Qingjin cough-relieving drink capable of preventing phlegm from forming and relieving cough and quality detection method thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly discloses a Qingjin cough-relieving decoction for preventing phlegm from forming and relieving cough and a quality detection method thereof. The Qingjin cough-relieving drink is prepared by decocting 12 medicinal materials including platycodon grandiflorum, schizonepeta, radix stemonae, cynanchum glaucescens, liquorice, pericarpium citri reticulatae, herba ardisiae japonicae and the like in water, and has the effects of ventilating the lung, relieving cough, reducing phlegm and relieving sore-throat. The quality detection method comprises the following steps: carrying out qualitative identification on pericarpium citri reticulatae, liquorice and cynanchum glaucescens in the Qingjin cough-relieving drink by adopting a thin-layer chromatography method, and determining the contents of hesperidin, bergenin, myricetin and gallic acid in the Qingjin cough-relieving drink by adopting a high-performance liquid chromatography method. The quality detection method is stable, feasible, high in specificity and accurate in detection result, and the quality of the Qingjin cough-relieving drink can be effectively and accurately evaluated and controlled by adopting the method. The extraction and preparation process of the Qingjin cough-relieving drink is optimized by adopting a Box-Behnken response surface method, and a quality detection method is established, so that technical support is provided for production and quality control of the Qingjin cough-relieving drink.

Description

Technical Field

[0001] The invention relates to a Qingjin Zhike drink for resolving phlegm and relieving cough and a quality detection method thereof, belonging to the technical field of traditional Chinese medicine. Background Art

[0002] Cough is a common respiratory disease that can occur alone or in conjunction with other respiratory diseases. Clinically, the main symptoms are coughing or coughing up sputum. In Traditional Chinese Medicine, with the continuous deepening of diagnosis and treatment methods, it is believed that the cause of the disease is no longer limited to external infection and internal injury, but is caused by a variety of factors: such as congenital constitution, environmental pollution, nose, pharynx, larynx, stomach, etc. In recent years, with the high incidence of respiratory diseases, the incidence of cough has continued to rise. Cough is a common clinical symptom. In the early stages of the disease, it does not cause much discomfort. However, if it is not treated with medication in time, long-term coughing can easily induce symptoms such as headaches, vomiting, musculoskeletal pain, and even syncope, which seriously affect the patient's sleep and daily life.

[0003] At present, Western medicine mainly uses central antitussives and peripheral antitussives to treat coughs in clinical practice. Compared with the two, central antitussives have the characteristics of rapid onset and strong efficacy, but they cannot be used for a long time due to the adverse reaction of respiratory depressant addiction. The overall efficacy of peripheral antitussives is relatively mild, and they mainly produce efficacy through one or more links, so they often need to be combined with other drugs to exert their therapeutic effects. In addition, Chinese medicine treatment for cough includes internal and external treatments, among which external treatments include acupuncture, massage, acupoint application, foot fumigation, etc. Its main function is to stimulate the corresponding acupoints to improve the patient's blood circulation, promote the recovery of organ function, and play an auxiliary treatment role, thereby further improving the efficacy of internal drugs. Despite the diversity of treatment methods, the current treatment of early cough is still in a scarce stage, and further exploration and improvement of effective therapeutic drugs and methods are needed.

[0004] In recent years, with the national policy of allowing medical institutions to implement independent pricing for their traditional Chinese medicine preparations and including qualified institutions' preparations within the scope of medical insurance reimbursement according to procedures, this has provided greater room for the development of in-hospital preparations. This has also accelerated the application of long-standing folk prescriptions into in-hospital preparations, enriching clinical medication regimens. In-hospital preparations are not only an important component of Traditional Chinese Medicine (TCM) but also a "nursery" and important source of new TCM drugs, holding significant significance for the inheritance and innovation of TCM. Qingjin Zhike Yin, a representative in-hospital preparation of the Guangxi International Zhuang Medical Hospital, is formulated with 12 medicinal ingredients and boasts excellent clinical efficacy. However, Qingjin Zhike Yin has long been used in the form of a decoction, which has led to numerous drawbacks during its use, limiting its clinical application. Furthermore, qualitative and quantitative testing of its active ingredients is currently lacking, and scientifically validated quality assurance methods are lacking. Therefore, further optimization of its preparation process and development of appropriate quality assurance methods are necessary to ensure the quality and efficacy of Qingjin Zhike Yin. Summary of the Invention

[0005] The present invention aims to address the deficiencies of the prior art and provide a Qingjin Zhike Decoction for resolving phlegm and relieving cough, and a quality detection method thereof. The Qingjin Zhike Decoction has the effects of relieving cough, resolving phlegm, relieving asthma, and inhibiting bacteria. The present invention optimizes the extraction and preparation process of the Qingjin Zhike Decoction using the Box-Behnken response surface methodology and establishes a quality detection method. The method can effectively and accurately evaluate and control the quality of the Qingjin Zhike Decoction, providing technical support for the production and quality control of the Qingjin Zhike Decoction.

[0006] The technical solution adopted in the present invention is as follows: A Qingjin Zhike drink for resolving phlegm and relieving cough, the Qingjin Zhike drink is prepared from the following raw materials in parts by weight: 8-15 parts of Platycodon grandiflorum; 8-15 parts of Schizonepeta tenuifolia; 12-18 parts of Stemona radix; 6-15 parts of Radix Phellodendri; 8-15 parts of Licorice root; 12-18 parts of Tangerine peel; 6-14 parts of Oroxylum indicum; 12-18 parts of Camellia sinensis; 6-15 parts of Amygdalus dulcis; 8-15 parts of Magnolia officinalis; 8-15 parts of Morus alba bark; 8-15 parts of Fritillaria thunbergii; The preparation method of the Qingjin Zhike Drink comprises the following steps: taking the above twelve medicinal materials by weight, soaking them in water for 30 to 60 minutes, then extracting them twice by decoction or heating reflux, adding 8 to 12 times the amount of water for the first extraction and extracting for 60 to 90 minutes, and adding 6 to 10 times the amount of water for the second extraction and extracting for 60 to 90 minutes, filtering, combining the filtrates, concentrating the filtrates to a concentrate with a relative density of 1.03 to 1.10 (60°C), filtering through a 200-mesh filter cloth, and placing the concentrate in a cool and shady storage overnight; taking the supernatant, adding sodium benzoate in an amount of 0.3% by weight of the supernatant, boiling for 30 minutes, adding water to make the volume constant to 1.225g of the crude drug per ml, cooling, and filling the concentrate.

[0007] A method for testing the quality of the Qingjin Zhike Drink for resolving phlegm and relieving cough as described above comprises determining the contents of hesperidin, bergenin, myricetin, and gallic acid in the Qingjin Zhike Drink by high performance liquid chromatography, the specific steps being as follows: (1) Preparation of test solution: Take 2 mL of the product, evaporate to dryness in a water bath, dissolve the residue in 50% methanol to 10 mL, centrifuge at 13,000 rpm for 5 min, take the supernatant, and filter with a 0.45 μm microporous membrane; (2) Preparation of mixed reference solution: Accurately weigh appropriate amounts of hesperidin, bergenin, myricetin, and gallic acid reference substances, place them in 5 mL volumetric flasks, add 50% methanol and ultrasonically dissolve them to prepare single reference solution with mass concentrations of 1.306 mg / mL, 1.36 mg / mL, 1.404 mg / mL, and 1.086 mg / mL, respectively. Take 0.09, 0.5, 0.4, and 1.3 mL of each of the above single reference solutions, place them in 5 mL volumetric flasks, add 50% methanol to prepare mixed reference solution with mass concentrations of 276.74 μL / mL, 135.37 μL / mL, 112.05 μL / mL, and 23.48 μL / mL, respectively; (3) Chromatographic conditions: X-Peonyx-C18 column (250 mm × 4.6 mm); acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution according to the following program: 0-12 min, 2%-7% A; 11-20 min, 7%-13% A; 20-31 min, 13%-15% A; 31-40 min, 15%-19% A; 40-50 min, 19%-20% A; 50-60 min, 20%-23% A; set the column temperature at 30 °C; the volume flow rate was 1.0 mL / min; the detection wavelength was 270 nm; the injection volume was 10 μl; (4) Take 10 μl of the mixed reference solution and the test solution respectively, inject them into the liquid chromatograph, and measure them according to the chromatographic conditions described above.

