, and 25% methanol as the extraction solvent are significantly smaller than the peak areas of the chromatograms of the sample extracted by ultrasonic extraction; therefore, ultrasonic extraction was selected as the extraction method.

[0162] Comparative Example 3

[0163] A methanol aqueous solution with a volume fraction of 75% was used for ultrasonic extraction for 30 minutes, 60 minutes and 120 minutes respectively, and the rest was the same as in Example 1.

[0164] Fig.10 This is the HPLC chromatogram of the test sample extracted with 75% methanol solvent for 30 minutes by ultrasonic; Fig.11The HPLC chromatogram of the test sample extracted by ultrasonication with 75% methanol solvent for 60 minutes; Fig.12 The HPLC chromatogram of the sample extracted with 75% methanol solvent for 120 minutes by ultrasonic extraction is shown in Figure 1. It can be seen that the peak areas of the chromatograms of the sample with extraction time of 30min and 60min are significantly smaller, the chromatograms of the sample with extraction time of 120min interfere with the determination, and the chromatograms of the sample with extraction time of 90min have better separation and do not interfere with the detection; therefore, the extraction time is selected as 90min ultrasonic extraction.

[0165] Comparative Example 4

[0166] The column temperature was 40°C, and the rest was the same as in Example 1.

[0167] Fig.13 The following is the HPLC chromatogram of the test sample at a column temperature of 40°C. By comparison, it can be seen that when the column temperature is 40°C, the retention time of the chromatographic peak in the chromatogram of the test sample is advanced, and the chromatographic peak separation effect is not as good as the chromatographic peak in the chromatogram of the test sample at a column temperature of 30°C, so the column temperature is selected to be 30°C.

[0168] Comparative Example 5

[0169] The chromatograms of the test samples under different mobile phase systems were investigated, and the mobile phase system in step C of Example 1 was changed to acetonitrile-water, specifically: acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and the gradient elution program was shown in Table 2. The rest was the same as Example 1.

[0170] Table 2 Comparative Example 5 Gradient elution program

[0171] Time (min) Mobile phase A% Mobile phase B% 0 2 98 9 7 93 21 7 93 26 12 88 33 12 88 36 14 86 42 20 80 46 20 80 49 45 55 55 45 55 55.5 2 98 60 2 98

[0172] Fig.14 The figure is a high performance liquid chromatogram of the test sample under the condition of acetonitrile-water as the mobile phase system. By comparing with the high performance liquid chromatogram of the test sample under the mobile phase system (i.e., acetonitrile-water-methanol) of Example 1, it can be seen that the separation of each chromatographic peak of the test sample chromatogram under the condition of acetonitrile-water as the mobile phase system cannot reach the baseline separation, and the separation of each chromatographic peak of the test sample chromatogram under the condition of acetonitrile-water-methanol as the mobile phase system basically meets the requirements and can achieve baseline separation, so the mobile phase system is selected as acetonitrile-water-methanol, that is, acetonitrile is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, and methanol is used as mobile phase C.

[0173] Comparative Example 6

[0174] Different mobile phase gradient elution programs were investigated, and the 68-minute mobile phase gradient elution program shown in Table 3 and the 67-minute mobile phase gradient elution program shown in Table 4 were used for gradient elution, and the rest was the same as in Example 1.

[0175] Table 3 Comparative Example 68 minutes mobile phase gradient elution program

[0176] Time (min) Mobile phase A% Mobile phase B% Mobile phase C% 0 1 97 2 9 6 92 2 21 6 92 2 26 11 87 2 33 11 87 2 36 13 85 2 42 19 79 2 46 19 79 2 49 44 54 2 55 44 54 2 62 89 9 2 62.5 1 97 2 68 1 97 2

[0177] Table 4 Comparative Example 67-minute mobile phase gradient elution program

[0178] Time (min) Mobile phase A% Mobile phase B% 0 2 98 12 7 93 21 12 88 36 14 86 42 20 80 45 25 75 55 45 55 60 45 55 61 2 98 67 2 98

[0179] Fig.15 It is the HPLC chromatogram of the test product under the condition of 68 minutes mobile phase gradient elution program; Fig.16 This is the HPLC chromatogram of the test product under the conditions of 67 minutes mobile phase gradient elution program.

[0180] The separation of the chromatographic peaks in the test sample chromatogram under the mobile phase gradient elution program condition of 67 minutes was not developed to the baseline separation, and the test sample chromatogram under the mobile phase gradient elution program condition of 68 minutes was basically consistent with the test sample chromatogram under the mobile phase gradient elution program condition of 60 minutes in Example 1, so the mobile phase gradient elution program condition of 60 minutes was selected.

[0181] Comparative Example 7

[0182] The test solution was detected at wavelengths of 235 nm, 255 nm, 275 nm, 305 nm and 325 nm, respectively, and the rest was the same as in Example 1.

