A Chinese medicine composition and its application

Abstract

The present invention proposes a traditional Chinese medicine composition, comprising an active ingredient prepared from goat horn or antelope horn, scutellaria baicalensis, fritillaria cirrhosa, liquorice, rhubarb, gypsum, bezoar and green stone, wherein the active ingredients include, by mass, more than 1600 parts of amino acids, more than 120 parts of gallic acid, more than 130 parts of liquiritin, more than 20 parts of liquiritigenin, more than 400 parts of baicalin, more than 40 parts of melaleucoside, more than 120 parts of wogonin, more than 280 parts of glycyrrhizic acid, more than 12 parts of chrysin-7-O-β-D-glucuronic acid, more than 50 parts of aloe-emodin-8-O-β-D-glucoside, more than 30 parts of chrysophanol-1-O-β-D-glucoside, more than 45 parts of chrysophanol-8-O-β-D-glucoside, more than 260 parts of hyodeoxycholic acid and more than 150 parts of cholic acid. The composition of the present invention has antipyretic, anti-inflammatory and antitussive effects.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, in particular to a traditional Chinese medicine compound preparation and application thereof. Background Art

[0002] Acute bronchitis (AB) is an inflammation of the bronchial mucosa caused by infection with pathogens such as viruses or bacteria. It is a common respiratory disease in pediatrics, often secondary to upper respiratory tract infections and often an early manifestation of pneumonia. Because it often affects both the trachea and bronchi, it is also known as acute tracheobronchitis. Clinically, it is characterized by cough or increased bronchial secretions. Basic pathological changes include congestion and edema of the tracheobronchial mucosa, mucus plug formation, increased secretions, epithelial cell damage and desquamation, and inflammatory cell infiltration. In recent years, the global incidence of AB has increased significantly, becoming a serious global public health issue. Recurrent AB can develop into chronic bronchitis and even further into emphysema or cor pulmonale, placing a heavy psychological and economic burden on patients. According to the World Health Organization, acute respiratory infections, particularly AB, are the leading cause of death in children under five years of age (excluding newborns).

[0003] Research both domestically and internationally indicates that the underlying pathological mechanism of acute bronchitis is airway inflammation, with multiple cytokines and inflammatory mediators involved in the process. An imbalance in the Th1 / Th2 cell ratio is central to its pathogenesis, ultimately triggering an inflammatory response in the airways. Currently, Western medicine treatment for acute bronchitis in children focuses on infection control, primarily using antibiotics for inflammation, supplemented by antipyretic and cough suppressant medications. Reportedly, in 2012, the most common indication for antibiotics in children was bronchitis (AB), accounting for approximately 25.6%. Indiscriminate use of antibiotics can lead to the development of drug-resistant bacteria and serious consequences such as superinfection. In recent years, the role of traditional Chinese medicine in treating acute bronchitis in children has gained increasing attention. Traditional Chinese medicine possesses multiple antiviral, antipyretic, and anti-inflammatory properties, and can fundamentally enhance the body's immune system, demonstrating unique advantages in addressing the underlying cause of the disease, strengthening the body's resistance and expelling pathogenic factors, and treating both the symptoms and the root causes. Traditional Chinese medicine (TCM) compound prescriptions are a primary form of TCM medication. Based on the concept of syndrome differentiation and the principle of formulating according to the principles of monarch, minister, assistant, and envoy, they select appropriate TCM decoction pieces and combine them according to specific proportions. They embody the TCM theory of syndrome differentiation and treatment. TCM compound preparations have complex chemical compositions. The safety, efficacy, and stable, controllable quality of TCM materials and their products depend on quality control of authentic, sourced herbs to ensure the content of active ingredients within a specified range. This allows for the development of safe and effective TCM patent preparations for the treatment of acute bronchitis in children. Patent application number: 02129192.6, discloses a traditional Chinese medicine composition for treating colds and acute and chronic bronchitis and its preparation method. The traditional Chinese medicine composition of the present invention is made of 8-12 weight parts of antelope horn, 30-60 weight parts of flat fritillaria, 20-40 weight parts of rhubarb, 10-20 weight parts of scutellaria, 10-20 weight parts of greenstone, 15-30 weight parts of gypsum, 5-15 weight parts of artificial bezoar, and 20-40 weight parts of liquorice as raw materials. It has a good therapeutic effect on colds and acute and chronic bronchitis.

[0004] However, for traditional Chinese medicine (TCM) compound prescriptions, most raw materials are derived from natural products. The quality of these materials is highly variable due to factors such as origin, growth period, harvest season, and planting and processing techniques. Even when the raw materials comply with pharmacopoeia regulations, differences in active ingredients from different origins and batches can still lead to unstable ingredients and poor quality consistency in different batches of preparations. Therefore, prescriptions based solely on the weight of the medicinal materials cannot meet the production requirements of modern Chinese patent medicines, severely restricting the stability and controllability of TCM clinical efficacy and the reproducibility and acceptance of modern research results. Furthermore, the active ingredients in complex TCM compound prescriptions are unclear. Which major ingredients significantly influence the efficacy? What is the optimal range for their content? Reasonable control of the range of different active ingredients is necessary to ensure both the stability of TCM preparation quality and the reliability of its efficacy. This is a key issue restricting the development of modern TCM. Summary of the Invention

[0005] The present invention aims to further study the traditional Chinese medicine compound composed of eight traditional Chinese medicines, namely, goat horn, scutellaria baicalensis, fritillaria balsamifera, liquorice, rhubarb, gypsum, bezoar and green stone. The traditional Chinese medicine compound is prepared by using medicinal materials from different origins and batches to conduct component analysis and efficacy tests. The focus is on the relationship between the main components with large fluctuations in content in the traditional Chinese medicine compound and the efficacy test results, to find out the specific active ingredients and appropriate content that can affect the efficacy, to screen out traditional Chinese medicine compositions with stronger activity, and to ensure the stable quality and reliable efficacy of traditional Chinese medicine products.