[0008] Furthermore, the quality detection method further comprises using thin layer chromatography to qualitatively identify tangerine peel, licorice and white radix scutellariae in Qingjinzhike drink, wherein the method of qualitatively identifying tangerine peel using thin layer chromatography comprises the following specific steps: (1) Solution preparation: Take 20 ml of Qingjin Zhike Drink, steam it until almost dry, add 30 ml of ethyl acetate solution, ultrasonicate for 30 min, take it out, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution; prepare a negative sample without tangerine peel according to the preparation method of Qingjin Zhike Drink, and then prepare a negative sample solution in the same way as above; take an appropriate amount of hesperidin reference substance, add methanol to make a solution containing 1 mg per 1 ml as the reference substance solution; (2) Take 3 μL of each of the test sample, negative sample and reference sample solutions and spot them on a silica gel G plate. Develop the plate with ethyl acetate-methanol-water (volume ratio 7.7:1.4:1) as the developing agent. Remove the plate, let it air dry, spray it with AlCl3 test solution, heat it at 105°C for 5 minutes, remove the plate and examine it under a UV lamp at 365 nm. In the chromatogram of the test sample, a spot of the same color appears at the corresponding position in the chromatogram of the reference sample, and there is no interference with the negative sample at the same position.

[0009] The method of qualitatively identifying licorice by thin layer chromatography comprises the following steps: (1) Solution preparation: Take 10 ml of Qingjin Zhike Drink, add 10 ml of methanol and shake to extract, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of purified water, extract once with 10 ml of ether, take the water layer, add 10 ml of ethyl acetate, extract twice, take the ethyl acetate layer, evaporate to dryness, add 1 ml of methanol to dissolve, and obtain the test solution; prepare a negative sample lacking licorice in the same way as the preparation method of Qingjin Zhike Drink, and then prepare a negative sample solution in the same way as above; take another licorice glycoside reference substance, add methanol to make a solution containing 1 mg per 1 ml as the reference solution. Take 1 g of licorice control medicinal material, add 20 ml of methanol, filter by ultrasonication for 30 minutes, take the filtrate and evaporate to dryness, and prepare it according to the preparation method of the test sample as the licorice control medicinal material solution.

[0010] (2) 3 μL each of the test solution, negative sample solution, and control medicinal material solution, and 1 μL of the control solution were spotted on the same silica gel G thin layer plate. The lower layer solvent was developed with chloroform-methanol-water (volume ratio 13:6:2). The plate was taken out, dried, sprayed with 10% sulfuric acid ethanol test solution, heated at 105°C until the spots were clearly colored, and inspected under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the control medicinal material and the control sample, and there was no interference from the negative sample at the same position.

[0011] The specific steps of using thin layer chromatography to qualitatively identify white pectin are as follows: (1) Solution preparation: Take 40 mL of Qingjin Zhike Drink, steam until almost dry, add 40 mL of 5% sodium carbonate solution, ultrasonically treat for 20 minutes, filter, place in a separatory funnel, shake and extract twice with ethyl acetate, each time 40 mL, combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 mL of ethyl acetate to obtain the test solution; prepare a negative sample lacking Baiqian according to the same method as the preparation of Qingjin Zhike Drink, and then prepare a negative sample solution in the same way as above; take another 3 g of Baiqian control medicinal material, add 50 mL of water and boil for 30 minutes, cool, filter, evaporate to dryness, add 40 mL of 5% sodium carbonate solution to the residue, ultrasonically dissolve it, place in a separatory funnel, shake and extract twice with ethyl acetate, each time 40 mL, combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 mL of ethyl acetate to obtain the Baiqian control medicinal material solution; (2) Pipette 8 μL of the test solution, 8 μL of the negative sample solution, and 1 μL of the control medicinal material solution onto the same silica gel G thin layer plate, use toluene-dichloromethane-acetone, volume ratio 4:4:0.6, as the developing solvent, take out, dry, and examine under ultraviolet light at 365 nm; in the chromatogram of the test sample, a spot of the same color appears at the corresponding position in the chromatogram of the control medicinal material, and there is no interference with the negative sample at the same position.

[0012] The Qingjinzhike Drink disclosed in the present invention is an in-hospital preparation of the Guangxi International Zhuang Medical Hospital. It is derived from the clinical experience of Shu Lili, chief physician of the Department of Pulmonary Diseases. The whole recipe consists of 12 medicinal materials and is mainly used to treat coughs caused by exogenous wind-cold and wind-heat. In the recipe, Platycodon grandiflorum is bitter in taste and slightly warm in nature, can promote the flow of lung qi, purge fire and dissipate cold, treat phlegm congestion, shortness of breath, nasal congestion and sore throat. Schizonepeta tenuifolia is pungent and warm, aromatic and dispersing, can dissipate rheumatism, clear the head and eyes, and benefit the throat, and is good at treating colds, headaches and coughs. Stemona radix is ​​sweet, bitter and slightly warm, can moisten the lungs, and treat coughs caused by lung heat. Whitehead atractylodes is pungent, sweet and slightly cold, good at relieving phlegm and relieving coughs, and treats coughs caused by excessive lung qi. Tangerine peel regulates the middle and quickens the diaphragm, guides stagnation and eliminates phlegm. Licorice clears heat, eliminates phlegm and relieves coughs, and harmonizes all the medicines; Oroxylum indicum relieves sore throat and opens voice, Morus alba bark purges the lungs and relieves asthma, promotes diuresis and reduces swelling, Ayurvedic tea and Fritillaria thunbergii clear heat, resolve phlegm and relieve coughs, and bitter almond and Magnolia officinalis promote the flow of lung qi. The combination of these herbs works together to clear the lungs and relieve coughs, while also resolving phlegm and relieving sore throats. Qingjin Zhike Yin is an oral preparation that can reduce cough frequency, promote phlegm discharge, prolong the incubation period of asthma, and reduce inflammatory responses, with excellent efficacy in resolving phlegm and relieving coughs.

[0013] The beneficial effects of the present invention are: 1. The Qingjin Zhike Decoction provided by the present invention is optimized to be a mixture consistent with the decoction in terms of administration and onset time, making it convenient for patients to carry and use in clinical practice. It has the advantages of rapid efficacy, small clinical dosage, portability, and good patient compliance, while maintaining a dosage consistent with the decoction. Furthermore, the present invention optimizes the extraction process of the Qingjin Zhike Decoction using a Box-Behnken response surface methodology combined with an AHP-EWM method, providing a stable and feasible preparation method for the Qingjin Zhike Decoction.

[0014] 2. The Qingjin Zhike Drink provided by the present invention has the effects of promoting lung function and relieving cough, resolving phlegm and relieving sore throat, and is mainly used to treat cough caused by exogenous wind-cold and wind-heat, providing a new medicine for the prevention and treatment of cough. The present invention has conducted a pharmacodynamic study, and the experimental results show that Qingjin Zhike Drink has the effects of relieving cough, resolving phlegm, relieving asthma and inhibiting bacteria, laying a solid theoretical foundation for the clinical application of Qingjin Zhike Drink.