[0183] Fig.17 It is a high performance liquid chromatogram at a wavelength of 235 nm; Fig.18 It is a high performance liquid chromatogram at a wavelength of 255 nm; Fig.19 It is a high performance liquid chromatogram at a wavelength of 275 nm; Fig. 20 It is a high performance liquid chromatogram at a wavelength of 305 nm; Fig.21 The HPLC chromatogram at 325 nm is shown in Figure 1. By comparing the HPLC chromatogram at 285 nm in Example 1, it can be seen that the number of chromatographic peaks and the peak area under 285 nm are more reasonable, so 285 nm is selected as the detection wavelength.

[0184] Example 2

[0185] Precision experiment

[0186] Take 0.5 g of Chinese medicine preparation granules (Shijueming Chinese medicine granules (batch number: 20231101, Shenyang Feilong Pharmaceutical Co., Ltd.)), accurately weigh, put into a 25 mL volumetric flask, add 15 mL of 75% methanol, ultrasonic (frequency is 40 kHz, power is 300 W) for 90 min, let it stand to room temperature, dilute to the scale with 75% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0187] The sample was injected 6 times continuously, and the relative retention time and relative peak area of ​​the common peak were calculated with luteolin as the reference peak. The results showed that the RSD values ​​of the relative retention time were all less than 2%, and the relative peak area RSD of the chromatographic peak 7 cassia bark was less than 5.0%; the peak area of ​​cassia bark was small, resulting in the RSD result of the peak area of ​​cassia bark was 5.90%, but its relative peak area percentage was between 90 and 110, which met the requirements, indicating that the precision of this method was good. Table 5 and Table 6 are the relative retention time results of the precision test and the relative peak area results of the precision test, respectively.

[0188] Table 5 Precision test relative retention time results

[0189]

[0190] Table 6 Precision test relative peak area results

[0191]

[0192]

[0193] Fig. 22 This is the overlapping chromatogram of 6 consecutive injections of the test solution in the precision test.

[0194] Example 3

[0195] Solution stability test

[0196] Take 0.5 g of Chinese medicine preparation granules (Shijueming Chinese medicine granules (batch number: 20231101, Shenyang Feilong Pharmaceutical Co., Ltd.)), accurately weigh, put into a 25 mL volumetric flask, add 15 mL of 75% methanol, ultrasonic (frequency is 40 kHz, power is 300 W) for 90 min, let it stand to room temperature, dilute to the scale with 75% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0197] The samples were injected at 0, 2, 4, 6, 8, and 16 hours, and luteolin was used as the reference peak to calculate the relative retention time and relative peak area of ​​the common peak. The results showed that the relative retention time RSD values ​​were all less than 2%, and the relative peak area RSD values ​​were all less than 5.0%, which met the requirements, indicating that the test solution was basically stable within 16 hours. Table 7 and Table 8 are the relative retention time results and relative peak area results of the solution stability test, respectively.

[0198] Table 7 Solution stability test relative retention time results

[0199]

[0200] Table 8 Solution stability test relative peak area results

[0201]

[0202]

[0203] Fig.23 This is the HPLC overlay chromatogram of the test solution for solution stability test.

[0204] Example 4

[0205] Repeatability experiment

[0206] Take 0.5 g of Chinese medicine preparation granules (Shijueming Chinese medicine granules (batch number: 20231101, Shenyang Feilong Pharmaceutical Co., Ltd.)), accurately weigh, put into a 25 mL volumetric flask, add 15 mL of 75% methanol, ultrasonic (frequency is 40 kHz, power is 300 W) for 90 min, let it stand to room temperature, dilute to the scale with 75% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0207] Six test sample solutions were prepared in parallel, and the samples were analyzed separately. The relative retention time and relative peak area of ​​the common peak were calculated with luteolin as the reference peak. The results showed that the relative retention time RSD values ​​were less than 2%, and the relative peak area RSD values ​​were less than 5.0%, indicating that the method had good repeatability. Table 9 and Table 10 are the relative retention time results of the repeatability test and the relative peak area results of the repeatability test, respectively.

[0208] Table 9 Relative retention time results of repeatability test

[0209]

[0210] Table 10 Relative peak area results of repeatability test

[0211]

[0212]

[0213] Fig.24 Overlapping HPLC chromatograms of the test solution for repeatability test.

[0214] Example 5

[0215] Establishment of characteristic spectrum of traditional Chinese medicine preparation of Semen Cassiae

[0216] Take 0.5 g of each of the Chinese medicine preparation granules (20230301, 20230302, 20230303, 20231101, 20231102, 20231103), accurately weigh, put into a 25 mL volumetric flask, add 15 mL of 75% methanol, ultrasonically (frequency is 40 kHz, power is 300 W) for 90 min, let stand to room temperature, dilute to the scale with 75% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0217] Take appropriate amount of caffeic acid, chlorogenic acid, liquiritin, luteolin, resveratrol, hesperidin and aurantium cassia seed reference substances respectively, add 50% methanol solution to dilute to prepare a mixed standard solution containing 10 μg per 1 mL.