[0006] In view of this, a traditional Chinese medicine composition is characterized in that it comprises active ingredients made from goat horn, scutellaria baicalensis, fritillaria brevis, licorice, rhubarb, gypsum, bezoar and green stone, and the active ingredients include, by mass: more than 1600 parts of amino acids, more than 120 parts of gallic acid, more than 130 parts of liquiritin, more than 20 parts of liquiritigenin, more than 400 parts of baicalin, more than 40 parts of melaleucoside, more than 120 parts of baicalin, more than 280 parts of glycyrrhizic acid, more than 12 parts of chrysin-7-O-β-D-glucuronic acid, more than 50 parts of aloe-emodin 8-O-β-D-glucoside, more than 30 parts of chrysophanol-1-O-β-D-glucoside, more than 45 parts of chrysophanol-8-O-β-D-glucoside, more than 260 parts of hyodeoxycholic acid, and more than 150 parts of cholic acid.

[0007] Preferably, 1610.45 to 1901.21 parts of amino acids, 120.27 to 167.01 parts of gallic acid, 136.22 to 172.23 parts of liquiritin, 21.23 to 37.12 parts of liquiritigenin, 411.30 to 542.50 parts of baicalin, 40.54 to 55.01 parts of melaleucoside, 121.11 to 148.22 parts of wogonin, 287.88 to 333.32 parts of glycyrrhizic acid, and 10 parts of baicalin. The content of the present invention is 12.87-24.11 parts of aloe-emodin-7-O-β-D-glucuronic acid, 50.11-55.21 parts of aloe-emodin-8-O-β-D-glucoside, 30.71-38.56 parts of chrysophanol-1-O-β-D-glucoside, 45.09-57.88 parts of chrysophanol-8-O-β-D-glucoside, 260.11-285.32 parts of hyodeoxycholic acid and 151.11-191.03 parts of cholic acid.

[0008] The Chinese medicine composition can be in an optional preparation form, and the dosage form of the Chinese medicine composition can be a pharmaceutically acceptable dosage form, including decoction, granules, capsules, soft capsules, pills, oral liquids, tinctures, syrups, suppositories, gels, sprays, and injections.

[0009] The preparation method comprises the following steps: taking 18.9 parts of goat horn, 9.45 parts of fritillaria brevis, 6.3 parts of rhubarb, 3.15 parts of scutellaria baicalensis, 3.15 parts of chlorophyll, 4.724 parts of gypsum, 1.89 parts of artificial bezoar and 6.3 parts of liquorice, grinding the goat horn into fine powder, adding water and sodium hydroxide to reflux and hydrolyze until almost completely dissolved, filtering, and concentrating the filtrate; grinding the chlorophyll and gypsum into coarse powder, heating and decocting with water, and concentrating the filtrate; extracting the bezoar by reflux with ethanol, and concentrating the filtrate; adding water to decoct the remaining medicinal materials, filtering, concentrating the filtrate, centrifuging, adding ethanol to the supernatant to precipitate, standing, taking the supernatant, filtering, recovering the ethanol under reduced pressure, concentrating, and combining the above concentrates.

[0010] The present invention also proposes the use of the active fraction of any of the above-mentioned traditional Chinese medicine compositions in the preparation of antipyretic, anti-inflammatory, antitussive and / or anti-new coronavirus drugs, such as the preparation of acute bronchitis drugs.

[0011] The term "use" refers to administering the extract to a subject suffering from a corresponding disease or a predisposition to the disease, for the purpose of imparting a therapeutic effect, such as curing, alleviating, modifying, affecting, ameliorating, or preventing the disease, its symptoms, or its predisposition. A person skilled in the art can readily determine a specific effective dosage based on the type of disease being treated, the route of administration, and the excipients used, and this dosage may vary depending on the concomitant use of other drugs.

[0012] The present invention utilizes three animal models: dry yeast-induced fever in rats, xylene-induced ear swelling in mice, and ammonia-induced cough in mice, and confirms that the above-mentioned traditional Chinese medicine composition can significantly reduce the increase in body temperature in rats caused by dry yeast; it has a significant inhibitory effect on acute inflammatory response, significantly prolongs the incubation period of cough in mice, reduces the number of coughs in mice, and effectively inhibits the replication of the new coronavirus in cells, and has significant antipyretic, anti-inflammatory, antitussive and / or anti-new coronavirus effects.

[0013] Moreover, the above-mentioned relationship between the content of ingredients also provides a basis for the quality control of the traditional Chinese medicine composition, that is, the present invention proposes a quality control method for the above-mentioned traditional Chinese medicine composition, which is characterized in that the active ingredients should meet the following requirements: more than 1,600 parts of amino acids, more than 120 parts of gallic acid, more than 130 parts of liquiritin, more than 20 parts of liquiritigenin, more than 400 parts of baicalin, more than 40 parts of melaleucoside, more than 120 parts of baicalin, more than 280 parts of glycyrrhizic acid, more than 12 parts of chrysin-7-O-β-D-glucuronic acid, more than 50 parts of aloe-emodin 8-O-β-D-glucoside, more than 30 parts of chrysophanol-1-O-β-D-glucoside, more than 45 parts of chrysophanol-8-O-β-D-glucoside, more than 260 parts of hyodeoxycholic acid, and more than 150 parts of cholic acid.

[0014] Furthermore, the active ingredients include 1610.45 to 1901.21 parts of amino acids, 120.27 to 167.01 parts of gallic acid, 136.22 to 172.23 parts of liquiritin, 21.23 to 37.12 parts of liquiritigenin, 411.30 to 542.50 parts of baicalin, 40.54 to 55.01 parts of melaleucoside, 121.11 to 148.22 parts of wogonin, and 287.88 to 333.32 parts of glycyrrhizic acid. parts, chrysin-7-O-β-D-glucuronic acid 12.87~24.11 parts, aloe-emodin 8-O-β-D-glucoside 50.11~55.21 parts, chrysophanol-1-O-β-D-glucoside 30.71~38.56 parts, chrysophanol-8-O-β-D-glucoside 45.09~57.88 parts, hyodeoxycholic acid 260.11~285.32 parts, and cholic acid 151.11~191.03 parts.