[0015] 3. The present invention also provides a quality detection method for Qingjin Zhike Drink, including using TLC to qualitatively identify dried tangerine peel, liquorice, and radix scutellariae in Qingjin Zhike Drink, and using HPLC to determine the content of hesperidin, bergenin, myricetin, and gallic acid. The provided TLC identification method is stable and feasible, with strong specificity. The TLC spots of dried tangerine peel, liquorice, and radix scutellariae are clear, and the negative results are interference-free. The provided HPLC determination method is simple and stable, with strong specificity, good precision and repeatability, and accurate test results. The quality detection method of the present invention can effectively and accurately evaluate and control the quality of Qingjin Zhike Drink, filling the technical gap in the quality detection of Qingjin Zhike Drink. The present invention provides technical support for the subsequent process production and quality control of Qingjin Zhike Drink, and provides a reference for the establishment of the quality standard of Qingjin Zhike Drink. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of soaking time on the comprehensive score; Figure 2 The effect of water addition on the overall score; Figure 3 the effect of extraction time on the overall score; Figure 4 The impact of extraction times on the overall score; Figure 5 Response surface plot of the interaction between factors; Figure 6 TLC chromatogram of dried tangerine peel. In the figure, 1 to 3 are test samples, 4 is a negative sample lacking dried tangerine peel, and 5 is a hesperidin reference substance (A is the standard point); Figure 7 Investigation of different temperatures of tangerine peel TLC. In the figure, 1 to 3 are test samples, 4 is a negative sample lacking tangerine peel, and 5 is a reference substance for hesperidin (A is the standard point); Figure 8 Investigation of different humidity levels of tangerine peel TLC. In the figure, 1 to 3 are test samples, 4 is a negative sample lacking tangerine peel, and 5 is a hesperidin reference substance (A is the standard point); Figure 9 Investigation of different sample amounts of tangerine peel TLC. In the figure, 1 to 3 are test samples, 4 is a negative sample lacking tangerine peel, and 5 is a hesperidin reference substance (A is a standard point); Figure 10 Investigation of silica gel G plates from different manufacturers for TLC of tangerine peel. In the figure, 1 to 3 are test samples, 4 is a negative sample lacking tangerine peel, and 5 is a hesperidin reference substance (A is a standard point); Figure 11 TLC chromatogram of licorice. In the figure, 1 to 3 are test samples, 4 is a negative sample lacking licorice, 5 is a reference substance for liquiritin, and 6 is a licorice herbal medicine (A is a standard point); Figure 12 Licorice TLC investigation at different temperatures. In the figure, 1 to 3 are test samples, 4 is a negative sample lacking licorice, and 5 is a reference substance (A is the standard point); Figure 13 Investigation of different humidity levels of licorice by TLC. In the figure, 1 to 3 are test samples, 4 is a negative sample lacking licorice, and 5 is a reference substance (A is the standard point); Figure 14 Investigation of different sample volumes in TLC of licorice. In the figure, 1 to 3 samples (sample volumes are 1, 3, and 5 μL, respectively). 4-negative sample lacking licorice, 5-liquiritin reference (A is the standard point); Figure 15 Investigation of different manufacturers of licorice TLC. In the figure, 1 to 3 are test samples, 4 is a negative sample lacking licorice, and 5 is a liquiritin reference substance (A is the standard point); Figure 16TLC chromatogram of Baiqian. In the figure, 1-3 are test samples, 4 is a negative sample lacking Baiqian, and 5 is a control herb of Baiqian (A is a standard point); Figure 17 Investigation of different temperatures of Baiqian TLC. In the figure, 1-3 are test samples, 4 is a negative sample lacking Baiqian, and 5 is a control herb for Baiqian (A is a standard point); Figure 18 Investigation of different humidity levels of Baiqian TLC. In the figure, 1-3 are test samples, 4 is a negative sample lacking Baiqian, and 5 is a control herb for Baiqian (A is a standard point); Figure 19 Investigation of different spot amounts of Baiqian TLC. In the figure, 1-3 are test samples (spot amounts are 4, 8, and 12 μL, respectively), 4 is a negative sample lacking Baiqian, and 5 is a control medicinal material for Baiqian (A is a standard spot); Figure 20 Investigation of different manufacturers’ silica gel G plates for TLC of Baiqian. In the figure, 1 to 3 are test samples, 4 is a negative sample lacking Baiqian, and 5 is a control herbal medicine for Baiqian (A is a standard point); Figure 21 HPLC profiles of the components, including 1-gallic acid, 2-bergenin, 3-myricetin, and 4-hesperidin; Figure 22 Effects of Qingjin Zhike Yin on the cough latency and cough frequency in mice; Figure 23 Effect of Qingjin Zhikeyin on phenol red secretion in mouse trachea; Figure 24 Effect of Qingjin Zhike Drink on the latent period of asthma. DETAILED DESCRIPTION Example 1

[0017] A Qingjin Zhike drink for relieving phlegm and relieving cough, the Qingjin Zhike drink is prepared from the following raw materials in parts by weight: 8 parts of Platycodon grandiflorum; 8 parts of Schizonepeta tenuifolia; 12 parts of Stemona radix; 6 parts of Radix Bletillae; 8 parts of Licorice root; 12 parts of Tangerine peel; 6 parts of Oroxylum indicum; 12 parts of Camellia sinensis; 6 parts of Amygdalus dulcis; 8 parts of Magnolia officinalis; 8 parts of Morus alba bark; 8 parts of Fritillaria thunbergii; the Qingjin Zhike drink is prepared by taking the above twelve medicinal materials in parts by weight, soaking them in water for 30 minutes, and then using a decoction extraction method to extract the 12 medicinal materials. Extract twice, add 8 times the amount of water for the first time and extract for 80 minutes, add 8 times the amount of water for the second time and extract for 75 minutes, filter, combine the filtrates, concentrate the filtrate to a concentrate with a relative density of 1.03-1.10 (60℃), filter through a 200-mesh filter cloth, and place it in a cool storage and let it stand overnight; take the supernatant and add 0.3% of the mass of the supernatant sodium benzoate, boil for 30 minutes, add water to make up the volume to 1.225g of crude drug per 1ml, cool and fill. Example 2

[0018] A Qingjin Zhike drink for relieving phlegm and relieving cough, the Qingjin Zhike drink is prepared from the following raw materials in parts by weight: 12 parts of Platycodon grandiflorum; 12 parts of Schizonepeta tenuifolia; 15 parts of Stemona radix; 10 parts of Radix Bletillae; 12 parts of Licorice root; 15 parts of Tangerine peel; 10 parts of Oroxylum indicum; 15 parts of Camellia sinensis; 10 parts of Amygdalus dulcis; 12 parts of Magnolia officinalis; 12 parts of Morus alba bark; 12 parts of Fritillaria thunbergii; the Qingjin Zhike drink is prepared by taking the above twelve medicinal materials in parts by weight, soaking them in water for 60 minutes, and then heating and reheating them. The extraction method of the stream is to extract twice, add 10 times the amount of water for the first time, extract for 60 minutes, add 6 times the amount of water for the second time, extract for 60 minutes, filter, combine the filtrates, concentrate the filtrate to a concentrate with a relative density of 1.03-1.10 (60°C), filter through a 200-mesh filter cloth, and place it in a cool storage and let it stand overnight; take the supernatant and add 0.3% of the mass of the supernatant sodium benzoate, boil for 30 minutes, add water to make up the volume to 1.225g of crude drug per 1ml, cool and fill. Example 3

[0019] A Qingjin Zhike drink for relieving phlegm and relieving cough is prepared from the following raw materials in parts by weight: 15 parts of Platycodon grandiflorum; 15 parts of Schizonepeta tenuifolia; 18 parts of Stemona radix; 15 parts of Radix Phellodendri; 15 parts of Licorice root; 18 parts of Tangerine peel; 14 parts of Oroxylum indicum; 18 parts of Camellia sinensis; 15 parts of Amygdalus dulcis; 15 parts of Magnolia officinalis; 15 parts of Morus alba bark; and 15 parts of Fritillaria thunbergii. The Qingjin Zhike drink is prepared by taking the above twelve medicinal materials in parts by weight, soaking them in water for 45 minutes, and then decocting them. The extraction method is as follows: extract twice, add 12 times the amount of water for the first time, extract for 90 minutes, add 10 times the amount of water for the second time, extract for 90 minutes, filter, combine the filtrates, concentrate the filtrate to a concentrate with a relative density of 1.03-1.10 (60°C), filter through a 200-mesh filter cloth, place in a cool storage and let stand overnight; take the supernatant and add 0.3% of the mass of the supernatant sodium benzoate, boil for 30 minutes, add water to make up the volume to 1.225g of crude drug per 1ml, cool and fill. Example 4

[0020] Optimization of extraction process and quality testing method of Qingjin Zhike Decoction 1. Materials 1.1 Instruments: Agilent 1220 high-performance liquid chromatograph (Agilent Technologies, USA); HWS-26 constant-temperature water bath (Shanghai Qixin Scientific Instrument Co., Ltd.); 98-1-B electric heating mantle (Tianjin Test Instrument Co., Ltd.); electric constant-temperature blast drying oven (Shanghai Yuejin Medical Instrument Co., Ltd.); 5425R high-speed refrigerated centrifuge (Eppendorf AG, Germany); ultrapure water machine (Nanning Bomei Biotechnology Co., Ltd., Guangxi); 1 / 100,000 electronic balance (Mettler-Toledo Technology Co., Ltd.).

[0021] 1.2 Reagents and drugs: Hesperidin (Batch No. 231115), bergenin (Batch No. 240319), myricetin (Batch No. 40309), and gallic acid (Batch No. 231206) reference substances were purchased from Chengdu Zhibiao Huachun Biotechnology Co., Ltd. Tangerine peel (lot number 20240501), Platycodon grandiflorum (lot number 20240701), Schizonepeta tenuifolia (lot number 20240301), Stemona tuberosa (lot number 20240301), Glycyrrhiza uralensis (lot number 20240701), Camellia davidiana (lot number 20240701), Apricot kernel (lot number 8240108601), Magnolia officinalis (lot number 20240702), Morus alba bark (lot number 2724004), Fritillaria thunbergii (lot number 20240602), Oroxylum indicum (lot number 20240801), and Radix Phellodendri (lot number 20240601) were purchased from Guangxi Xianzhu Traditional Chinese Medicine Technology Co., Ltd. Acetonitrile (Fisher Company, USA, batch number F24O3M201) and phosphoric acid were of chromatographic grade; silica gel G plates (Qingdao Ocean Chemical Co., Ltd., batch number 20231122; Yantai Xinnuo New Materials Co., Ltd., batch number 20230907; Yantai Jiangyou Silica Gel Development Co., Ltd., batch number 20211227; Yantai Huayang New Materials Technology Co., Ltd., batch number 20181109).

