Radix astragali and ginseng palpitation relieving granules for treating ventricular premature beat and quality detection method thereof

Abstract

The invention discloses radix astragali and radix ginseng palpitation relieving granules and a quality detection method thereof. The optimal forming process of the radix astragali and radix ginseng palpitation relieving granules is screened out through a large number of experiments. According to the invention, the optimal thin-layer chromatography development condition is screened out, and qualitative identification research is carried out on salvia miltiorrhiza, radix astragali preparata and ligusticum wallichii in the quality detection method of the radix astragali, radix astragali and radix astragali palpitation relieving granules. According to the present invention, the optimal fluidity composition, the gradient elution mode and other chromatographic conditions are screened through a large number of experiments, the fingerprint detection method with characteristics of high precision, good stability and high accuracy is established, and the contents of the effective components such as calycosin-7-glucoside, ferulic acid, dihydroquercetin, ammonium glycyrrhizinate, nardosinone and the like can be simultaneously determined; and a quality detection method is provided for ensuring the safety and the effectiveness.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a quality testing method for Astragalus and Ginseng Pingji Granules and the method thereof. Background Technology

[0002] Qishen Pingji Granules are made from roasted astragalus, codonopsis, ophiopogon, asparagus, salvia miltiorrhiza, chuanxiong, poria cocos, prepared pinellia, and nard. Previous research reports [Zhang Linning, Liu Chunling, et al., Clinical study on the treatment of ventricular premature beats with the method of invigorating qi, nourishing yin, promoting blood circulation and resolving phlegm. Nanjing University of Chinese Medicine, 2019], through a randomized controlled trial, collected 53 patients with ventricular premature beats from the outpatient and inpatient departments of the Department of Cardiology of Jiangsu Provincial Hospital of Traditional Chinese Medicine, and randomly divided them into an experimental group and a control group. There were no significant differences between the two groups in terms of gender, age, comorbidities and Lown's classification of ventricular premature beats. In addition to treating the primary disease, the control group received beta-blockers, while the experimental group received a traditional Chinese medicine decoction (15g Astragalus membranaceus, 10g Codonopsis pilosula, 10g Asparagus cochinchinensis, 10g Ophiopogon japonicus, 15g Salvia miltiorrhiza, 15g Ligusticum chuanxiong, 15g Poria cocos, 10g Pinellia ternata, 10g Nardostachys jatamansi) in addition to beta-blockers. One course of treatment lasted 4 weeks. The total effective score after treatment was lower in the experimental group than in the control group. Regarding individual TCM scores, both groups showed improvement in symptoms such as palpitations, chest tightness, shortness of breath, fatigue, dry mouth, dry throat, and insomnia after treatment. The average heart rate in both groups decreased after treatment compared to before treatment. Both the experimental and control groups showed improvement in the number, frequency, and average heart rate of premature ventricular contractions (PVCs), but the experimental group showed better overall efficacy than the control group. After treatment, the experimental group showed better results than the control group in terms of Myerburg classification and heart rate variability.

[0003] This clinical study confirms that Chief Physician Liu Chunling's experience formula for treating ventricular premature beats (PVCs) based on the principle of "tonifying qi and nourishing yin, promoting blood circulation and removing blood stasis" can significantly improve the clinical symptoms of patients with PVCs of the qi and yin deficiency and phlegm-blood stasis type. It can also reduce the number and frequency of PVCs, lower the Myerburg grade, improve the morphological grade of PVCs, and improve heart rate variability. Furthermore, this method has good safety in treating PVCs and has no obvious adverse reactions.

[0004] Currently, there are few quality testing methods for Qishen Pingji Granules, which cannot comprehensively and objectively test them. This invention combines thin-layer chromatography identification, fingerprinting, and content determination methods to conduct comprehensive quality testing of Qishen Pingji Granules, which is of great significance for controlling its clinical efficacy and ensuring its safety. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a Qishen Pingji granule and an objective and comprehensive quality testing method for it.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A type of Qishen Pingji granules for treating premature ventricular contractions is prepared by the following method: Take roasted Astragalus membranaceus, Codonopsis pilosula, Ophiopogon japonicus, Asparagus cochinchinensis, Salvia miltiorrhiza, Ligusticum chuanxiong, Poria cocos, Pinellia ternata, and Nardostachys chinensis. Add 6 to 10 times the amount of water, soak them first, then decoct and extract for 20 to 120 minutes. Filter, concentrate the filtrate under reduced pressure, take an appropriate amount of dextrin, place it in a fluidized bed, spray dry, and granulate to obtain the final product.

[0007] As a preferred embodiment, the Qishen Pingji Granules for treating premature ventricular contractions described above are prepared by the following method: Take 15g of roasted Astragalus membranaceus, 10g of Codonopsis pilosula, 10g of Ophiopogon japonicus, 10g of Asparagus cochinchinensis, 15g of Salvia miltiorrhiza, 15g of Ligusticum chuanxiong, 15g of Poria cocos, 10g of Pinellia ternata, and 10g of Nardostachys jatamansi. Add water and decoct twice. For the first decoction, add 10 times the amount of water, soak for 60 minutes, decoct for 30 minutes, and filter. For the second decoction, add 8 times the amount of water, decoct for 20 minutes, and filter. Concentrate the filtrate under reduced pressure to a relative density of 1.15~1.20 at 60℃. Take an appropriate amount of dextrin and place it in a fluidized bed. Set the inlet air temperature to 90℃~100℃. When the material temperature rises to 60℃, start the liquid injection. Control the liquid injection rate at 80~150 r / min. The atomization pressure is 0.2 MPa externally and 0.15 MPa internally. After spraying, continue drying at 60℃~70℃ to obtain granules.