[0015] The present invention also proposes a method for detecting the active part of the Chinese medicine composition as described above, wherein the active part of the Chinese medicine composition is detected by HPLC, and the chromatographic conditions of the HPLC detection include: COSMOSIL-C with a specification of 4.6 mm × 250 mm, 5.0 μm 18 Chromatographic column; mobile phase: methanol (B)-acid water (A) (containing 0.1% formic acid), gradient elution: 0-15min: 10-40% B, 15-70min: 40-70% B, 70-90min: 70-100% B. DETAILED DESCRIPTION

[0016] As mentioned above, the present invention aims to provide an active fraction of a traditional Chinese medicine composition, a preparation method, an application, and a quality control method. The following will be described in detail with reference to the experimental examples.

[0017] It is particularly important to note that similar substitutions and modifications made to the present invention will be obvious to those skilled in the art and are considered to be included in the present invention. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments.

[0018] Unless otherwise specified, the present invention was carried out under conventional conditions or those recommended by the manufacturer. Reagents or instruments used without manufacturer's indication were all commercially available conventional products.

[0019] Example: Preparation of Chinese medicine composition

[0020] A total of 15 batches of Chinese medicine compositions were prepared by selecting medicinal materials from different origins and batches. Specifically, the following steps were taken: 94.5g of goat horn, 47.25g of Fritillaria cirrhosa, 31.50g of Rhubarb, 15.75g of Scutellaria baicalensis, 15.75g of celery, 23.62g of gypsum, 9.45g of artificial bezoar, and 31.50g of liquorice; the goat horn was crushed into fine powder, and refluxed with water and sodium hydroxide to hydrolyze until almost completely dissolved, filtered, and the filtrate was concentrated; the celery and gypsum were crushed into coarse powder, heated and decocted with water, and the filtrate was concentrated; the bezoar was refluxed with ethanol, and the filtrate was concentrated. The remaining medicinal materials are decocted with water, filtered, the filtrate is concentrated, centrifuged, ethanol is added to the supernatant to precipitate, the mixture is allowed to stand, the supernatant is collected, filtered, the ethanol is recovered under reduced pressure, and concentrated. The above concentrates are combined and mixed with 0.2% stevia, water is added to 1000 ml, stirred, boiled for 40 minutes, refrigerated for not less than 48 hours, filtered, the filtrate is added with water to 1000 ml, the pH value is adjusted to 8.0-9.0, potted, sterilized, and prepared into an oral solution. Among them, the goat horn medicinal material is sourced from Zhejiang, Hebei, Jiangsu, Inner Mongolia, etc.; the flat fritillaria medicinal material is selected from Liaoning, Jilin, Heilongjiang, etc.; the rhubarb medicinal material is selected from Gansu, Qinghai, Chongqing, etc.; the scutellaria medicinal material is sourced from Shandong, Shaanxi, Shanxi, Gansu, etc.; the licorice medicinal material is sourced from Inner Mongolia, Ningxia, Gansu, Xinjiang, etc.

[0021] Content detection

[0022] In the present invention, the content of the active ingredient in the traditional Chinese medicine composition is determined by an automatic amino acid analyzer and a multi-wavelength high performance liquid chromatography-ultraviolet vacuum wave-evaporative light scattering detector (HPLC-UVD-ELSD). The specific method is as follows:

[0023] Content Determination Ⅰ Gallic acid, liquiritin, liquiritigenin, baicalin, melaleucoside, wogonin, glycyrrhizic acid, chrysin-7-O-β-D-glucuronic acid, aloe-emodin 8-O-β-D-glucoside, chrysophanol-1-O-β-D-glucoside, chrysophanol-8-O-β-D-glucoside, hyodeoxycholic acid and cholic acid were determined by HPLC method specified in the Chinese Pharmacopoeia.

[0024] Liquid chromatography conditions and system suitability test COSMOSIL-C 18 Chromatographic column (4.6 mm × 250 mm, 5.0 μm); mobile phase: methanol (B)-acid water (A) (containing 0.1% formic acid), gradient elution: 0-15 min: 10-40% B, 15-70 min: 40-70% B, 70-90 min: 70-100% B; flow rate 1.0 mL / min, column temperature 35°C, ELSD gas flow rate 2.0 L / min, drift tube temperature 115°C, gain value 4.0.

[0025] Preparation of reference solution (1) Preparation of single standard solution: accurately weigh 8.39 mg, 6.89 mg, 10.8 mg, 4.24 mg, 4.49 mg, 2.36 mg, 6.00 mg and 9.40 mg of glycyrrhizin, glycyrrhizic acid, hyodeoxycholic acid, cholic acid, melaleucoside, chrysin-7-O-β-D-glucuronic acid, glycyrrhizin and gallic acid respectively, place them in a 5.0 mL volumetric flask, add about 3.0 mL of methanol, dissolve them by ultrasonication, and dilute to 5.0 mL with methanol to obtain reference solution with concentrations of 1678 μg / mL, 1378 μg / mL, 2160 μg / mL, 848 μg / mL, 898 μg / mL, 472 μg / mL, 1200 μg / mL and 1880 μg / mL respectively; accurately weigh 8.39 mg, 6.89 mg, 10.8 mg, 4.24 mg, 4.49 mg, 2.36 mg, 6.00 mg and 9.40 mg of glycyrrhizin, glycyrrhizic acid, hyodeoxycholic acid, cholic acid, melaleucoside, chrysin-7-O-β-D-glucuronic acid, glycyrrhizin and gallic acid respectively, place them in a 5.0 mL volumetric flask, add about 3.0 mL of methanol, dissolve them by ultrasonication, and dilute to 5.0 mL with methanol to obtain reference solution with concentrations of 1678 μg / mL, 1378 μg / mL, 2160 μg / mL, 848 μg / mL, 898 μg / mL, 472 μg / mL, 1200 μg / mL and 12.08 mg and 5.54 mg of baicalin and wogonin were accurately weighed and placed in a 10.0 mL volumetric flask. About 7.0 mL of methanol was added, and after ultrasonic dissolution, the volume was adjusted to 10.0 mL with methanol to obtain reference solutions with concentrations of 1208 μg / mL and 554 μg / mL, respectively. 2.08 mg, 4.51 and 4.71 mg of aloe-emodin 8-O-β-D-glucoside, chrysophanol-1-O-β-D-glucoside and chrysophanol 8-O-β-D-glucoside were accurately weighed respectively, placed in a 20.0 mL volumetric flask, about 17.0 mL of methanol was added, and after ultrasonic dissolution, the volume was adjusted to 20.0 mL with methanol to obtain reference solutions with concentrations of 104 μg / mL, 225.5 μg / mL and 235.5 μg / mL, respectively. (2) Preparation of mixed reference solution: Accurately pipette different volumes of each monomer reference solution (1.0 mL each of glycyrrhizin, hyodeoxycholic acid, melaleucoside, and chrysin-7-O-β-D-glucuronic acid; 0.5 mL each of gallic acid and glycyrrhizin; 5.0 mL of baicalin, 4.0 mL of aloe-emodin 8-O-β-D-glucoside; 2.0 mL of wogonin; 2.5 mL of glycyrrhizic acid; 3.0 mL of chrysophanol-1-O-β-D-glucoside; 1.5 mL each of cholic acid and chrysophanol-8-O-β-D-glucoside) into a 25 mL volumetric flask and dilute to the mark.