[0022] 2 Extraction process optimization 2.1 Chromatographic conditions: X-Peonyx-C18 column (250 mm × 4.6 mm); mobile phase: acetonitrile (A)-0.1% phosphoric acid (B), gradient elution (0–12 min, 2%–7% A; 11–20 min, 7%–13% A; 20–31 min, 13%–15% A; 31–40 min, 15%–19% A; 40–50 min, 19%–20% A; 50–60 min, 20%–23% A); column temperature: 30°C; flow rate: 1.0 mL / min; detection wavelength: 270 nm; injection volume: 10 μl.

[0023] 2.2 Determination of dry cream rate: Accurately pipette 20 mL of the extract into a constant weight evaporating dish, evaporate to dryness in a water bath, and dry at 105°C for 5 h. Remove the extract and cool in a desiccator for 1 h. Weigh the extract and repeat the procedure twice. The difference between the two results should be within the range of 0-5 mg. Calculate the dry cream rate. The formula is: dry cream rate = [(G1 / V1) × (V2 / G2)] × 100%, where G1 is the dry cream mass, G2 is the medicinal material mass, V1 is the sample volume, and V2 is the total volume of the medicinal solution.

[0024] 2.3 Determination of comprehensive weight coefficient by AHP-EWM method 2.3.1 AHP ​​Method: Based on the compatibility patterns and potency of the medicinal ingredients in the Qingjin Zhike Decoction, the order of the five indicators was determined as hesperidin > bergenin > myricetin = gallic acid > paste yield. A judgment matrix was established (see Table 1), and a hierarchical analysis was performed using the SPSS PRO data science analysis platform (https: / / www.spsspro.com). The results showed that the weight coefficients (ωAHPij) for hesperidin, bergenin, myricetin, gallic acid, and paste yield were 0.4017, 0.2442, 0.1373, 0.1373, and 0.0794, respectively, and the consistency ratio factor (CR) was less than 0.10, demonstrating the effectiveness of these weight coefficients.

[0025]

[0026] 2.3.2 EWM method: Referring to the data analysis method of Zeng Hairong et al., the EWM weighting steps are followed to standardize the data, convert the original data into probabilities, calculate and determine the entropy value of each indicator, and finally obtain the weight coefficient (ωEWMij) of each indicator component.

[0027] 2.3.3 Determining the Comprehensive Weight Coefficient Using the AHP-EWM Method: The AHP method reflects the subjective evaluation of each indicator component by experts and is subject to significant subjective influence. The EWM method, by weighting the data using entropy, reflects the objectivity of each evaluation indicator. Combining the two methods may reduce the bias inherent in single weighting methods and provide more comprehensive and scientific results. The formula for the comprehensive weight coefficient ω is: ωcomprehensive = ωAHPijωEWMij / ∑ωAHPijωEWMij, where i represents the i-th trial and j represents the evaluation indicator.

[0028] 2.3.4 Comprehensive score calculation Comprehensive score = (hesperidin content / maximum hesperidin content) × ω hesperidin comprehensive + (bergenin content / maximum bergenin content) × ω bergenin comprehensive + (myricetin / maximum myricetin content) × ω myricetin comprehensive + (gallic acid / maximum gallic acid content) × ω gallic acid comprehensive + (dry paste rate / maximum dry paste rate content) × ω dry paste rate comprehensive.

[0029] 2.4 Single-factor experiment The raw material prescription of Qingjin Zhike Drink is: Platycodon 12g, Schizonepeta 12g, Stemona 15g, Bletilla striata 10g, Licorice 12g, Tangerine peel 15g, Oroxylum indica 10g, Camellia sinensis 15g, Bitter Apricot 10g, Magnolia officinalis 12g, Morus alba bark 12g, Fritillaria thunbergii 12g.

[0030] 2.4.1 Soaking time: Weigh 4 portions of medicinal materials of the daily prescription amount, add 10 times of water of the daily prescription amount respectively, soak for 30, 60, 90 and 120 minutes, reflux extraction once, time is 1 hour, filter and concentrate by rotary evaporation, take a 500mL volumetric flask to make up the volume; accurately aspirate 2mL of the concentrate, evaporate to dryness in a water bath, add 50% methanol to dissolve the residue, make up the volume to 10mL, centrifuge (13000 r / min) for 5min, take the supernatant, filter with a 0.45μm microporous filter membrane and then inject. The weight coefficients of soaking time obtained by EWM method were 0.1998, 0.1884, 0.1836, 0.2354 and 0.1928 respectively. The comprehensive weight coefficients of hesperidin, bergenin, myricetin, gallic acid and dry paste rate obtained by AHP-EWM method were 0.4031, 0.2311, 0.1266, 0.1623 and 0.0769 respectively. The comprehensive scores were calculated and the results are shown in the table. Figure 1 It can be seen that the comprehensive score is the largest when the immersion time is 60 minutes. In order to facilitate the experiment and save time, the immersion time is fixed at 60 minutes.

[0031] 2.4.2 Amount of water added: Weigh 5 portions of medicinal materials in a single-day prescription, add 6, 8, 10, 12, and 14 times the daily prescription amount of water in turn, soak for 1 hour, heat and reflux once for 1 hour, filter and concentrate by rotary evaporation, take a 500mL volumetric flask to the volume; accurately aspirate 2mL of the concentrate, evaporate to dryness in a water bath, add 50% methanol to dissolve the residue, adjust the volume to 10mL, centrifuge (13000 r / min) for 5min, take the supernatant, filter with a 0.45μm microporous filter membrane and then inject. The weight coefficients of water addition obtained by EWM method were 0.1439, 0.2671, 0.2226, 0.1976 and 0.1688 respectively. The comprehensive weight coefficients of hesperidin, bergenin, myricetin, gallic acid and dry paste rate obtained by AHP-EWM method were 0.2977, 0.3360, 0.1575, 0.1340 and 0.0691 respectively. The comprehensive scores were calculated and the results are shown in the table. Figure 2 It can be seen that the comprehensive score is the largest when the water addition amount is 8 times, so 8 times the water addition amount is selected as the factor level center point of the Box-Behnken response surface method.

[0032] 2.4.3 Extraction time: Weigh 5 portions of the daily prescription amount of medicinal materials, add 8 times the daily prescription amount of water to each, soak for 1 hour, heat and reflux once for 1 hour, filter and concentrate by rotary evaporation, take a 500mL volumetric flask to the volume; accurately aspirate 2mL of the concentrate, evaporate to dryness in a water bath, add 50% methanol to dissolve the residue, adjust the volume to 10mL, centrifuge (13000 r / min) for 5min, take the supernatant, filter with a 0.45μm microporous filter membrane and then inject. The weight coefficients of water addition obtained by EWM method were 0.1727, 0.1926, 0.2394, 0.2585 and 0.1368 respectively. The comprehensive weight coefficients of hesperidin, bergenin, myricetin, gallic acid and dry paste rate obtained by AHP-EWM method were 0.3547, 0.2403, 0.1680, 0.1814 and 0.0555 respectively. The comprehensive scores were calculated and the results are shown in the table. Figure 3 It can be seen that the comprehensive score is the largest when the extraction time is 90 minutes, so the extraction time of 90 minutes is selected as the center point of the Box-Behnken response surface method factor level.

[0033] 2.4.4 Number of extractions: Weigh 4 doses of the daily prescription of medicinal material, add 8 times the amount of water of the daily prescription and soak for 1 hour, heat and reflux 1, 2, 3, and 4 times respectively, with each extraction time of 90 minutes, filter, combine the filtrates and concentrate them by rotary evaporation, and take a 500mL volumetric flask to the volume; accurately aspirate 2mL of the concentrate, evaporate to dryness in a water bath, add 50% methanol to dissolve the residue, make up to 10mL, centrifuge (13000 r / min) for 5 minutes, take the supernatant, filter it with a 0.45μm microporous filter membrane and inject it. The weight coefficients of water addition obtained by EWM method were 0.1686, 0.1955, 0.2538, 0.2091 and 0.1730 respectively. The comprehensive weight coefficients of hesperidin, bergenin, myricetin, gallic acid and dry paste rate obtained by AHP-EWM method were 0.3513, 0.2476, 0.1808, 0.1490 and 0.0713 respectively. The comprehensive score was calculated. The results are shown in the table. Figure 4 Therefore, the comprehensive score is the largest when the number of extractions is 2, so reflux extraction 2 times is selected as the level center point of the Box-Behnken response surface method factor.