[0008] The above-mentioned Qi-Shen-Ping-Ji Granules contain Astragalus membranaceus (Huang Qi), which is sweet and slightly warm in nature, and invigorates the spleen and replenishes Qi, strengthening the exterior. Codonopsis pilosula (Dang Shen) is sweet and neutral in nature, and can invigorate the spleen and stomach, moisten the lungs and generate fluids. These two herbs are the principal herbs, working synergistically to replenish heart Qi. Asparagus cochinchinensis (Tian Men Dong) is cold in nature, sweet and slightly bitter in taste, and can clear heat and fire, generate fluids and quench thirst. Ophiopogon japonicus (Mai Men Dong) is slightly cold in nature, slightly bitter and sweet in taste, and enters the heart, lung and stomach meridians. It can clear the heart and moisten the lungs, nourish Yin and generate fluids. Asparagus cochinchinensis and Ophiopogon japonicus together nourish the Yin and blood of the heart. Salvia miltiorrhiza (Zi Dan Shen) is slightly cold in nature, bitter in taste, and enters the heart meridian, reaching the internal organs and resolving stagnation. Ligusticum chuanxiong (Chuan Xiong) is pungent, warm, fragrant and dry, moving without lingering. These two herbs are used together to clear heat and cool the blood to relieve irritability, and to invigorate blood and nourish blood to calm palpitations. Poria cocos (Fu Ling) invigorates the spleen, promotes diuresis and eliminates dampness, calms the mind and soothes the nerves, and is often used for phlegm retention, dizziness and palpitations, restlessness, palpitations and insomnia. Pinellia ternata is pungent and warm in nature, and toxic. It enters the spleen, stomach, and lung meridians, and has the functions of drying dampness and resolving phlegm, relieving nausea and vomiting, and reducing swelling and dissipating nodules. Asparagus cochinchinensis, Ophiopogon japonicus, Salvia miltiorrhiza, Ligusticum chuanxiong, Poria cocos, and Pinellia ternata are used as assistant herbs. Nardostachys chinensis is used as an adjuvant herb; it is pungent and sweet in nature, and warm in nature. The Compendium of Materia Medica records that it "regulates the original qi and removes qi stagnation," and can regulate qi, relieve pain, and invigorate the spleen. The herbs in this formula tonify qi and yin to treat the root cause, and invigorate blood and resolve phlegm to treat the symptoms. They tonify without causing stagnation, remove blood stasis and promote new blood production. The formula is meticulously formulated and closely matches the pathogenesis, demonstrating excellent clinical efficacy in treating premature ventricular contractions.

[0009] A quality detection method for Astragalus and Ginseng Pingji Granules includes thin-layer chromatography qualitative identification, fingerprinting and content determination; The aforementioned thin-layer chromatography qualitative identification includes thin-layer identification of processed Astragalus membranaceus, and the specific steps are as follows: (1) Take Astragalus and Ginseng Pingji Granules, dissolve them in water, extract them by shaking with water-saturated n-butanol solution, combine the n-butanol solutions, wash them with ammonia solution, discard the ammonia solution, evaporate to dryness, dissolve the residue in methanol, and use it as the test solution. (2) Prepare granules without processed Astragalus membranaceus using the same method as above, and prepare a negative control solution of processed Astragalus membranaceus using the same method as in step (1); (3) Take another roasted Astragalus membranaceus reference material, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, and prepare the roasted Astragalus membranaceus reference material solution in the same way as in step (1); (4) Take the above three solutions and spot them on the same silica gel G thin layer plate. Use the upper layer of n-butanol-ethyl acetate-dilute ammonia as the developing solvent. Develop, remove, and air dry. Spray with 10% sulfuric acid ethanol solution. Heat until the spots are clearly visible and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material. The specific steps for thin-layer chromatography identification of Salvia miltiorrhiza are as follows: (1) Take the Astragalus and Ginseng Pingji Granules, dissolve them in water, adjust the pH to acidic with hydrochloric acid, extract with ethyl acetate, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in methanol, and use it as the test solution. (2) Prepare granules without danshen medicinal material using the same method as above, and prepare danshen negative control solution using the same method as in step (1); (3) Take another Danshen reference material and prepare a Danshen reference material solution in the same way as in step (1); (4) Take the above three solutions and spot them on the same silica gel G thin layer plate respectively. Use dichloromethane-ethyl acetate-formic acid as the developing solvent, develop, remove, air dry, spray with 5% vanillin sulfuric acid solution, and heat until the spots are clear; in the chromatogram of the test sample, spots of the same color are displayed at the corresponding positions as in the chromatogram of the reference medicinal material. The specific steps for thin-layer chromatography identification of Ligusticum chuanxiong are as follows: (1) Take Astragalus and Codonopsis pilosula granules, dissolve them in water, extract them with ethyl acetate, combine the extracts, evaporate them to dryness in a water bath, dissolve the residue in methanol, and use it as the test solution; (2) Prepare granules without Ligusticum chuanxiong using the same method as above, and prepare a negative control solution of Ligusticum chuanxiong using the same method as in step (1); (3) Take another reference herb, add water, decoct, filter, extract the filtrate with ethyl acetate, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in methanol, and prepare a reference herb solution; (4) Take the above two solutions and spot them on the same silica gel GF254 thin layer plate. Use n-hexane-ethyl acetate as the developing solvent, develop, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