[0026] Preparation of the test solution: Accurately pipette 1.0 mL of the different batches of the traditional Chinese medicine composition prepared in Example 1 into a 5.0 mL volumetric flask, add about 4.0 mL of distilled water, mix by ultrasonication, and make up to 5.0 mL with distilled water. Take 1.0 mL, filter through a 0.45 μm microporous membrane, and take the filtrate to obtain the solution.

[0027] Assay: Accurately pipette 10 μL of each reference solution and test solution into a liquid chromatograph for determination. Record the chromatogram and the peak areas of the 13 components listed above. Hyodeoxycholic acid and cholic acid were linearly regressed using the natural logarithm of mass concentration (X) as the abscissa and the natural logarithm of peak area (Y) as the ordinate. For the remaining 11 components, linear regression was performed using mass concentration (X, μg / mL) as the abscissa and peak area (Y) as the ordinate. Specific content of each batch is shown in Table 1.

[0028] Table 1 Determination of the contents of liquiritin, glycyrrhizic acid, hyodeoxycholic acid, cholic acid, melaleucoside, chrysin-7-O-β-D-glucuronic acid, liquiritin, gallic acid, baicalin, wogonin, aloe-emodin-8-O-β-D-glucoside, chrysophanol-1-O-β-D-glucoside, and chrysophanol-8-O-β-D-glucoside in 15 batches of traditional Chinese medicine compositions (μg / mL)

[0029]

[0030]

[0031] Content Determination II Amino Acids

[0032] Liquid chromatography conditions and system suitability test: Chromatographic column: Hitachi 855-4507 ion exchange column; Mobile phase: Citric acid buffer solution; Detector: Fluorescence detector; Elution pump flow rate: 0.35 mL min -1 ; Derivatization pump flow rate: 0.30 mL min -1 ; Reaction column temperature: 135℃ (programmed temperature); Detection wavelength: 570nm, 440nm; Injection volume: 20μL; Analysis time: 148min.

[0033] Preparation of reference solution: Accurately weigh 36.535 mg of L-glutamine reference substance, place it in a 100 mL volumetric flask, add appropriate amount of water, sonicate for 5 minutes to dissolve it, and dilute to the scale with water to obtain 2.5 μmol / ml L-glutamine reference substance stock solution; accurately pipette 400 μL each of amino acid mixed standard solution type B, AN-II type, L-aspartic acid standard solution and L-glutamine reference substance stock solution, place it in a 10 mL volumetric flask, add appropriate amount of 0.02 mol / L hydrochloric acid to dilute it, sonicate for 5 minutes to mix it, and dilute to the scale with 0.02 mol / L hydrochloric acid to obtain 100 nmol / mL amino acid standard solution, which is standard solution 1.

[0034] Preparation of standard curve solution:

[0035] Accurately pipette 6 mL of standard solution 1 and dilute to 10 mL with 0.02 mol / L hydrochloric acid to obtain standard solution 2 containing 60 nmol / mL of amino acid standard.

[0036] Accurately pipette 5 mL of standard solution 2 and dilute to 10 mL with 0.02 mol / L hydrochloric acid to obtain standard solution 3 containing 30 nmol / mL of amino acid standard.

[0037] Accurately pipette 5 mL of standard solution 3 and dilute to 10 mL with 0.02 mol / L hydrochloric acid to obtain standard solution 4 containing 15 nmol / mL of amino acid standard.

[0038] Accurately pipette 2 mL of standard solution 1 and dilute to 10 mL with 0.02 mol / L hydrochloric acid to obtain standard solution 5 containing 20 nmol / mL of amino acid standard.

[0039] Accurately pipette 5 mL of standard solution 1 and dilute to 100 mL with 0.02 mol / L hydrochloric acid to obtain standard solution 6 containing 5 nmol / mL of amino acid standard.

[0040] Prepare the test solution according to the sample pretreatment requirements of the amino acid analyzer. Take 2 mL of the finished product and add an equal volume of 5% sulfosalicylic acid. Refrigerate at -4°C for 24 hours. Centrifuge at 12,000 rpm for 10 minutes. Dilute the supernatant with an equal volume of 0.02 mol / mL HCl to prepare the test solution.

[0041] The assay method involves preparing a series of standard solutions of varying concentrations. Samples were injected sequentially according to liquid chromatography analysis conditions, and the chromatographic peak areas of the quantitative components were recorded and analyzed. Linear regression was performed on the quantitative amino acid components, with mass concentration (μg / mL) as the abscissa and peak area as the ordinate. The regression equation, correlation coefficient, and linear range for each amino acid were obtained. Specific content for each batch is shown in Table 2.

[0042] Table 2 Determination results of total amino acid content in different batches of Chinese medicine compositions

[0043]

[0044]

[0045] Efficacy experiments

[0046] Experimental study of 15 batches of traditional Chinese medicine compositions on three animal models: dry yeast-induced fever in rats, xylene-induced ear swelling in mice, and ammonia-induced cough in mice

[0047] 1. Experimental Materials

[0048] 1.1 Drugs and reagents

[0049] The Chinese medicine composition AO of the present invention has a total of 15 batches and is self-made by Jiangsu Kangyuan Pharmaceutical Co., Ltd.; enteric-coated aspirin tablets, specification 100 mg / tablet; dioxetine hydrochloride granules, specification 1.5 mg / bag; dry yeast, specification 15 g / bag; xylene, ammonia water.