[0034] 2.5 Box-Behnken response surface experiment: According to the results of single-factor experiment, extraction time (A), amount of water added (B), and number of extractions (C) were used as influencing factors, and the comprehensive score (Y) of hesperidin, bergenin, myricetin, gallic acid content and dry paste rate was used as the evaluation index. The design was carried out using Design-Expert 13.0 software. The results are shown in Tables 2 and 3. After data analysis, the quadratic regression equation was Y=93.12-2.43A+1.18B+0.80C-1.14AB-4.20AC-0.3975BC-9.69A2 -4.99B 2 -10.98C 2 (R 2 =0.9431). The regression model variance results are shown in Table 4. P < 0.05 indicates that the model is highly significant; the lack-of-fit term is > 0.05, indicating no significant difference, indicating that the model fits well. The above data demonstrate that unknown factors have little impact on the experimental data, with A > B > C representing the degree of influence of each factor on the comprehensive score.

[0035]

[0036]

[0037]

[0038] Response surface analysis results (see Figure 5 The above data show that the optimal extraction conditions for Qingjin Zhike Drink are: 8 times the amount of water added, 86 minutes of extraction time, 2 extraction times, and a comprehensive score of 93.40 points.

[0039] Three prescription quantities of medicinal materials were accurately weighed, and verification tests were carried out according to the above optimal extraction process. The results are shown in Table 5. The results show that the average comprehensive score of 97.96 is greater than the predicted comprehensive score of 93.40, indicating that the extraction process is stable and feasible.

[0040]

[0041] 3. Establishment of quality inspection methods 3.1 Properties: This product is a brown liquid with a light fragrance and a slightly bitter taste with a sweet aftertaste.

[0042] 3.2 Qualitative identification by thin layer chromatography (TLC) 3.2.1 Tangerine peel 3.2.1.1 Solution Preparation: Prepare three batches of Qingjin Zhike Drink samples (241101, 241102, and 241103) according to the optimal extraction conditions under "2.5." Take 20 ml of the sample and evaporate to near dryness. Add 30 ml of ethyl acetate and sonicate for 30 min. Remove, filter, and evaporate the filtrate to dryness. Dissolve the residue in 1 ml of methanol to obtain the test solution. Prepare the negative sample solution using the same method. Separately, take an appropriate amount of hesperidin reference standard and add methanol to a solution containing 1 mg per ml as the reference standard solution.

[0043] 3.2.1.2 Specificity test: Take 3 μL of each of the test sample, negative sample and reference sample solution under “3.2.1.1” and spot them on a silica gel G plate. Develop with ethyl acetate-methanol-water (7.7:1.4:1). Remove the plate, dry it in the air, spray it with AlCl3 test solution, heat it at 105℃ for 5 minutes, remove it and examine it under ultraviolet light (365nm). The results are shown in the table. Figure 6 The experimental results showed that in the chromatogram of the test sample, a spot of the same color appeared at the corresponding position in the chromatogram of the reference sample, and there was no interference in the negative sample at the same position.

[0044] 3.2.1.3 Methodological Investigation Temperature: Spot the sample according to the conditions in "3.2.1.2". Place the silica gel G plate under low temperature (5℃), high temperature (45℃) and room temperature (25℃) conditions. The results are shown in Figure 7 .

[0045] Humidity: Spot the sample according to the conditions in "3.2.1.2". Place the silica gel G plate under low humidity (40%), high humidity (90%) and normal humidity (60%) conditions. The results are shown in Figure 8 .

[0046] Spotting volume: Spot the sample according to the conditions in “3.2.1.2”, take 1, 3, and 5 μL of the test sample respectively, and the results are shown in Figure 9 .

[0047] Different manufacturers: According to the conditions under "3.2.1.2", three different manufacturers' silica gel G plates were used for spotting. The results are shown in Figure 10 .

[0048] The above experimental results prove that the TLC qualitative identification method of tangerine peel shows the same color spots at the same position under different temperature, humidity and silica gel plates from different manufacturers, and there is no interference in the negative test, which shows that this method is stable and feasible.

[0049] 3.2.2 Licorice 3.2.2.1 Solution Preparation: Prepare three batches of Qingjin Zhike Drink samples (241101, 241102, and 241103) according to the optimal extraction conditions under "2.5." Take 10 ml of the sample and add 10 ml of methanol for extraction by shaking. Filter and evaporate the filtrate to dryness. Dissolve the residue in 10 ml of purified water and extract once with 10 ml of ether. Take the aqueous layer and add 10 ml of ethyl acetate, extract twice. Take the ethyl acetate layer, evaporate to dryness, and dissolve in 1 ml of methanol to obtain the test solution. Prepare the negative solution using the same method as above. Separately, prepare a 1 mg / ml solution of the liquiritin reference standard (Glycyrrhizin). Take 1 g of the Glycyrrhizin reference medicinal material (Glycyrrhizin) and add 20 ml of methanol. Ultrasonicate for 30 minutes and filter. After evaporation of the filtrate, prepare the Glycyrrhizin reference medicinal material solution according to the preparation method for the test sample.

[0050] 3.2.2.2 Specificity test: Pipette 3 μL each of the test solution, negative solution, and control medicinal material solution under "3.2.2.1", and 1 μL of the control solution onto the same silica gel G thin layer plate, use chloroform-methanol-water (13:6:2) as the lower solvent as the developing agent, remove the plate, dry it, spray it with 10% sulfuric acid ethanol test solution, heat it at 105℃ until the spots are clearly colored, and inspect it under sunlight. The results are shown in the figure. Figure 11 The experimental results showed that in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the control medicinal materials and the reference sample, and there was no interference in the negative sample at the same position.

[0051] 3.2.2.3 Methodological investigation: Temperature: Spot samples according to the conditions in "3.2.2.2". Develop the silica gel G plate at low temperature (5°C), high temperature (45°C), and room temperature (25°C). The results are shown in Figure 12 .

[0052] Humidity: Spot samples according to the conditions in 3.2.2.2. Place the silica gel G plate under low humidity (40%), high humidity (90%), and normal humidity (60%) conditions. The results are shown in Figure 13 .

[0053] Spotting volume: Spot the sample according to the conditions under "3.2.2.2". Take 1, 3, and 5 μL of the test sample for spotting respectively. The results are shown in Figure 14 .

[0054] Different manufacturers: According to the conditions under "3.2.2.2", three different manufacturers' silica gel G plates were used for spotting. The results are shown in Figure 15 .

[0055] The above experimental results show that the TLC qualitative identification method of licorice shows the same color spots at the same position under different temperature, humidity and silica gel plates from different manufacturers, and there is no interference in the negative test, which shows that this method is stable and feasible.

[0056] 3.2.3 Bai Qian 3.2.3.1 Solution Preparation: Prepare three batches of Qingjin Zhike Drink samples (241101, 241102, and 241103) according to the optimal extraction conditions under "2.5." Take 40 mL of each sample and evaporate to near dryness. Add 40 mL of 5% sodium carbonate solution, sonicate for 20 minutes, filter, and transfer to a separatory funnel. Extract twice with ethyl acetate (40 mL each time) by shaking. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 mL of ethyl acetate to obtain the test solution. Prepare the negative solution using the same method. Separately, take 3 g of the Baiqian control herb, add 50 mL of water, and boil for 30 minutes. Cool, filter, and evaporate to dryness. Add 40 mL of 5% sodium carbonate solution to the residue, sonicate, and transfer to a separatory funnel. Extract twice with ethyl acetate (40 mL each time) by shaking. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 mL of ethyl acetate to obtain the Baiqian control herb solution.

[0057] 3.2.3.2 Specificity test: Take 8μL of the test solution and negative solution under "3.2.3.1" and 1μL of the control medicinal material solution, and spot them on the same silica gel G thin layer plate. Use toluene-dichloromethane-acetone (4:4:0.6) as the developing solvent. Take out, dry, and examine under ultraviolet light (365nm). The results are shown in the figure. Figure 16 The experimental results showed that in the chromatogram of the test sample, a spot of the same color appeared at the corresponding position in the chromatogram of the control medicinal material, and there was no interference in the negative sample at the same position.

[0058] 3.2.3.3 Methodological Investigation Temperature: Spot the sample according to the conditions in 3.2.3.2. Place the silica gel G plate under low temperature (5°C), high temperature (45°C), and room temperature (25°C) conditions. The results are shown in Figure 17 .

[0059] Humidity: Spot samples according to the conditions in 3.2.3.2. Place the silica gel G plate under low humidity (40%), high humidity (90%), and normal humidity (60%) conditions. The results are shown in the table below. Figure 18 .

[0060] Spotting volume: Spot the sample according to the conditions under "3.2.3.2". Take 4, 8, and 12 μL of the test sample for spotting respectively. The results are shown in Figure 19 .

[0061] Different manufacturers: According to the conditions under "3.2.3.2", three different manufacturers' silica gel G plates were used for spotting. The results are shown in Figure 20 .