[0010] As a preferred embodiment, the quality detection method for Astragalus and Ginseng Pingji Granules described above includes thin-layer chromatography qualitative identification, which includes thin-layer identification of processed Astragalus membranaceus. The specific steps are as follows: (1) Take Astragalus and Ginseng Pingji Granules, add water to dissolve and filter. Take 40 mL of the filtrate and extract it 4 times with water-saturated n-butanol solution, 40 mL each time. Combine the n-butanol solutions and wash them 2 times with ammonia solution, 40 mL each time. Discard the ammonia solution and evaporate it to dryness in an evaporating dish. Add 1 mL of methanol to dissolve the residue and use it as the test solution. (2) Prepare granules without processed Astragalus membranaceus using the same method as above, and prepare a negative control solution of processed Astragalus membranaceus using the same method as in step (1); (3) Take another 3 g of roasted Astragalus reference material, add 50 mL of methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, add 30 mL of water to dissolve the residue, and prepare the roasted Astragalus reference material solution according to the same method as in step (1); (4) Take 10 µL of the above three solutions and spot them on the same silica gel G thin layer plate. Use the upper layer solution of n-butanol-ethyl acetate-10% dilute ammonia (volume ratio 4:1:5) as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly visible, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material. The specific steps for thin-layer chromatography identification of Salvia miltiorrhiza are as follows: (1) Take the Astragalus and Ginseng Pingji Granules, add water to dissolve them, take 40 mL of the filtrate, adjust the pH to 2 with 10% hydrochloric acid, extract with ethyl acetate 3 times, 20 mL each time; combine the extracts, evaporate to dryness in a water bath, add 5 mL of methanol to dissolve the residue, and use it as the test solution. (2) Prepare granules without danshen medicinal material using the same method as above, and prepare danshen negative control solution using the same method as in step (1); (3) Take another Danshen reference material and prepare a Danshen reference material solution in the same way as in step (1); (4) Take 10 µL of the above three solutions and spot them on the same silica gel G thin layer plate. Use dichloromethane-ethyl acetate-formic acid with a volume ratio of 5:4:2 as the developing solvent. Develop, remove, air dry, spray with 5% vanillin sulfuric acid solution, and heat at 105℃ until the spots are clear. In the chromatogram of the test sample, spots of the same color are displayed at the corresponding positions as in the chromatogram of the reference medicinal material. The specific steps for thin-layer chromatography identification of Ligusticum chuanxiong are as follows: (1) Take Astragalus and Codonopsis pilosula granules, dissolve them in water, take 40 mL of the filtrate, extract twice with ethyl acetate, 40 mL each time, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in 2 mL of methanol, and use it as the test solution. (2) Prepare granules without Ligusticum chuanxiong using the same method as above, and prepare a negative control solution of Ligusticum chuanxiong using the same method as in step (1); (3) Take another 2 g of Ligusticum chuanxiong reference material, add 100 mL of water, decoct for 0.5 h, filter, extract twice with 40 mL of ethyl acetate each time, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in 2 mL of methanol to prepare Ligusticum chuanxiong reference material solution; (4) Take the above three solutions and spot them on the same silica gel GF254 thin layer plate. Use hexane-ethyl acetate with a volume ratio of 3:1 as the developing solvent, develop, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

[0011] As a preferred option, the quality detection method for Qishen Pingji granules described above, including fingerprint analysis and content determination, includes the following steps: (1) Preparation of the test solution Take 15 batches of Astragalus and Ginseng Pingji Granules, dissolve them in water, filter, accurately pipette 1 mL, dilute to 2 mL volumetric flask with methanol, shake to mix, and filter through a 0.22 μm microporous membrane to obtain 15 batches of test solution S1~S15. (2) Preparation of reference solution Accurately weigh appropriate amounts of five reference standards: verbascoside, ferulic acid, dihydroquercetin, ammonium glycyrrhizate, and nardostachysone. Place them in a volumetric flask, dissolve them in methanol, and dilute to the mark to prepare a mixed reference standard solution. Before use, dilute with methanol to prepare a series of mixed reference standard solutions of the corresponding concentrations. (3) Establishment of the standard curve equation Inject the series of mixed reference solutions of step (2) into the high performance liquid chromatograph in sequence and record the chromatograms; plot the standard curve equation with concentration on the x-axis and peak area on the y-axis. (4) Establishment of fingerprint spectrum Fifteen batches of test solutions from step (1) were sequentially injected into a high-performance liquid chromatograph, and chromatograms were recorded. Then, the fingerprint chromatograms of the test solutions were exported and imported into the 2012 version of the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System. Chromatographic peaks present in the chromatograms of different batches of test solutions were selected as common peaks. The relative retention time and relative peak area of ​​each common peak were calculated using the average value method to generate the reference fingerprint chromatogram. The chemical composition of the peaks in the reference fingerprint chromatogram was labeled according to the retention time of the chromatogram of the mixed reference solution. Content determination Based on the standard curve equation in step (3), and according to the retention time, substitute the peak area of ​​the test solution into the standard curve equation to calculate the content of each compound in the test solution.

[0012] As a preferred embodiment, the quality detection method of the above-described Astragalus and Ginseng Pingji Granules is characterized in that, in step (2), the preparation of the reference solution is as follows: accurately weigh appropriate amounts of five reference standards, namely, isoflavone glycoside, ferulic acid, dihydroquercetin, ammonium glycyrrhizate, and nardoxylonone, place them in a 10 mL volumetric flask, dissolve them in methanol and dilute to the mark to prepare a mixed reference solution with mass concentrations of 104.75, 197.37, 59.63, 987.65, and 103.48 µg / mL; before use, dilute with methanol to prepare the corresponding mixed reference solution of the five concentrations.