[0050] 1.2 Main equipment

[0051] 15-D03A thermometer, BS224S electronic analytical balance, mouse ear punch, YLS-8A multifunctional cough and wheezing induction instrument.

[0052] 1.3 Experimental animals

[0053] SD rats, SPF grade, male, weighing 180-220 g; KM mice, SPF grade, male, weighing 18-22 g; ICR mice, SPF grade, half male and half female, weighing 18-22 g.

[0054] 2. Methods

[0055] 2.1 Effects of different batches of Chinese herbal medicine compositions on fever induced by dry yeast in rats

[0056] (1) Experimental methods

[0057] Male Sprague-Dawley rats, weighing 180-220 g, were acclimated for 7 days. The ambient temperature was maintained at 20-25°C during the experiment. Rectal temperature was measured daily at 10:00 AM for two consecutive days to allow the rats to acclimate to this procedure. The average of these two temperatures was used as the basal body temperature. Rats with a body temperature within the range of 37.0-38.6°C and a fluctuation of <0.5°C were selected for the experiment. Each rat was subcutaneously injected with 10 ml / kg of a 20% dry yeast suspension (preparation: 120 g of dry yeast was weighed, placed in a mortar, and gradually ground into a uniform suspension with distilled water. The final volume was adjusted to 600 mL, prepared immediately before use). Six hours after yeast suspension injection, the rats' body temperatures were measured. 170 rats with a body temperature rise between 1.0 and 2.2°C were selected and randomly divided into 17 groups based on body temperature. That is, (1) model group: distilled water, (2) TCM composition A-O group: 3.34g crude drug / kg; (3) control drug group: aspirin suspension, 0.20g / kg. Each group was gavaged with the corresponding solvent or drug, and rectal temperature was measured 1h, 2h, 3h, 4h, and 5h after administration, and the difference between body temperature at each time period after administration and basal body temperature was calculated.

[0058] (2) Experimental results

[0059] Compared with the model group, all treatment groups reduced the dry yeast-induced increase in rat body temperature at 2, 3, 4, and 5 hours after administration. The differences were more pronounced in the TCM composition groups B, C, D, H, I, J, and N compared with the model group. The results are shown in Table 3.

[0060] Table 3 Effects of different batches of Chinese medicine compositions on fever induced by dry yeast in rats ( n=10)

[0061]

[0062]

[0063] Compared with the model group: * P<0.05, ** P<0.01.

[0064] 2.2 Effects of different batches of Chinese herbal medicine compositions on xylene-induced ear swelling in mice

[0065] (1) Experimental methods

[0066] Male KM mice, weighing 18-22 g, were acclimated for 4 days. The ambient temperature was maintained at 20-25°C during the experiment. They were randomly divided into 17 groups, each with 10 mice: (1) model group: administered with distilled water; (2) Chinese medicine composition A-O groups: administered with 4.83 g crude drug / kg; and (3) positive group: administered with aspirin suspension at a dose of 0.20 g / kg. Mice in each group were gavaged once daily for 5 consecutive days. One hour after the last administration, 0.03 ml of xylene was applied to both sides of the right ear of the mouse, with the left ear serving as a control. One hour later, the mouse was sacrificed by cervical dislocation. An 8 mm punch was used to punch out round ear pieces from the same part of both ears of the mouse. The pieces were accurately weighed, and the difference between the weight of the right ear piece and the weight of the left ear piece was calculated as the degree of swelling.

[0067] (2) Experimental results

[0068] Compared with the model group, all drug-treated groups could inhibit xylene-induced ear swelling in mice, with significant differences observed in groups B, C, D, H, I, M, N, and O. The results are shown in Table 4.

[0069] Table 4 Effects of different batches of Chinese medicine compositions on xylene-induced ear swelling in mice ( n=10)

[0070]

[0071] Compared with the model group: *P<0.05, **P<0.01.

[0072] 2.3 Effects of different batches of Chinese herbal medicine compositions on ammonia-induced cough in mice

[0073] (1) Experimental methods

[0074] ICR mice weighing 18-22 g, half male and half female, were pre-selected and the qualified mice were reared for 3 days (animals with a latent period of less than 1 min and more than 3 typical coughs within 1 min were classified as "coughing" sensitive animals; otherwise, they were eliminated as non-coughing animals). They were randomly divided into 17 groups, namely (1) model group: distilled water; (2) Chinese medicine composition A-O groups: 4.83 g crude drug / kg; (3) positive group: dioxetine hydrochloride suspension, 1.79 mg / kg. Each group of mice was gavage-administered once a day for 5 consecutive days. One hour after the last administration, the mice were placed in a glass bell jar of a multifunctional cough-inducing and wheezing instrument. 25-28% ammonia water was sprayed into the bell jar for 3 seconds. The cough latency and the number of coughs within 3 minutes of the mice were then observed and recorded. The cough standard was: abdominal muscle contraction, wide mouth opening, and sometimes coughing sound.

[0075] (2) Experimental results

[0076] Compared with the model group, each drug-treated group could significantly prolong the latent period of cough in mice and / or significantly reduce the number of coughs in mice. Among them, the Chinese medicine compositions B, C, D, F, I, K, and N showed significant differences compared with the model group. The results are shown in Table 5.

[0077] Table 5 Effects of different batches of Chinese medicine compositions on ammonia-induced cough in mice ( n=10)

[0078]

[0079]

[0080] Compared with the model group: *P<0.05, **P<0.01.

[0081] 2.4 Effects of different batches of Chinese herbal medicine compositions against the novel coronavirus

[0082] (1) Experimental materials

[0083] Test drugs: different batches of Chinese medicine compositions (A-O).

[0084] VeroE6 cells: preserved by the Pathogen Center of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences.

[0085] 2019-nCoV virus: titer 10 5 TCID 50 / ml, stored at -80℃ by the Pathogen Center of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences. The virus titer used was 100TCID 50 .