[0062] The above experimental results prove that the TLC qualitative identification method of white qian shows the same color spots at the same position under different temperature, humidity and silica gel plates from different manufacturers, and there is no interference in the negative test, which shows that the method is stable and feasible.

[0063] 3.3 High-performance liquid chromatography (HPLC) content determination 3.3.1 Preparation of test solution: Take 2 mL of Qingjin Zhike Drink (241101), evaporate to dryness in a water bath, dissolve the residue in 50% methanol and make up to 10 mL, centrifuge (13000 r / min) for 5 min, take the supernatant, and filter with a 0.45 μm microporous membrane.

[0064] 3.3.2 Preparation of mixed reference solution: Accurately weigh appropriate amounts of hesperidin, bergenin, myricetin, and gallic acid reference substances, place them in 5 mL volumetric flasks, add 50% methanol and sonicate to dissolve them, to prepare single reference substance solutions with mass concentrations of 1.306 mg / mL, 1.36 mg / mL, 1.404 mg / mL, and 1.086 mg / mL, respectively. Take 0.09, 0.5, 0.4, and 1.3 mL of each of the above single reference substance solutions, respectively, place them in 5 mL volumetric flasks, add 50% methanol to prepare mixed reference substance solutions with mass concentrations of 276.74 μL / mL, 135.37 μL / mL, 112.05 μL / mL, and 23.48 μL / mL, respectively.

[0065] 3.3.3 Specificity test: Take appropriate amount of test sample and reference sample solution, inject and measure under the chromatographic conditions of "2.1", and the results are shown in Figure 21 .

[0066] 3.3.4 Linear relationship investigation: 0.2, 0.4, 0.8, 1.2, and 1.6 mL of the mixed reference solution were respectively drawn into a 2 mL volumetric flask, and 50% methanol was added to the volume. The sample was injected and measured under the chromatographic conditions of "2.1". The regression equations for hesperidin were Y = 6.913X + 12.17 (R 2 =0.9990), with a good linear relationship in the range of 27.674~221.393μL / mL; Bergenin Y=14.672X-1.912(R 2 =0.9991), and the linear relationship was good in the range of 13.537~108.302μL / mL; myricetin Y=15.032X-25.398(R 2 =0.9992), with a good linear relationship in the range of 11.205~89.640μL / mL; Gallic acid Y=31.02X-10.833(R 2 =0.9991), and had a good linear relationship in the range of 2.348 to 18.784 μL / mL.

[0067] 3.3.5 Precision test: Take an appropriate amount of the mixed reference solution and inject it six times in succession under the chromatographic conditions in “2.1”. The results show that the RSDs of the peak areas of hesperidin, bergenin, myricetin and gallic acid are 0.30%, 0.28%, 0.26% and 0.13%, respectively, indicating that the precision of the instrument is good.

[0068] 3.3.6 Stability test: Take 2 mL of this product (batch number 2411102) and prepare the test solution according to the method in "3.3.1". Sampling and detection are carried out at 0, 6, 8, 12, 16, and 28 h under the chromatographic conditions in "2.1". The results show that the peak areas of hesperidin, bergenin, myricetin, and gallic acid are 1.15%, 1.27%, 1.19%, and 1.23%, respectively.

[0069] 3.3.7 Repeatability test: Take 6 portions of this product (batch number 2411102), each 2 mL, and prepare the test solution according to the method under "3.3.1". The samples were injected and tested under the chromatographic conditions under "2.1". The results showed that the RSDs of the peak areas of hesperidin, bergenin, myricetin, and gallic acid were 0.99%, 1.81%, 1.54%, and 1.69%, respectively, indicating that the method has good repeatability.

[0070] 3.3.8 Sample recovery test: Pipette 0.2 mL of the aqueous extract with known content of each component and add it to the reference solution at 100% level. Prepare 6 test sample solutions in parallel using the method under "3.3.1". Samples were injected and tested under the chromatographic conditions under "2.1". The results showed that the average sample recoveries of hesperidin, bergenin, myricetin and gallic acid were 96.12%, 97.54%, 97.33% and 96.76%, respectively, and the RSDs were 1.22%, 1.79%, 1.46% and 1.87%, respectively.

[0071] 4 Discussion Qingjin Zhike Decoction is composed of 12 Chinese medicinal herbs with numerous and complex chemical compositions, making it challenging to select specific indicators and use thin-layer chromatography to identify these herbs for qualitative identification. Platycodon grandiflorum, licorice, and bitter almond index components platycodin, liquiritin, and amygdalin were not detected in multiple tests. Fritillaria thunbergii index component fritillariae var. thunbergii requires evaporative light scattering (ELS), a complex process that can lead to significant errors. Considering that Qingjin Zhike Decoction is an aqueous extract, and the main components of Stemona radix are alkaloids, which are slightly soluble in water and unstable when exposed to heat; magnolol and honokiol, index components of Magnolia officinalis, are both poorly soluble in water; and pulegone, an index component of Schizonepeta tenuifolia, is highly volatile and has a low total solubility in water, these index components were not selected to maximize the detection of components in the aqueous extract.

[0072] During TLC qualitative identification, multiple experiments were conducted on herbs such as Platycodon grandiflorum, Schizonepeta tenuifolia, Camellia davidiana, and Amygdalus amygdalus, all of which showed negative interference or blank plate results. Taking into account factors such as experimental stability, specificity, and reproducibility, this study selected hesperidin, bergenin, myricetin, gallic acid, and dry extract yield as indicator components. A stable and feasible preparation process for this prescription was established by combining AHP-EWM and Box-Behnken response surface methodology. A preliminary quality control method for Qingjin Zhike Drink was established using TLC qualitative identification and HPLC content determination. Example 5

[0073] 1. Pharmacological Study on Qingjin Zhike Drink 1 Materials and Methods 1.1 Materials 1.1.1 Animals: KM mice (SPF grade) were purchased from the Laboratory Animal Center of the Guangxi Zhuang Autonomous Region Institute for Drug Control, production license number SCXK-Gui 2022-0001. Hartley rats and guinea pigs (standard grade) were purchased from Hunan Taiping Biotechnology Co., Ltd., production license number SCXK-Xiang 2023-0005. Laboratory Animal Use License Number: SYXK-Gui 2023-0005. During the experiment, animals were housed in separate cages in a standard environment at a temperature of 21–22°C and a relative humidity of 54–64%. All animals had free access to water and food. 1.1.2 Drugs: Qingjin Zhike Drink (raw material prescription: Platycodon grandiflorum 12 g, Schizonepeta tenuifolia 12 g, Stemona 15 g, Bletilla striata 10 g, Licorice root 12 g, Tangerine peel 15 g, Oroxylum indica 10 g, Camellia sinensis 15 g, Bitter Apricot kernel 10 g, Magnolia officinalis 12 g, Morus alba bark 12 g, Fritillaria thunbergii 12 g) was provided by the Zhuang and Yao Medicine Preparation Center of Guangxi International Zhuang Medicine Hospital (Batch No.: 20241001); Jizhi Syrup was purchased from Chongqing Fuling Pharmaceutical Factory Co., Ltd. of Taiji Group (Batch No.: 24040739).

[0074] 1.1.3 Reagents: ammonia water (Guangdong Guanghua Science and Technology Co., Ltd., batch number: 20240118), phenol red (Shanghai Reagent Factory No. 3, batch number: 930224), sodium bicarbonate (Tianjin Guangfu Science and Technology Development Co., Ltd., batch number: 20210524), histamine phosphate (Shanghai MacLean Biochemical Technology Co., Ltd., batch number: C15963818), hydrolyzed casein peptone (MH) broth, MH agar medium, 0.5% sterile TTC solution (Guangdong Huankai Microbiological Technology Co., Ltd., batch numbers: 240104A30, 1110191, 241217P10), Pseudomonas aeruginosa CMCC(B)10104 ( Pseudomonas aeruginosa ), Staphylococcus aureus CMCC(B)26003 ( Staphylococcus aureus ), Escherichia coli CMCC(B)44102 ( Escherichia coli)(China Food and Drug Inspection Institute, batch numbers: 2a23-2, 5a31-1, 3a27-1).

[0075] 1.1.4 Instruments: Rotary evaporator (Heidolph, Germany, model: Advantage ML / G3); electronic balance (Changshu Shuangjie Testing Instrument Factory, model: JJ2000A); electronic balance (Mettler-Toledo Group, model: PL3001-S); precision balance (Mettler-Toledo Group, model: XSR304); multifunctional microplate reader (Tecan, Switzerland, model: INFINITE M NANO+); biochemical incubator (Puhexi Co., Ltd., model: PHcbi); intelligent high-pressure sterilizer (Zhiwei (Xiamen) Instrument Co., Ltd., model: GR110DF); bacterial enrichment shaker (Eppendorf, Germany, model: Innova 40); colony counter (Hangzhou Xunshu Technology Co., Ltd., model: MF3); Class B2 biological safety cabinet (Taicang Yisigao Medical Technology Co., Ltd., model: AB2-6S1).