[0013] As a preferred embodiment, the quality detection method for the above-described Astragalus and Ginseng Pingji Granules is characterized in that the chromatographic conditions for steps (3) and (4) are as follows: Chromatographic column: Phenomen Luna C18 column, 250 mm × 4.6 mm, 5 μm; flow rate: 1.00 mL / min; injection volume: 10 μL; column temperature: 30 ℃; mobile phase: acetonitrile (phase A) - 0.1% phosphoric acid aqueous solution (phase B); detection wavelength: 260 nm. nm, gradient elution: 0~10min, 2%A~12%A; 10~15min, 12%A~17%A; 15~23min, 17%A~23%A; 23~31min, 23%A; 31~38min, 23%A~32%A; 38~48min, 32%A~33%A; 48~51min, 33%A~36%A; 51~53min, 36%A~40%A; 53~58min, 40%A~42%A; 58~68min, 42%A~50%A; 68~75min, 50%A~62%A; 75~85min, 62%A~70%A; 85~90min, 70%A~80%A.

[0014] As a preferred option, the quality detection method for the above-mentioned Astragalus and Ginseng Pingji Granules has 20 common peaks in the established fingerprint spectrum. After comparison with the reference standard, peak 5 is identified as isoflavone glycoside, peak 6 as ferulic acid, peak 7 as dihydroquercetin, peak 18 as ammonium glycyrrhizate, and peak 19 as nardinone.

[0015] As a preferred option, the quality testing method for the above-mentioned Astragalus and Ginseng Pingji Granules has the following standard curve equation: .

[0016] The application of the Astragalus and Ginseng Pingji Granules described in this invention in the preparation of drugs for treating premature ventricular contractions.

[0017] Chromatographic condition screening experiment: Screening of mobile phase composition (see below for appropriate description) Because the quality testing method for Qishen Pingji Granules contains multiple Chinese medicinal herbs such as Astragalus membranaceus (processed), Codonopsis pilosula, Ophiopogon japonicus, Asparagus cochinchinensis, Salvia miltiorrhiza, Ligusticum chuanxiong, Poria cocos, Pinellia ternata (processed), and Nardostachys chinensis, the components are relatively complex. In order to achieve good separation during chromatographic separation, this invention conducted a large number of screening experiments on chromatographic conditions. This invention screened various mobile phases such as acetonitrile-0.1% phosphoric acid water, acetonitrile-0.2% phosphoric acid water, acetonitrile-0.3% formic acid water, and methanol-water. Comparative experiments showed that using acetonitrile-0.1% phosphoric acid water as the mobile phase resulted in the best separation of each chromatographic peak. Therefore, this invention selected acetonitrile-0.1% phosphoric acid water as the mobile phase.

[0018] 2. This invention screened different gradient elution methods. Qishen Pingji granules contain various components with similar polarities, such as triterpenoids, flavonoids, and quinones, which are difficult to separate using a single elution method. This invention optimized different gradient elution methods, ultimately obtaining chromatograms with good separation, such as... Figure 1 .

[0019] The beneficial effects of this invention are: 1. This invention has screened out the optimal molding process for Astragalus and Ginseng Pingji Granules through a large number of experiments.

[0020] 2. Quality Standards Research Section This invention screened the optimal thin-layer chromatography development conditions and conducted qualitative identification studies on Danshen, processed Astragalus membranaceus, and Ligusticum chuanxiong in the quality detection method of Qishen Pingji granules. Through extensive experiments, the optimal chromatographic conditions, including flow composition and gradient elution mode, were screened, establishing a fingerprint chromatographic detection method with high precision, stability, and accuracy. This method can simultaneously determine the content of active ingredients such as verbascoside, ferulic acid, dihydroquercetin, ammonium glycyrrhizate, and nardoxylonone, providing a quality detection method to ensure its safety and effectiveness. Attached Figure Description

[0021] Figure 1 TLC image of Astragalus membranaceus (processed with honey).

[0022] Figure 2 TLC image of Salvia miltiorrhiza.

[0023] Figure 3 for Figure 2 TLC diagram of Ligusticum chuanxiong.

[0024] Figure 4 The HPLC chromatogram of the test solution of Qishen Pingji Granules.

[0025] Figure 5 This is the HPLC chromatogram of the mixed reference solution.

[0026] Figure 6 Fingerprint chromatograms of 15 batches of Qishen Pingji Granules. Detailed Implementation

[0027] The embodiments of the present invention will be described in detail below with reference to examples. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. After reading this invention, any modifications of the present invention by those skilled in the art to various equivalent forms fall within the scope defined by the appended claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0028] Example 1 A type of Qishen Pingji granules for treating premature ventricular contractions is prepared by the following method: Take 15g of roasted Astragalus membranaceus, 10g of Codonopsis pilosula, 10g of Ophiopogon japonicus, 10g of Asparagus cochinchinensis, 15g of Salvia miltiorrhiza, 15g of Ligusticum chuanxiong, 15g of Poria cocos, 10g of Pinellia ternata, and 10g of Nardostachys chinensis. Add water and decoct twice. For the first decoction, add 10 times the amount of water, soak for 60 minutes, decoct for 30 minutes, and filter. For the second decoction, add 8 times the amount of water, decoct for 20 minutes, and filter. Concentrate the filtrate under reduced pressure to a relative density of 1.15~1.20 at 60℃. Take an appropriate amount of dextrin and place it in a fluidized bed. Set the inlet air temperature to 90℃~100℃. When the material temperature rises to 60℃, start the liquid injection. Control the liquid injection rate at 80~150 r / min. The atomization pressure is 0.2 MPa externally and 0.15 MPa internally. After spraying, continue drying at 60℃~70℃ to obtain granules.