[0086] (2) Experimental methods

[0087] Sterile 96-well culture plates were added with 200 μl of 5 × 10 4 Vero E6 cells were cultured at 37°C, 5% CO2 for 24 hours; the test drug was diluted to three concentrations (1:10,000, 1:20,000, and 1:50,000), with five replicates of each concentration and 100 μl per well. An equal volume of 100 TCID50 virus was then added to each well for 1 hour; after 1 hour, the cell culture medium in the 96-well culture plate was discarded and the above mixture was added; a cell control, a blank control (solvent control), and a virus control (negative control) were also set up; the cells were incubated in a 37°C, 5% CO2 incubator for 4-5 days; the cytopathic effect (CPE) was observed under an optical microscope, and complete cell lesions were recorded as "++++", 75% lesions were recorded as "+++", 50% lesions were recorded as "++", 25% lesions were recorded as "+", and no lesions were recorded as "-".

[0088] (3) Experimental conditions: All the above experimental operations were completed in a BSL-3 laboratory.

[0089] (4) Result judgment: The concentration at which the virus is effectively inhibited is when the cells do not show CPE; when CPE appears, it is ineffective.

[0090] (5) Experimental results:

[0091] The drug group of the present invention was set at three concentrations, all of which were able to effectively inhibit the replication of 2019-nCoV in cells. The results are shown in Table 1.

[0092] Table 1. Effects of different Chinese herbal medicine compositions against 2019-nCoV

[0093]

[0094]

[0095] (6) Conclusion:

[0096] According to the screening results at the cellular level, the Chinese medicine compositions B, C, D, I, and N according to the present invention can inhibit the replication of 2019-nCoV in cells at dilution concentrations of 1:10000, 1:20000, and 1:50000, indicating that they have good in vitro anti-2019-nCoV activity.

[0097] 3. Conclusion

[0098] In three animal models—dry yeast-induced fever in rats, xylene-induced ear swelling in mice, and ammonia-induced cough in mice—and in studies against the novel coronavirus, different batches of the Chinese herbal compositions significantly reduced the yeast-induced rise in rat body temperature, significantly inhibited the acute inflammatory response, significantly prolonged the incubation period of coughing in mice, reduced the frequency of coughing in mice, and effectively inhibited the intracellular replication of the novel coronavirus. Overall, Chinese herbal compositions B, C, D, I, and N were more effective in antipyretic, anti-inflammatory, and antitussive effects, as well as in the fight against the novel coronavirus.

[0099] According to the specific component contents of different batches of Chinese medicine compositions B, C, D, I, and N, the preferred content ranges can be summarized, namely: amino acids 1610.45-1901.21 μg / mL, gallic acid 120.27-167.01 μg / mL, liquiritin 136.22-172.23 μg / mL, liquiritigenin 21.23-37.12 μg / mL, baicalin 411.30-542.50 μg / mL, melaleucoside 40.54-55.01 μg / mL, and wogonin 121.11-148.22 μg / mL. Glycyrrhizic acid 287.88~333.32μg / mL, chrysin-7-O-β-D-glucuronic acid 12.87~24.11μg / mL, aloe-emodin 8-O-β-D-glucoside 50.11~55.21μg / mL, chrysophanol-1-O-β-D-glucoside 30.71~38.56μg / mL, chrysophanol-8-O-β-D-glucoside 45.09~57.88μg / mL, hyodeoxycholic acid 260.11~285.32μg / mL, and cholic acid 151.11~191.03μg / mL.

[0100] Screening and verification of the content range of main ingredients

[0101] A total of 7 batches of Chinese medicine compositions were prepared by selecting medicinal materials from different origins and batches. The contents of each component are shown in Tables 6 and 7.

[0102] Table 6 Determination of the contents of glycyrrhizin, glycyrrhizic acid, hyodeoxycholic acid, cholic acid, melaleucoside, chrysin-7-O-β-D-glucuronic acid, glycyrrhizin, gallic acid, baicalin, wogonin, aloe-emodin-8-O-β-D-glucoside, chrysophanol-1-O-β-D-glucoside and chrysophanol-8-O-β-D-glucoside in 7 batches of traditional Chinese medicine compositions

[0103]

[0104]

[0105] Table 7 Determination results of total amino acid content in different batches of Chinese medicine compositions

[0106] Different batches Total amino acid content (μg / mL) P 563.77 Q 1333.02 R 1701.55 S 1801.99 T 1099.89 U 1888.80 V 1555.98

[0107] Efficacy verification experiment

[0108] 1. Method

[0109] 1.1 Effects of different batches of Chinese herbal medicine compositions on fever induced by dry yeast in rats

[0110] (1) Experimental methods

[0111] Male SD rats, weighing 180-220g, were acclimated for 7 days. The ambient temperature was maintained at 20-25°C during the experiment. Rectal temperature was measured once daily at 10:00 AM for two consecutive days to allow the rats to acclimate to this procedure. The average of these two temperatures was used as the basal body temperature. Rats with a body temperature within the range of 37.0-38.6°C and a fluctuation of <0.5°C were selected for the experiment. Each rat was subcutaneously injected with 10ml / kg of a 20% dry yeast suspension (preparation method: 90g of dry yeast was weighed, placed in a mortar, and gradually ground into a uniform suspension with distilled water. The final volume was adjusted to 450ml and prepared immediately before use). Six hours after yeast suspension injection, the rats' body temperatures were measured. Ninety rats whose body temperatures rose between 1.0 and 2.2°C were selected and randomly divided into nine groups based on body temperature. The following were used: (1) model group: distilled water; (2) Chinese herbal medicine combination P-V group: 3.34 g crude drug / kg; (3) control group: 0.20 g / kg aspirin suspension. Each group was gavaged with the corresponding solvent or drug, and rectal temperature was measured 1, 2, 3, 4, and 5 hours after administration. The difference between body temperature at each time period and basal body temperature was calculated.

[0112] (2) Experimental results

[0113] Compared with the model group, all drug-administered groups were able to reduce the dry yeast-induced increase in rat body temperature after administration. The TCM composition R, S, and U groups showed significant differences compared with the model group at 2, 3, 4, and 5 hours. The results are shown in Table 8.

[0114] Table 8 Effects of different batches of Chinese medicine compositions on fever induced by dry yeast in rats ( n=10)

[0115]

[0116] Compared with the model group: * P<0.05, ** P<0.01.