[0076] 1.2 Methods 1.2.1 Antitussive Effect of Qingjin Zhike Decoction: Sixty mice were randomly divided into a blank control group, a low-dose Qingjin Zhike Decoction group (0.612 g crude drug / ml), a medium-dose Qingjin Zhike Decoction group (1.225 g crude drug / ml), a high-dose Qingjin Zhike Decoction group (2.45 g crude drug / ml), and a positive control group, with 12 mice in each group. The blank control group received the same volume of purified water, while the positive control group received Jizhi Syrup. Each group received a gavage volume of 20 ml / kg once daily for 7 days. One hour after the last dose, the mice were placed in a 500 ml conical flask. 0.3 ml of concentrated ammonia solution was poured into a 200 mg cotton ball, which was then placed in the conical flask. The mice were removed immediately after 2 minutes. Coughing was assessed by vigorous abdominal muscle contraction, shoulder and back shrug, and mouth opening. The latency to cough and the number of coughs within 2 minutes after the placement of the cotton ball were observed and recorded.

[0077] 1.2.2 Expectorant Effect of Qingjin Zhike Decoction: Sixty mice (half male and half female) were randomly divided into a blank control group, a low-dose Qingjin Zhike Decoction group (0.612 g crude drug / ml), a medium-dose Qingjin Zhike Decoction group (1.225 g crude drug / ml), a high-dose Qingjin Zhike Decoction group (2.45 g crude drug / ml), and a positive control group, with 12 mice in each group. The blank control group received the same volume of purified water, while the positive control group received Jizhi Syrup. Each group received 20 ml / kg of oral administration once daily for 7 days. Thirty minutes after the last administration, phenol red was injected intraperitoneally at 5 mg / 10 g body weight. Half an hour after the injection of phenol red, the animals were killed, the tissues around the trachea were stripped off, and a section of trachea from the thyroid cartilage to the tracheal branch was cut and placed in a test tube containing 2 ml of 5% sodium bicarbonate-normal saline. The absorbance was detected at a wavelength of 546 nm using an enzyme reader, and the phenol red excretion amount (μg / ml) was calculated by substituting it into the standard curve.

[0078] 1.2.3 Antiasthmatic Effect of Qingjin Zhike Drink 1.2.3.1 Preselection: 100 guinea pigs, half male and half female, were placed in an asthma induction chamber and sprayed with 2 mg / ml histamine phosphate for 15 seconds. After a certain latency period following inhalation of the solution, the animals developed an asthmatic reaction. Asthmatic reactions are categorized into four levels: Level I: Accelerated breathing, Level II: Dyspnea, Level III: Convulsions, and Level IV: Falls. Level III or IV reactions generally do not last more than 150 seconds. Those lasting longer than 150 seconds are considered insensitive and are not selected. As soon as an animal develops a convulsion, the chamber door should be opened and the animal removed to prevent suffocation.

[0079] 1.2.3.2 Experimental Procedure: Fifty pre-selected guinea pigs were randomly divided into a blank control group, a low-dose Qingjin Zhike Yin group (0.612 g crude drug / ml), a medium-dose Qingjin Zhike Yin group (1.225 g crude drug / ml), a high-dose Qingjin Zhike Yin group (2.45 g crude drug / ml), and a positive control group, with 12 mice in each group. The blank control group received the same volume of purified water, while the positive control group received Jizhi syrup. Each group received a gavage volume of 10 ml / kg once daily for 7 days. One hour after the last dose, the guinea pigs were placed in an asthma induction device and inhaled with 2 mg / ml histamine phosphate for 15 seconds. The latency time was recorded from the start of inhalation to the onset of symptoms (convulsions, falls).

[0080] 1.2.4 Antibacterial effect of Qingjin Zhike Drink Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were inoculated into hydrolyzed casein peptone (MH) broth and incubated in a biochemical incubator at 37°C for 11 h. The bacterial concentration was determined by turbidimetry and adjusted to 10 6CFU / mL for future use. The test drug solution was continuously diluted using the two-fold dilution method to 1000mg crude drug / mL, 500mg crude drug / mL, 250mg crude drug / mL, 125mg crude drug / mL, 62.5mg crude drug / mL, 31.25mg crude drug / mL, 15.625mg crude drug / mL, 7.812mg crude drug / mL, 3.906mg crude drug / mL, and 1.953mg crude drug / mL. Accurately pipette 50μL crude drug / well of each concentration gradient drug solution and add it to the 96-well cell culture plate in turn, and then add an equal volume of 10 6 CFU / mL bacterial suspensions were prepared, and bacterial growth control wells and drug solution control wells were set up. After incubation at 37°C in a biochemical incubator for 24 hours, TTC solution (0.5%) was added and observed. The minimum inhibitory concentration (MIC) was determined as the lowest drug concentration at which the solution did not turn red. Cultures from each sterile growth well were streaked onto MH agar plates and incubated at 37°C in a biochemical incubator for 24 hours. The minimum bactericidal concentration (MBC) was determined as the lowest drug concentration at which no colonies grew.

[0081] 1.3 Statistical methods The data were statistically analyzed using the one-way analysis of variance-LSD method in SPSS Statistics 25.0 software. The measurement data were expressed as mean ± standard deviation (x¯±s). P <0.05 indicated that the difference was statistically significant.

[0082] 2 Results 2.1 Results of the study on the efficacy of Qingjin Zhike Yin in relieving cough: Compared with the blank control group, the middle and high dose groups of Qingjin Zhike Yin and the positive control group all showed extremely significant inhibitory effects ( P <0.01). Although there was a certain improvement trend in the low-dose group, the difference was not statistically significant ( P >0.05). See Table 6, Figure 22 .

[0083]

[0084] 2.2 Results of the study on the efficacy of Qingjin Zhike Yin in reducing phlegm: Compared with the blank control group, the phenol red excretion in the medium-dose Qingjin Zhike Yin group was significantly increased ( P <0.05, while the high-dose group and the positive control group showed extremely significant promoting effects ( P <0.01). Although the low-dose group showed a certain improvement trend, the difference did not reach statistical significance ( P >0.05). See Table 7, Figure 23 .

[0085] .

[0086] 2.3 The results of the study on the efficacy of Qingjin Zhike Yin in relieving asthma: Compared with the blank control group, the high-dose Qingjin Zhike Yin group and the positive control group showed a very significant effect in prolonging the incubation period of asthma ( P <0.01). Although there was a certain tendency of prolongation in the medium-dose group and the low-dose group, the difference did not reach statistical significance ( P >0.05). See Table 8, Figure 24 .

[0087]

[0088] 2.4 Results of the study on the antibacterial efficacy of Qingjin Zhike Drink: The experimental results showed that Qingjin Zhike Drink exhibited different degrees of antibacterial activity against the test strains, among which the antibacterial effect against Staphylococcus aureus was the most significant, see Table 9.

[0089]

[0090] 3 Discussion Pharmacodynamic research is a prerequisite for the clinical promotion of Qingjin Zhike Decoction. This study focused on its antitussive, expectorant, asthmatic, and antibacterial effects. The results demonstrated that Qingjin Zhike Decoction exhibited significant therapeutic effects in these areas. These findings provide a solid theoretical foundation for its clinical application.

[0091] 2. Acute and subacute toxicity evaluation of Qingjin Zhike Drink The present invention also studies the acute and subacute toxicity of the Qingjin Zhike Drink when administered orally.

[0092] Test drug: Qingjin Zhike Drink (batch number 20241013), provided by Guangxi International Zhuang Medicine Hospital. The clinical oral dosage is 2 bags per day, each bag is 100 mL, and one bag is equivalent to 73.5 g of the original medicinal material.

[0093] Methods: Twenty KM mice, half male and half female, were gavage-administered twice daily with the maximum dose of Qingjin Zhike Yin (equivalent to 259.2 g / kg of the original medicinal material, 106 times the clinical daily dose). The mice's health and mortality were observed during and 14 days after administration. In the subacute toxicity study, 120 SD rats were randomly divided into a blank control group and high-, medium-, and low-dose Qingjin Zhike Yin groups (54.4 g / kg, 27.2 g / kg, and 13.6 g / kg, respectively, (147 g / kg, 73.5 g / kg, and 36.75 g / kg of the original medicinal material, equivalent to 60, 30, and 15 times the human clinical daily dose). Each group was treated with Qingjin Zhike Yin for 30 consecutive days. During and after drug withdrawal, changes in body weight and food intake, hematological and blood biochemical parameters, urine, and organ indices were measured and recorded.