[0029] Example 2: Thin-layer chromatography qualitative identification method for Astragalus and Ginseng Pingji Granules, comprising the following steps: Thin-layer chromatography identification of processed Astragalus membranaceus: (1) Take Astragalus and Ginseng Pingji Granules, add water to dissolve and filter. Take 40 mL of the filtrate and extract it 4 times with water-saturated n-butanol solution, 40 mL each time. Combine the n-butanol solutions and wash them 2 times with ammonia solution, 40 mL each time. Discard the ammonia solution and evaporate it to dryness in an evaporating dish. Add 1 mL of methanol to dissolve the residue and use it as the test solution. (2) Prepare granules without processed Astragalus membranaceus according to the method of Example 1, and prepare a negative control solution of processed Astragalus membranaceus according to the same method as in step (1); (3) Take another 3 g of roasted Astragalus reference material, add 50 mL of methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, add 30 mL of water to dissolve the residue, and prepare the roasted Astragalus reference material solution according to the same method as in step (1); (4) Take 10 µL of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use the upper layer of a solution with a volume ratio of 4:1:5 n-butanol-ethyl acetate-10% dilute ammonia as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 °C until the spots are clearly visible. Examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material. Figure 1 As shown.

[0030] The specific steps for thin-layer chromatography identification of Salvia miltiorrhiza are as follows: (1) Take the Astragalus and Ginseng Pingji Granules, add water to dissolve them, take 40 mL of the filtrate, adjust the pH to 2 with 10% hydrochloric acid, extract with ethyl acetate 3 times, 20 mL each time; combine the extracts, evaporate to dryness in a water bath, add 5 mL of methanol to dissolve the residue, and use it as the test solution. (2) Prepare granules without danshen medicinal material according to the same method as in Example 1, and prepare danshen negative control solution according to the same method as in step (1); (3) Take another Danshen reference material and prepare a Danshen reference material solution in the same way as in step (1); (4) Apply 10 µL of each of the above three solutions to the same silica gel G thin-layer plate. Develop the plate using a 5:4:2 volume ratio of dichloromethane-ethyl acetate-formic acid as the developing solvent. Remove the plate, air dry, spray with 5% vanillin-sulfuric acid solution, and heat at 105°C until the spots are clear. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material. Figure 2 As shown.

[0031] The specific steps for thin-layer chromatography identification of Ligusticum chuanxiong are as follows: (1) Take Astragalus and Ginseng Pingji Granules, dissolve them in water, take 40 mL of the filtrate, extract twice with ethyl acetate, 40 mL each time, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in 2 mL of methanol, and use it as the test solution. (2) Prepare granules without Ligusticum chuanxiong using the same method as in Example 1, and prepare a negative control solution of Ligusticum chuanxiong using the same method as in step (1); (3) Take another 2 g of Ligusticum chuanxiong reference material, add 100 mL of water, decoct for 0.5 h, filter, extract twice with 40 mL of ethyl acetate each time, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in 2 mL of methanol to prepare Ligusticum chuanxiong reference material solution; (4) Apply the above three solutions separately to the same silica gel GF254 thin-layer plate, develop using a volume ratio of 3:1 hexane-ethyl acetate as the developing solvent, remove, air dry, and examine under a 365 nm UV lamp; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material. Figure 3 As shown.

[0032] Example 3: The fingerprint spectrum and content determination of Qishen Pingji Granules include the following steps: (1) Preparation of the test solution Take 15 batches of Astragalus and Ginseng Pingji Granules, dissolve them in water, filter, accurately pipette 1 mL, dilute to 2 mL volumetric flask with methanol, shake to mix, and filter through a 0.22 μm microporous membrane to obtain 15 batches of test solution S1~S15. (2) Preparation of reference solution Accurately weigh appropriate amounts of five reference standards: verbascoside, ferulic acid, dihydroquercetin, ammonium glycyrrhizate, and nardostachysone. Place them in a 10 mL volumetric flask, dissolve them in methanol, and dilute to the mark to prepare mixed reference standard solutions with mass concentrations of 104.75, 197.37, 59.63, 987.65, and 103.48 µg / mL, respectively. Before use, dilute with methanol to prepare the corresponding mixed reference standard solutions of the five concentrations.

[0033] (3) Establishment of the standard curve equation The series of concentration mixed reference solutions from step (2) were sequentially injected into the high-performance liquid chromatograph, and the chromatograms were recorded. The chromatographic conditions were as follows: column: Phenomen Luna C18 column, 250 mm × 4.6 mm, 5 μm; flow rate: 1.00 mL / min; injection volume: 10 μL; column temperature: 30 ℃; mobile phase: acetonitrile (phase A) - 0.1% phosphoric acid aqueous solution (phase B); detection wavelength: 260 nm. nm, gradient elution: 0~10min, 2%A~12%A; 10~15min, 12%A~17%A; 15~23min, 17%A~23%A; 23~31min, 23%A; 31~38min, 23%A~32%A; 38~48min, 32%A~33%A; 48~51min, 33%A~36%A; 51~53min, 36%A~40%A; 53~58min, 40%A~42%A; 58~68min, 42%A~50%A; 68~75min, 50%A~62%A; 75~85min, 62%A~70%A; 85~90min, 70%A~80%A.