[0218] HPLC conditions:

[0219] Chromatographic column: Thermo Hypersil GOLD C 18 The column is 100 mm long, with an inner diameter of 2.1 mm, octadecylsilane bonded silica gel as the filler, and the filler particle size is 1.9 μm;

[0220] Column temperature: 30°C;

[0221] Detection wavelength: 285nm;

[0222] Injection volume: 10 μL;

[0223] Injection concentration of test solution: 0.02g / mL;

[0224] Mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, methanol as mobile phase C, the flow rate of the mobile phase is 0.3 mL / min;

[0225] The elution method is gradient elution, and the elution program of the gradient elution is shown in Table 1.

[0226] Determination:

[0227] Accurately pipette 10 μL of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph, and measure to obtain the liquid chromatogram of the test solution and the liquid chromatogram of the reference solution respectively.

[0228] Liquid chromatography of the reference solution Figure 1As shown, Figure 1 Peak 1 is caffeic acid, peak 2 is chlorogenic acid, peak 3 is liquiritin, peak 4 is luteolin, peak 5 is resveratrol, peak 6 is hesperidin, and peak 7 is aurantium dulcisin.

[0229] Fig.25 The HPLC overlapping chromatograms of 6 batches of test sample solutions are as follows; it can be seen that in the chromatograms of the 6 batches of test sample solutions, the positions of the 7 common peaks of caffeic acid, chlorogenic acid, liquiritin, luteolin, resveratrol, hesperidin and aurantiacum are basically consistent (the RSD of the relative retention time of the chromatographic peaks at the same position are all within 5%), and the characteristic spectrum of the Chinese medicine compound preparation can be established. Taking the chromatographic peak of luteolin No. 4 as a reference, Peak 1 in the characteristic spectrum is caffeic acid; Peak 2 is chlorogenic acid; Peak 3 is liquiritin; Peak 5 is resveratrol; Peak 6 is hesperidin; and Peak 7 is aurantiacum.

[0230] Example 6

[0231] According to the method of Example 5, the positive control chromatograms of Cassia seed, Schizonepeta, Chrysanthemum and Licorice in the Chinese medicine compound preparation were respectively determined. By analyzing the chromatographic peaks in the characteristic spectrum of the Chinese medicine compound preparation and the positive control chromatogram of each medicinal material, the retention times of the chromatographic peaks were compared, and finally the attribution peaks of the common peaks in the characteristic spectrum of the Chinese medicine compound preparation were confirmed. The results are shown in Table 11.

[0232] Table 11 Attribution of common peaks in the characteristic spectrum of traditional Chinese medicine compound preparations

[0233] Peak Retention time (min) Compound Medicinal materials 1 10.851 Caffeic acid Nepeta 2 11.303 Chlorogenic acid chrysanthemum 3 29.515 Licorice glycoside Licorice 4 34.086 Luteolin chrysanthemum 5 39.428 Resveratrol chrysanthemum 6 41.591 Hesperidin Nepeta 7 51.22 Cassia aurantium Cassia seed

[0234] Fig.26 It is the HPLC overlapping chromatogram of 4 Chinese medicinal materials positive control and mixed reference solution and test solution; Fig. 27 This is the HPLC chromatogram of Cassia seed positive control; Fig.28 This is the HPLC chromatogram of Nepeta tenuifolia positive control; Fig.29 This is the HPLC chromatogram of the chrysanthemum positive control; Fig.30 This is the HPLC chromatogram of the positive control of licorice (Note: Fig. 27 and Fig.28 Most of the smaller impurity peaks are close to each other and do not interfere with the positioning of the corresponding reference peaks).