[0094] Results: In the acute toxicity test, Qingjin Zhike Yin had no obvious toxicity or lethality to mice. In the subacute toxicity test, Qingjin Zhike Yin had no significant effect on rats' food intake and urine index. The body weight of female rats in the high and medium dose groups of Qingjin Zhike Yin was higher than that in the control group ( P <0.05 or P <0.01), the body weight of male rats was lower than that of the control group ( P <0.05 or P <0.01). After 30 days of drug withdrawal, the high-dose group had significant differences in both female and male rats compared with the control group. In addition, the serum ALT and GGT values ​​in the high- and medium-dose groups were significantly higher than those in the control group ( P <0.01 or P <0.05), the liver and kidney coefficients were significantly increased compared with the control group ( P <0.01), and showed a recovery trend 30 days after drug withdrawal; no obvious abnormalities were found in the histopathological examination of the above conditions.

[0095] Conclusion: The oral toxicity test of Qingjin Zhike Drink showed that the no-observable adverse effect level was greater than 259.2 g / kg, indicating that it was highly safe.

Claims

1. A Qingjinzhike drink for resolving phlegm and relieving cough, characterized in that: The Qingjinzhike drink is prepared from the following raw materials in parts by weight: 8-15 parts of Platycodon grandiflorum; 8-15 parts of Schizonepeta tenuifolia; 12-18 parts of Stemona radix; 6-15 parts of Radix Bletillae; 8-15 parts of Licorice root; 12-18 parts of Tangerine peel; 6-14 parts of Oroxylum indicum; 12-18 parts of Camellia sinensis; 6-15 parts of Bitter Apricot kernel; 8-15 parts of Magnolia officinalis; 8-15 parts of Morus alba bark; 8-15 parts of Fritillaria thunbergii; The preparation method of the Qingjin Zhike Drink comprises the following steps: taking the above twelve medicinal materials by weight, soaking them in water for 30 to 60 minutes, then extracting them twice by decoction or heating reflux, adding 8 to 12 times the amount of water for the first extraction and extracting for 60 to 90 minutes, and adding 6 to 10 times the amount of water for the second extraction and extracting for 60 to 90 minutes, filtering, combining the filtrates, concentrating the filtrates to a concentrated solution with a relative density of 1.03 to 1.10 at 60°C, filtering through a 200-mesh filter cloth, and placing the solution in a cool and shady storage to stand overnight; taking the supernatant, adding sodium benzoate in an amount of 0.3% by weight of the supernatant, boiling for 30 minutes, adding water to make the volume constant so that each ml contains 1.225 g of the crude drug, cooling, and filling the solution to obtain the product.

2. A quality testing method for the Qingjin Zhike Drink for resolving phlegm and relieving cough according to claim 1, characterized in that: The method comprises the following steps: determining the contents of hesperidin, bergenin, myricetin and gallic acid in Qingjin Zhike Drink by high performance liquid chromatography. (1) Preparation of test solution: Take 2 mL of the product, evaporate to dryness in a water bath, dissolve the residue in 50% methanol to 10 mL, centrifuge at 13,000 rpm for 5 min, take the supernatant, and filter with a 0.45 μm microporous membrane; (2) Preparation of mixed reference solution: Accurately weigh appropriate amounts of hesperidin, bergenin, myricetin, and gallic acid reference substances, place them in 5 mL volumetric flasks, add 50% methanol and ultrasonically dissolve them to prepare single reference solution with mass concentrations of 1.306 mg / mL, 1.36 mg / mL, 1.404 mg / mL, and 1.086 mg / mL, respectively. Take 0.09, 0.5, 0.4, and 1.3 mL of each of the above single reference solutions, place them in 5 mL volumetric flasks, add 50% methanol to prepare mixed reference solution with mass concentrations of 276.74 μL / mL, 135.37 μL / mL, 112.05 μL / mL, and 23.48 μL / mL, respectively; (3) Chromatographic conditions: X-Peonyx-C18 column; acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution according to the following program: 0-12 min, 2%-7% A; 11-20 min, 7%-13% A; 20-31 min, 13%-15% A; 31-40 min, 15%-19% A; 40-50 min, 19%-20% A; 50-60 min, 20%-23% A; set the column temperature at 30°C; the volume flow rate was 1.0 mL / min; the detection wavelength was 270 nm; the injection volume was 10 μl; (4) Take 10 μl of the mixed reference solution and the test solution respectively, inject them into the liquid chromatograph, and measure them according to the chromatographic conditions described above.

3. The quality inspection method of the Qingjin Zhike Drink for resolving phlegm and relieving cough according to claim 2, characterized in that: The quality detection method further comprises using thin layer chromatography to qualitatively identify tangerine peel, liquorice and white radix scutellariae in Qingjinzhike drink, wherein the method of qualitatively identifying tangerine peel by thin layer chromatography comprises the following specific steps: (1) Solution preparation: Take 20 ml of Qingjin Zhike Drink, steam it until almost dry, add 30 ml of ethyl acetate solution, ultrasonicate for 30 min, take it out, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution; prepare a negative sample without tangerine peel according to the preparation method of Qingjin Zhike Drink, and then prepare a negative sample solution in the same way as above; take an appropriate amount of hesperidin reference substance, add methanol to make a solution containing 1 mg per 1 ml as the reference substance solution; (2) Take 3 μL of each of the test sample, negative sample and reference sample solutions and spot them on a silica gel G plate. Develop the plate with ethyl acetate-methanol-water (volume ratio 7.7:1.4:1) as the developing agent. Remove the plate, let it air dry, spray it with AlCl3 test solution, heat it at 105°C for 5 minutes, remove the plate and examine it under a UV lamp at 365 nm. In the chromatogram of the test sample, a spot of the same color appears at the corresponding position in the chromatogram of the reference sample, and there is no interference with the negative sample at the same position.

4. The quality inspection method of the Qingjin Zhike Drink for resolving phlegm and relieving cough according to claim 3, characterized in that: The method of qualitatively identifying licorice by thin layer chromatography comprises the following steps: (1) Solution preparation: Take 10 ml of Qingjin Zhike Drink, add 10 ml of methanol and shake to extract, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of purified water, extract once with 10 ml of ether, take the water layer, add 10 ml of ethyl acetate, extract twice, take the ethyl acetate layer, evaporate to dryness, add 1 ml of methanol to dissolve, and obtain the test solution; prepare a negative sample lacking licorice in the same way as the preparation method of Qingjin Zhike Drink, and then prepare a negative sample solution in the same way as above; take another licorice glycoside reference substance, add methanol to make a solution containing 1 mg per 1 ml as the reference solution. Take 1 g of licorice control medicinal material, add 20 ml of methanol, filter by ultrasonication for 30 minutes, take the filtrate and evaporate it to dryness, and prepare it according to the preparation method of the test sample as the licorice control medicinal material solution; (2) 3 μL each of the test solution, negative sample solution, and control medicinal material solution, and 1 μL of the control solution were spotted on the same silica gel G thin layer plate. The lower layer solvent was developed with chloroform-methanol-water (volume ratio 13:6:2). The plate was taken out, dried, sprayed with 10% sulfuric acid ethanol test solution, heated at 105°C until the spots were clearly colored, and inspected under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the control medicinal material and the control sample, and there was no interference from the negative sample at the same position.

5. The quality inspection method of the Qingjin Zhike Drink for resolving phlegm and relieving cough according to claim 3, characterized in that: The method of qualitatively identifying white pectin by thin layer chromatography is as follows: (1) Solution preparation: Take 40 mL of Qingjin Zhike Drink, steam until almost dry, add 40 mL of 5% sodium carbonate solution, ultrasonically treat for 20 minutes, filter, place in a separatory funnel, shake and extract twice with ethyl acetate, each time 40 mL, combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 mL of ethyl acetate to obtain the test solution; prepare a negative sample lacking Baiqian according to the same method as the preparation of Qingjin Zhike Drink, and then prepare a negative sample solution in the same way as above; take another 3 g of Baiqian original medicinal material, add 50 mL of water and boil for 30 minutes, cool, filter, evaporate to dryness, add 40 mL of 5% sodium carbonate solution to the residue, ultrasonically dissolve it, place in a separatory funnel, shake and extract twice with ethyl acetate, each time 40 mL, combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 mL of ethyl acetate to obtain the control medicinal material solution; (2) Pipette 8 μL of the test solution, negative sample solution, and 1 μL of the control medicinal material solution onto the same silica gel G thin layer plate, use toluene-dichloromethane-acetone, volume ratio 4:4:0.6, as the developing solvent, take out, dry, and examine under ultraviolet light at 365 nm; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatograms of the control medicinal material and the reference sample, and there is no interference with the negative sample at the same position.

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