[0034] like Figure 5 The standard curve equation is plotted with concentration on the x-axis and peak area on the y-axis as shown in Table 1 below. Table 1

[0035] (4) Establishment of fingerprint spectrum Take 15 batches of test solution from step (1) and inject them sequentially into the high-performance liquid chromatograph under the same chromatographic conditions as in step (3), and record the chromatograms, such as... Figure 4 Then, the fingerprint chromatogram of the test solution was exported and imported into the 2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System; chromatographic peaks present in the chromatograms of different batches of test solutions were selected as common peaks; the reference fingerprint chromatogram was generated using the average value calculation method, such as... Figure 6 The relative retention time and relative peak area of ​​each common peak were calculated. Using the reference chromatogram (S1) as a reference, the similarity of 15 batches were measured to be 0.999, 1.000, 0.999, 0.999, 1.000, 0.999, 1.000, 0.999, 0.999, 0.999, 1.000, 0.999, 1.000, 1.000, and 1.000, all above 0.95, indicating that each batch of standard decoction has good homogeneity and stability. The chemical components of the peaks in the reference fingerprint chromatogram were labeled according to the retention time of the chromatogram of the mixed reference solution. There were 20 common peaks in the established fingerprint chromatogram. After comparison with the reference, peak 5 was identified as verbascoside, peak 6 as ferulic acid, peak 7 as dihydroquercetin, peak 18 as ammonium glycyrrhizate, and peak 19 as nardinone.

[0036] Content determination According to the standard curve equation in step (3), based on the retention time, substitute the peak area of ​​the test solution into the standard curve equation to calculate the content of each active ingredient (μg∙mL-1). The content determination result (μg∙g-1) = active ingredient content (μg∙mL-1) * 2 (the volume of the sample in mL) / 1 (the volume of the sample in mL) * the volume of the solution in mL / the total mass of the medicinal material in g).

[0037] The content determination results are shown in Table 2. The results show that the average contents of verbascoside, ferulic acid, dihydroquercetin, glycyrrhizic acid ammonium, and nardostachysone are 60.44, 95.50, 30.19, 403.95, and 52.14, respectively.

[0038] Table 2. Content of 5 components in 15 batches of Astragalus, Ginseng and Panax notoginseng. .

[0039] Example 4 Methodological Investigation 1. Precision test The test solution was prepared using Astragalus and Ginseng Pingji (S13 batch). The solution was injected six times consecutively under the chromatographic conditions of Example 3. Using ammonium glycyrrhizate (peak 18) as the reference peak, the relative retention time RSD of each common peak (common peak of the reference standard) was less than 0.7%, and the relative peak area RSD was less than 1.9%, indicating that the instrument precision was good.

[0040] 2. Stability test The test solution was prepared using Astragalus and Ginseng Pingji (S13 batch). The solution was injected and measured at 0, 2, 4, 8, 12 and 24 h according to the chromatographic conditions of Example 3. Using ammonium glycyrrhizate (peak 18) as the reference peak, the relative retention time RSD of each common peak (common peak of the reference standard) was less than 0.5% and the relative peak area RSD was less than 2.0%, indicating that the solution had good stability within 24 h.

[0041] 3. Repeatability test Six parallel test solutions of Astragalus and Ginseng Antiseptic (batch S13) were prepared and injected for determination under the chromatographic conditions of Example 3. Using ammonium glycyrrhizate (peak 18) as the reference peak, the relative retention time RSD of each common peak (common peak of the reference standard) was less than 0.7%, and the relative peak area RSD was less than 2.0%, indicating good repeatability of the method. Specificity test 4. Recovery test Six 1 mL aliquots of the Astragalus, Ginseng, and Panax notoginseng test solution with known contents were precisely pipetted into each aliquot. Appropriate amounts of reference solutions of vernix caseosa, ferulic acid, dihydroquercetin, ammonium glycyrrhizate, and nardostachysone were added to each aliquot, mixed thoroughly, and injected under the chromatographic conditions described in Example 3. The recoveries were calculated. The results showed that the average recoveries of vernix caseosa, ferulic acid, dihydroquercetin, ammonium glycyrrhizate, and nardostachysone were 100.2%, 100.7%, 104.5%, 101.6%, and 103.9%, respectively, with RSDs of 1.34%, 0.50%, 1.87%, 1.22%, and 0.32%, respectively.

[0042] Table 3 Results of the spiking recovery test (n=6) .

[0043] The above methodological test results show that the fingerprint spectrum and content determination method established in this invention has high precision and accuracy, and good repeatability and stability.

Claims

1. A Qishen Pingji Granule for treating premature ventricular contractions, characterized in that, It is prepared by the following method: Take roasted Astragalus membranaceus, Codonopsis pilosula, Ophiopogon japonicus, Asparagus cochinchinensis, Salvia miltiorrhiza, Ligusticum chuanxiong, Poria cocos, Pinellia ternata, and Nardostachys chinensis. Add 6 to 10 times the amount of water, soak them first, then decoct and extract for 20 to 120 minutes. Filter, concentrate the filtrate under reduced pressure, take an appropriate amount of dextrin, place it in a fluidized bed, spray dry, and granulate to obtain the final product.