[0117] 1.2 Effects of different batches of Chinese herbal medicine compositions on xylene-induced ear swelling in mice

[0118] (1) Experimental methods

[0119] Male KM mice, weighing 18-22 g, were acclimated for 4 days. The ambient temperature was maintained at 20-25°C during the experiment. They were randomly divided into 9 groups, each with 10 mice: (1) model group: administered with distilled water; (2) Chinese medicine composition P-V group: administered with 4.83 g crude drug / kg; and (3) positive group: administered with aspirin suspension at a dose of 0.20 g / kg. Mice in each group were gavaged once daily for 5 consecutive days. One hour after the last administration, 0.03 ml of xylene was applied to both sides of the right ear of the mouse, with the left ear serving as a control. One hour later, the mouse was killed by cervical dislocation. An 8 mm punch was used to punch out round ear pieces from the same part of both ears of the mouse. The two ears were accurately weighed, and the difference between the weight of the right ear piece and the weight of the left ear piece was calculated as the degree of swelling.

[0120] (2) Experimental results

[0121] Compared with the model group, all drug-treated groups could inhibit xylene-induced ear swelling in mice, and the R, S, T, and U groups of Chinese medicine compositions showed significant differences compared with the model group.

[0122] Table 9 Effects of different batches of Chinese medicine compositions on xylene-induced ear swelling in mice ( n=10)

[0123]

[0124] Compared with the model group: *P<0.05, **P<0.01.

[0125] 1.3 Effects of different batches of Chinese herbal medicine compositions on ammonia-induced cough in mice

[0126] (1) Experimental methods

[0127] ICR mice weighing 18-22 g, half male and half female, were pre-selected and the qualified mice were reared for 3 days (animals with a latent period of less than 1 min and more than 3 typical coughs within 1 min were classified as "coughing" sensitive animals, otherwise they were eliminated as non-coughing animals). They were randomly divided into 9 groups, 10 mice each, namely (1) model group: administered with distilled water, (2) Chinese medicine composition P-V group: 4.83 g crude drug / kg; (3) positive group: administered with dioxetine hydrochloride suspension at a dose of 1.79 mg / kg. Each group of mice was gavaged once a day for 5 consecutive days. One hour after the last administration, the mice were placed in a glass bell jar of a multifunctional cough-inducing and wheezing instrument. 25-28% ammonia water was sprayed into the bell jar for 3 seconds. The cough latency and the number of coughs within 3 minutes of the mice were then observed and recorded. The coughing standard was: abdominal muscle contraction, wide mouth opening, and sometimes coughing sounds.

[0128] (2) Experimental results

[0129] Compared with the model group, each drug-treated group could significantly prolong the latent period of coughing in mice and / or significantly reduce the number of coughing times in mice. Among them, the Chinese medicine compositions Q, R, S and U showed significant differences compared with the model group. The results are shown in Table 10.

[0130] Table 10 Effects of different batches of Chinese medicine compositions on ammonia-induced cough in mice ( n=10)

[0131]

[0132]

[0133] Compared with the model group: *P<0.05, **P<0.01.

[0134] 1.4 Effects of different batches of Chinese herbal medicine compositions against the novel coronavirus

[0135] (1) Experimental materials

[0136] Test drugs: different batches of Chinese medicine compositions (P~V).

[0137] VeroE6 cells: preserved by the Pathogen Center of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences.

[0138] 2019-nCoV virus: titer 10 5 TCID 50 / ml, stored at -80℃ by the Pathogen Center of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences. The virus titer used was 100TCID 50 .

[0139] (2) Experimental methods

[0140] Sterile 96-well culture plates were added with 200 μl of 5 × 10 4 Vero E6 cells were cultured at 37°C, 5% CO2 for 24 hours; the test drug was diluted to three concentrations (1:10,000, 1:20,000, and 1:50,000), with five replicates of each concentration and 100 μl per well. An equal volume of 100 TCID50 virus was then added to each well for 1 hour; after 1 hour, the cell culture medium in the 96-well culture plate was discarded and the above mixture was added; a cell control, a blank control (solvent control), and a virus control (negative control) were also set up; the cells were incubated in a 37°C, 5% CO2 incubator for 4-5 days; the cytopathic effect (CPE) was observed under an optical microscope, and complete cell lesions were recorded as "++++", 75% lesions were recorded as "+++", 50% lesions were recorded as "++", 25% lesions were recorded as "+", and no lesions were recorded as "-".

[0141] (3) Experimental conditions: All the above experimental operations were completed in a BSL-3 laboratory.

[0142] (4) Result judgment: The concentration at which the virus is effectively inhibited is when the cells do not show CPE; when CPE appears, it is ineffective.

[0143] (5) Experimental results:

[0144] The drug group of the present invention was set at three concentrations, all of which were able to effectively inhibit the replication of 2019-nCoV in cells. The results are shown in Table 1.

[0145] Table 1. Effects of different Chinese herbal medicine compositions against 2019-nCoV

[0146]

[0147] (6) Conclusion:

[0148] According to the screening results at the cellular level, the Chinese medicine compositions R, S, and U according to the present invention can inhibit the replication of 2019-nCoV in cells at dilution concentrations of 1:10000, 1:20000, and 1:50000, indicating that they have good in vitro anti-2019-nCoV activity.

[0149] 2. Conclusion

[0150] In three animal models—dry yeast-induced fever in rats, xylene-induced ear swelling in mice, and ammonia-induced cough in mice—the R, S, and U groups of the seven different batches of TCM compositions demonstrated significant antipyretic, anti-inflammatory, antitussive, and novel coronavirus-inhibiting effects. Animal trials have shown that TCM compositions with these active ingredient content characteristics exhibit stable and excellent therapeutic effects. These active ingredient content or ratio characteristics can be used to control the quality of active ingredients in compositions made from medicinal materials from different origins and batches, which is crucial for ensuring the stable quality and reliable efficacy of TCM preparations.

[0151] The above is merely an exemplary description of the embodiments of the present invention. It should be pointed out that a person skilled in the art may make several improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered as within the scope of protection of the present invention.