[0235] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for establishing a characteristic spectrum of a compound Chinese medicine preparation of Semen Cassiae, characterized in that: The following steps are involved: The compound Chinese medicine preparation of Shijueming is mixed with methanol aqueous solution for ultrasonic extraction to obtain a test solution; the volume fraction of the methanol aqueous solution is 75%; the raw materials for preparing the compound Chinese medicine preparation of Shijueming include Shijueming, Cassia seed, Nepeta tenuifolia, chrysanthemum and liquorice; The test solution is subjected to high performance liquid chromatography to obtain a liquid chromatogram of the test sample, the liquid chromatogram of the test sample is compared with a predetermined reference spectrum, and common peaks are selected from the liquid chromatogram of the test sample to obtain a characteristic spectrum of the Shijueming compound Chinese medicine preparation; the reference spectrum is a liquid chromatogram obtained under the same high performance liquid chromatography detection conditions of a reference solution, and the reference solution contains reference substances of caffeic acid, chlorogenic acid, liquiritin, luteolin, resveratrol, hesperidin and citrus cassia bark; The conditions for the high performance liquid chromatography detection include: The chromatographic column is Thermo Hypersil GOLD C 18 ; The column temperature was 30°C; The detection wavelength is 285nm; The injection volume was 10 μL; The mobile phase includes mobile phase A, mobile phase B and mobile phase C, wherein the mobile phase A is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution with a volume fraction of 0.1%, and the mobile phase C is methanol; The flow rate of the mobile phase is 0.3 mL / min; The elution program is a gradient elution program; The gradient elution procedure is: 0-9min: the volume percentage of mobile phase A increases from 1% to 6% at a constant rate, the volume percentage of mobile phase B decreases from 97% to 92% at a constant rate, and the volume percentage of mobile phase C remains at 2%; 9-21min: the volume percentage of mobile phase A is maintained at 6%, the volume percentage of mobile phase B is maintained at 92%, and the volume percentage of mobile phase C is maintained at 2%; 21-26 min: the volume percentage of mobile phase A increased from 6% to 11% at a constant rate, the volume percentage of mobile phase B decreased from 92% to 87% at a constant rate, and the volume percentage of mobile phase C remained at 2%; 26-33 min: the volume percentage of mobile phase A was maintained at 11%, the volume percentage of mobile phase B was maintained at 87%, and the volume percentage of mobile phase C was maintained at 2%; 33-36min: the volume percentage of mobile phase A increased from 11% to 13% at a constant rate, the volume percentage of mobile phase B decreased from 87% to 85% at a constant rate, and the volume percentage of mobile phase C was maintained at 2%; 36-42min: the volume percentage of mobile phase A increased from 13% to 19% at a constant rate, the volume percentage of mobile phase B decreased from 85% to 79% at a constant rate, and the volume percentage of mobile phase C was maintained at 2%; 42-46 min: the volume percentage of mobile phase A was maintained at 19%, the volume percentage of mobile phase B was maintained at 79%, and the volume percentage of mobile phase C was maintained at 2%; 46-49min: the volume percentage of mobile phase A increased from 19% to 44% at a constant rate, the volume percentage of mobile phase B decreased from 79% to 54% at a constant rate, and the volume percentage of mobile phase C remained at 2%; 49-55min: the volume percentage of mobile phase A is maintained at 44%, the volume percentage of mobile phase B is maintained at 54%, and the volume percentage of mobile phase C is maintained at 2%; 55-55.5min: the volume percentage of mobile phase A was uniformly decreased from 44% to 1%, the volume percentage of mobile phase B was uniformly increased from 54% to 97%, and the volume percentage of mobile phase C was maintained at 2%; 55.5-60 min: the volume percentage of mobile phase A was maintained at 1%, the volume percentage of mobile phase B was maintained at 97%, and the volume percentage of mobile phase C was maintained at 2%.

2. The method according to claim 1, characterized in that The dosage ratio of the compound Chinese medicinal preparation of Semen Cassiae to the methanol aqueous solution is 0.5 g: 15-20 mL.

3. The method according to claim 1, characterized in that The frequency of the ultrasonic extraction is 40kHz and the power is 300W.

4. The method according to claim 1 or 3, characterized in that: The ultrasonic extraction time is 90 minutes.

5. The method according to claim 1, characterized in that The concentration of each reference substance in the reference substance solution was 10 μg / mL.

6. The method according to claim 1, characterized in that The column length of the chromatographic column is 100 mm and the inner diameter is 2.1 mm.

7. The method according to claim 1, characterized in that The concentration of the test solution is 0.02 g / mL.

8. The method according to claim 1, characterized in that The amount of the stone cassia seed is 250 parts by mass, the amount of the cassia seed is 250 parts by mass, the amount of the schizonepeta tenuifolia is 250 parts by mass, the amount of the chrysanthemum is 250 parts by mass, and the amount of the liquorice is 125 parts by mass.

9. The method according to claim 8, characterized in that The preparation method of the compound Chinese medicine preparation of Semen Cassiae comprises the following steps: Add water to Cassiae Semen, Cassia Seed, Nepeta Tenuifolia, Chrysanthemum and Licorice and boil twice, filter the decoctions obtained from the two decoctions respectively, and combine the filtrates; The combined filtrate is concentrated and then maltodextrin is added, followed by granulation and drying to obtain the compound Chinese medicine preparation of Semen Cassiae.

10. The method according to claim 9, characterized in that The amount of water added for the first decoction is 12 times the mass of the raw materials, and the amount of water added for the second decoction is 10 times the mass of the raw materials. The time for the first decoction is 2 hours, and the time for the second decoction is 1.5 hours.

Images