2. The Astragalus and Ginseng Stabilizing Granules for treating premature ventricular contractions according to claim 1, characterized in that, It is prepared by the following method: Take 15g of roasted Astragalus membranaceus, 10g of Codonopsis pilosula, 10g of Ophiopogon japonicus, 10g of Asparagus cochinchinensis, 15g of Salvia miltiorrhiza, 15g of Ligusticum chuanxiong, 15g of Poria cocos, 10g of Pinellia ternata, and 10g of Nardostachys jatamansi. Add water and decoct twice. For the first decoction, add 10 times the amount of water, soak for 60 minutes, decoct for 30 minutes, and filter. For the second decoction, add 8 times the amount of water, decoct for 20 minutes, and filter. Concentrate the filtrate under reduced pressure to a relative density of 1.15~1.20 at 60℃. Take an appropriate amount of dextrin and place it in a fluidized bed. Set the inlet air temperature to 90℃~100℃. When the material temperature rises to 60℃, start the liquid injection. Control the liquid injection rate at 80~150 r / min. The atomization pressure is 0.2 MPa externally and 0.15 MPa internally. After spraying, continue drying at 60℃~70℃ to obtain granules.

3. The quality testing method for Astragalus and Ginseng Pingji Granules according to claim 2, characterized in that, It includes thin-layer chromatography qualitative identification, fingerprinting, and content determination; The aforementioned thin-layer chromatography qualitative identification includes thin-layer identification of processed Astragalus membranaceus, and the specific steps are as follows: (1) Take Astragalus and Ginseng Pingji Granules, dissolve them in water, extract them by shaking with water-saturated n-butanol solution, combine the n-butanol solutions, wash them with ammonia solution, discard the ammonia solution, evaporate to dryness, dissolve the residue in methanol, and use it as the test solution. (2) Prepare granules without processed Astragalus membranaceus using the same method as in claim 2, and prepare a negative control solution of processed Astragalus membranaceus using the same method as in step (1); (3) Take another roasted Astragalus membranaceus reference material, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, and prepare the roasted Astragalus membranaceus reference material solution in the same way as in step (1); (4) Take the above three solutions and spot them on the same silica gel G thin layer plate. Use the upper layer of n-butanol-ethyl acetate-dilute ammonia as the developing solvent. Develop, remove, and air dry. Spray with 10% sulfuric acid ethanol solution. Heat until the spots are clearly visible and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material. The specific steps for thin-layer chromatography identification of Salvia miltiorrhiza are as follows: (1) Take the Astragalus and Ginseng Pingji Granules, dissolve them in water, adjust the pH to acidic with hydrochloric acid, extract with ethyl acetate, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in methanol, and use it as the test solution. (2) Prepare granules without danshen medicinal material according to the same method as in claim 2, and prepare danshen negative control solution according to the same method as in step (1); (3) Take another Danshen reference material and prepare a Danshen reference material solution in the same way as in step (1); (4) Take the above three solutions and spot them on the same silica gel G thin layer plate respectively. Use dichloromethane-ethyl acetate-formic acid as the developing solvent, develop, remove, air dry, spray with 5% vanillin sulfuric acid solution, and heat until the spots are clear; in the chromatogram of the test sample, spots of the same color are displayed at the corresponding positions as in the chromatogram of the reference medicinal material. The specific steps for thin-layer chromatography identification of Ligusticum chuanxiong are as follows: (1) Take Astragalus and Codonopsis pilosula granules, dissolve them in water, extract them with ethyl acetate, combine the extracts, evaporate them to dryness in a water bath, dissolve the residue in methanol, and use it as the test solution; (2) Prepare granules without Ligusticum chuanxiong using the same method as in claim 2, and prepare a negative control solution of Ligusticum chuanxiong using the same method as in step (1); (3) Take another reference herb, add water, decoct, filter, extract the filtrate with ethyl acetate, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in methanol, and prepare a reference herb solution; (4) Take the above two solutions and spot them on the same silica gel GF254 thin layer plate. Use n-hexane-ethyl acetate as the developing solvent, develop, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

4. The quality testing method for Astragalus and Ginseng Pingji Granules according to claim 3, characterized in that, The aforementioned thin-layer chromatography qualitative identification includes thin-layer identification of processed Astragalus membranaceus, and the specific steps are as follows: (1) Take Astragalus and Ginseng Pingji Granules, add water to dissolve and filter. Take 40 mL of the filtrate and extract it 4 times with water-saturated n-butanol solution, 40 mL each time. Combine the n-butanol solutions and wash them 2 times with ammonia solution, 40 mL each time. Discard the ammonia solution and evaporate it to dryness in an evaporating dish. Add 1 mL of methanol to dissolve the residue and use it as the test solution. (2) Prepare granules without processed Astragalus membranaceus using the same method as in claim 2, and prepare a negative control solution of processed Astragalus membranaceus using the same method as in step (1); (3) Take another 3 g of roasted Astragalus reference material, add 50 mL of methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, add 30 mL of water to dissolve the residue, and prepare the roasted Astragalus reference material solution according to the same method as in step (1); (4) Take 10 µL of the above three solutions and spot them on the same silica gel G thin layer plate. Use the upper layer solution of n-butanol-ethyl acetate-10% dilute ammonia (volume ratio 4:1:5) as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly visible, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material. The specific steps for thin-layer chromatography identification of Salvia miltiorrhiza are as follows: (1) Take the Astragalus and Ginseng Pingji Granules, add water to dissolve them, take 40 mL of the filtrate, adjust the pH to 2 with 10% hydrochloric acid, extract with ethyl acetate 3 times, 20 mL each time; combine the extracts, evaporate to dryness in a water bath, add 5 mL of methanol to dissolve the residue, and use it as the test solution. (2) Prepare granules without danshen medicinal material according to the same method as in claim 2, and prepare danshen negative control solution according to the same method as in step (1); (3) Take another Danshen reference material and prepare a Danshen reference material solution in the same way as in step (1); (4) Take 10 µL of the above three solutions and spot them on the same silica gel G thin layer plate. Use dichloromethane-ethyl acetate-formic acid with a volume ratio of 5:4:2 as the developing solvent. Develop, remove, air dry, spray with 5% vanillin sulfuric acid solution, and heat at 105℃ until the spots are clear. In the chromatogram of the test sample, spots of the same color are displayed at the corresponding positions as in the chromatogram of the reference medicinal material. The specific steps for thin-layer chromatography identification of Ligusticum chuanxiong are as follows: (1) Take Astragalus and Codonopsis pilosula granules, dissolve them in water, take 40 mL of the filtrate, extract twice with ethyl acetate, 40 mL each time, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in 2 mL of methanol, and use it as the test solution. (2) Prepare granules without Ligusticum chuanxiong using the same method as in claim 2, and prepare a negative control solution of Ligusticum chuanxiong using the same method as in step (1); (3) Take another 2 g of Ligusticum chuanxiong reference material, add 100 mL of water, decoct for 0.5 h, filter, extract twice with 40 mL of ethyl acetate each time, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in 2 mL of methanol to prepare Ligusticum chuanxiong reference material solution; (4) Take the above three solutions and spot them on the same silica gel GF254 thin layer plate. Use hexane-ethyl acetate with a volume ratio of 3:1 as the developing solvent, develop, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