Claims

1. A Chinese medicine composition, characterized in that The invention comprises an active ingredient prepared from goat horn, scutellaria baicalensis, fritillaria brevis, liquorice, rhubarb, gypsum, bezoar and green stone, wherein the active ingredient comprises, by mass, 1610.45-1901.21 parts of amino acids, 120.27-167.01 parts of gallic acid, 136.22-172.23 parts of liquiritin, 21.23-37.12 parts of liquiritigenin, 411.30-542.50 parts of baicalin, 40.54-55.01 parts of melaleucoside, 121.11-148.22 parts of wogonin, 287.88-333.32 parts of glycyrrhizic acid, chrysin-7- O - β -D-glucuronic acid 12.87~24.11 parts, aloe-emodin 8- O - β -D-glucoside 50.11~55.21 parts, chrysophanol-1- O - β -D-glucoside 30.71~38.56 parts, chrysophanol-8- O - β -D-glucoside 45.09~57.88 parts, hyodeoxycholic acid 260.11~285.32 parts, cholic acid 151.11~191.03 parts.

2. The Chinese medicine composition according to claim 1, characterized in that The traditional Chinese medicine composition is in an optional preparation form.

3. The Chinese medicine composition according to claim 2, characterized in that The preparation is in the form of decoction, granules, capsules, pills, oral liquid, tincture, syrup, suppository, gel, spray and injection.

4. The Chinese medicine composition according to claim 1, characterized in that The method for detecting the active ingredients of the Chinese medicine composition is to perform HPLC detection, and the chromatographic conditions of the HPLC detection include: COSMOSIL-C with a specification of 4.6 mm×250 mm, 5.0 μm 18 Chromatographic column; mobile phase: methanol B - acid water A containing 0.1% formic acid, gradient elution: 0~15 min: 10~40% B, 15~70 min: 40~70% B, 70~90 min: 70~100% B; flow rate 0.8~1.2 mL / min, column temperature 30~40 ℃.

5. A Chinese medicine composition, characterized in that The invention comprises an active ingredient prepared from goat horn, scutellaria baicalensis, fritillaria brevis, liquorice, rhubarb, gypsum, bezoar and green stone, wherein the active ingredient comprises, by mass, 1610.45-1901.21 parts of amino acids, 120.27-167.01 parts of gallic acid, 136.22-172.23 parts of liquiritin, 21.23-37.12 parts of liquiritigenin, 411.30-542.50 parts of baicalin, 40.54-55.01 parts of melaleucoside, 121.11-148.22 parts of wogonin, 287.88-333.32 parts of glycyrrhizic acid, chrysin-7- O - β -D-glucuronic acid 12.87~24.11 parts, aloe-emodin 8- O - β -D-glucoside 50.11~55.21 parts, chrysophanol-1- O - β -D-glucoside 30.71~38.56 parts, chrysophanol-8- O - β -D-glucoside 45.09~57.88 parts, hyodeoxycholic acid 260.11~285.32 parts, cholic acid 151.11~191.03 parts, The preparation method of the traditional Chinese medicine composition comprises the following steps: taking 18.9 parts of goat horn, 9.45 parts of Fritillaria cirrhosa, 6.3 parts of rhubarb, 3.15 parts of scutellaria baicalensis, 3.15 parts of chlorophyll, 4.724 parts of gypsum, 1.89 parts of artificial bezoar, and 6.3 parts of liquorice; crushing the goat horn into fine powder, adding water and sodium hydroxide to reflux and hydrolyze until almost completely dissolved, filtering, and concentrating the filtrate; crushing the chlorophyll and gypsum into coarse powder, heating and decocting with water, and concentrating the filtrate; extracting the bezoar with ethanol by reflux, and concentrating the filtrate; adding water to decoct the remaining medicinal materials, filtering, concentrating the filtrate, centrifuging, adding ethanol to the supernatant to precipitate, standing, taking the supernatant, filtering, recovering ethanol under reduced pressure, concentrating, and combining the above concentrates.

6. A method for preparing the Chinese medicine composition according to claim 1, characterized in that: The method comprises the following steps: taking 18.9 parts of goat horn, 9.45 parts of fritillaria brevis, 6.3 parts of rhubarb, 3.15 parts of scutellaria baicalensis, 3.15 parts of chlorophyll, 4.724 parts of gypsum, 1.89 parts of artificial bezoar and 6.3 parts of liquorice, crushing the goat horn into fine powder, adding water and sodium hydroxide to reflux and hydrolyze until almost completely dissolved, filtering, and concentrating the filtrate; crushing the chlorophyll and gypsum into coarse powder, heating and decocting with water, and concentrating the filtrate; extracting the bezoar by reflux with ethanol, and concentrating the filtrate; adding water to decoct the remaining medicinal materials, filtering, concentrating the filtrate, centrifuging, adding ethanol to the supernatant to precipitate, standing, taking the supernatant, filtering, recovering the ethanol under reduced pressure, concentrating, and combining the above concentrates.

7. The preparation method according to claim 6, characterized in that The traditional Chinese medicine composition is an oral liquid, and the preparation method comprises: combining the concentrated liquid and mixing it with 0.2% stevia, adding water to 1000 mL, stirring evenly, boiling for 40 minutes, refrigerating for not less than 48 hours, filtering, adding water to the filtrate to 1000 mL, adjusting the pH value to 8.0-9.0, potting, and sterilizing; the traditional Chinese medicine composition comprises 94.5 g of goat horn, 47.25 g of flat fritillaria, 31.50 g of rhubarb, 15.75 g of scutellaria, 15.75 g of green stone, 23.62 g of gypsum, 9.45 g of artificial bezoar, and 31.50 g of liquorice.

8. Use of the traditional Chinese medicine composition according to any one of claims 1 to 4 or the traditional Chinese medicine composition according to claim 5 in the preparation of antipyretic, anti-inflammatory, antitussive and / or anti-new coronavirus drugs.

9. A method for detecting the active ingredient of the traditional Chinese medicine composition according to claim 1, characterized in that: HPLC detection was performed, and the chromatographic conditions of the HPLC detection included: COSMOSIL-C with a size of 4.6 mm×250 mm, 5.0 μm 18 Chromatographic column; mobile phase: methanol B - acid water A containing 0.1% formic acid, gradient elution: 0~15 min: 10~40% B, 15~70 min: 40~70% B, 70~90 min: 70~100% B; flow rate 0.8~1.2 mL / min, column temperature 30~40 ℃.

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