5. The quality testing method for Qishen Pingji granules according to claim 3, characterized in that, Fingerprint spectroscopy and content determination include the following steps: (1) Preparation of the test solution Take 15 batches of Astragalus and Ginseng Pingji Granules, dissolve them in water, filter, accurately pipette 1 mL, dilute to 2 mL volumetric flask with methanol, shake to mix, and filter through a 0.22 μm microporous membrane to obtain 15 batches of test solution S1~S15. (2) Preparation of reference solution Accurately weigh appropriate amounts of five reference standards: verbascoside, ferulic acid, dihydroquercetin, ammonium glycyrrhizate, and nardostachysone. Place them in a volumetric flask, dissolve them in methanol, and dilute to the mark to prepare a mixed reference standard solution. Before use, dilute with methanol to prepare a series of mixed reference standard solutions of the corresponding concentrations. (3) Establishment of the standard curve equation Inject the series of mixed reference solutions of step (2) into the high performance liquid chromatograph in sequence and record the chromatograms; plot the standard curve equation with concentration on the x-axis and peak area on the y-axis. (4) Establishment of fingerprint spectrum Fifteen batches of test solutions from step (1) were sequentially injected into a high-performance liquid chromatograph, and chromatograms were recorded. Then, the fingerprint chromatograms of the test solutions were exported and imported into the 2012 version of the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System. Chromatographic peaks present in the chromatograms of different batches of test solutions were selected as common peaks. The relative retention time and relative peak area of ​​each common peak were calculated using the average value method to generate the reference fingerprint chromatogram. The chemical composition of the peaks in the reference fingerprint chromatogram was labeled according to the retention time of the chromatogram of the mixed reference solution. Content determination Based on the standard curve equation in step (3), and according to the retention time, substitute the peak area of ​​the test solution into the standard curve equation to calculate the content of each compound in the test solution.

6. The quality testing method for Astragalus and Ginseng Pingji Granules according to claim 5, characterized in that, Step (2) Preparation of reference solutions: Accurately weigh appropriate amounts of five reference standards: verbascoside, ferulic acid, dihydroquercetin, ammonium glycyrrhizate, and nardostachysone. Place them in a 10 mL volumetric flask, dissolve them in methanol, and dilute to the mark to prepare mixed reference solutions with mass concentrations of 104.75, 197.37, 59.63, 987.65, and 103.48 µg / mL, respectively. Dilute with methanol to prepare the corresponding mixed reference solutions of the five concentrations.

7. The quality testing method for Astragalus and Ginseng Pingji Granules according to claim 5, characterized in that, The chromatographic conditions for steps (3) and (4) are as follows: Chromatographic column: Phenomen Luna C18 column, 250 mm × 4.6 mm, 5 μm; flow rate: 1.00 mL / min; injection volume: 10 μL; column temperature: 30 ℃; mobile phase: acetonitrile (phase A) - 0.1% phosphoric acid aqueous solution (phase B); detection wavelength: 260 nm. nm, gradient elution: 0~10min, 2%A~12%A; 10~15min, 12%A~17%A; 15~23min, 17%A~23%A; 23~31min, 23%A; 31~38min, 23%A~32%A; 38~48min, 32%A~33%A; 48~51min, 33%A~36%A; 51~53min, 36%A~40%A; 53~58min, 40%A~42%A; 58~68min, 42%A~50%A; 68~75min, 50%A~62%A; 75~85min, 62%A~70%A; 85~90min, 70%A~80%A.

8. The quality testing method for Astragalus and Ginseng Pingji Granules according to claim 5, characterized in that, The established fingerprint spectrum contained 20 common peaks. After comparison with the reference standard, peak 5 was identified as verbascoside, peak 6 as ferulic acid, peak 7 as dihydroquercetin, peak 18 as ammonium glycyrrhizate, and peak 19 as nardinone.

9. The quality testing method for Qishen Pingji granules according to claim 5, characterized in that, The equation of the standard curve is 。 10. The use of the Astragalus and Ginseng Relief Granules according to claim 1 or 2 in the preparation of a medicament for treating premature ventricular contractions.

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