Application of pharmaceutical composition in preparation of medicine for preventing and treating cold

Abstract

The invention discloses application of a pharmaceutical composition in preparation of a medicine for preventing and treating cold, and relates to the technical field of new application of traditional Chinese medicines. The medicine has the effects of clearing heat, resolving dampness, ventilating lung and relieving cough, and integrates the antipyretic effect, the analgesic effect and the antiviral effect; the traditional Chinese medicine composition can be used for effectively relieving symptoms such as pulmonary congestion, alveolar thickening and obvious inflammatory cell infiltration caused by viral infection, relieving lung injury and improving the life prolonging rate, and provides a pharmacological basis for clinical application of the traditional Chinese medicine composition.

Description

Technical Field

[0001] The present invention relates to the technical field of new applications of traditional Chinese medicines, and in particular to an application of a pharmaceutical composition in the preparation of a medicine for preventing and treating colds. Background Art

[0002] Cold is the most common respiratory infectious disease, of which 70%-80% is caused by viral infection and a small part is caused by bacterial infection. Influenza (flu for short) refers to a severe acute respiratory disease caused by influenza virus, which includes respiratory infection, and its symptoms include high fever, headache, general pain, fatigue and weakness, and in some cases, extreme exhaustion, nasal congestion, sneezing, sore throat, chest discomfort, cough, shortness of breath, bronchitis, pneumonia, and death in severe cases. Among them, influenza A virus is a common influenza virus, which has a high mutation rate, general susceptibility of the population, and a relatively high morbidity and mortality rate, which seriously endangers human life and health.

[0003] Bacterial colds are respiratory diseases caused by bacterial infection, such as Streptococcus pneumoniae, Haemophilus influenzae, etc.; the symptoms of bacterial colds are usually more serious, including high fever, cough, sputum, etc., and may even cause serious complications such as pneumonia.

[0004] The clinical application of anti-cold drugs has limited indications, slow onset of effect, and side effects such as salivation and drowsiness, and the clinical efficacy is not ideal.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide an application of a pharmaceutical composition in the preparation of a medicine for preventing and treating colds, so as to solve the above technical problems.

[0007] The present invention is achieved in that:

[0008] In the first aspect, an embodiment of the present invention provides an application of a pharmaceutical composition in the preparation of a medicine for preventing and treating colds. The pharmaceutical composition includes the following components, by weight: 10-20 parts of bupleurum, 20-25 parts of artemisia annua, 10-20 parts of kudzu vine, 10-20 parts of bitter almonds, 10-20 parts of forsythia, 10-20 parts of mulberry leaves, 10-20 parts of scutellaria, 5-10 parts of cardamom, 10-20 parts of talc and 10-20 parts of Poria.

[0009] In a second aspect, an embodiment of the present invention provides a use of the aforementioned pharmaceutical composition in the preparation of a drug for fighting colds.

[0010] The present invention has the following beneficial effects:

[0011] The pharmaceutical composition provided by the present invention is used in the preparation of a medicine for preventing and treating colds. The medicine has the effects of clearing away heat and dampness, promoting lung function and relieving cough; it can reduce the level of inflammatory cytokines in the serum of a febrile body and has an antipyretic effect on fever; in addition, it also has analgesic and antiviral effects; it can effectively inhibit the replication of viruses at the gene level and the protein level, and can also inhibit the release of viral particles; it can effectively alleviate the symptoms of pulmonary congestion, alveolar thickening and obvious inflammatory cell infiltration caused by viral infection, reduce lung damage, and improve the life extension rate, providing a pharmacological basis for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0013] Figure 1 The changes in body temperature of rats;

[0014] Figure 2 The temperature changes of rabbits;

[0015] Figure 3 This is a schematic diagram of the drug toxicity results of Qingxing Granules on MDCK cells;

[0016] Figure 4 Schematic diagram of the viral inhibition effect of Qingxing Granules under different administration modes;

[0017] Figure 5 It is a schematic diagram of the plaque formation test results;

[0018] Figure 6 This is a schematic diagram of the effect of Qingxing Granules on NP gene expression in A549 cells;

[0019] Figure 7 This is the curve of the survival rate and weight change of mice after infection with the H1N1 virus;

[0020] Figure 8 This is a schematic diagram of lung consolidation and pathological observation results of mice infected with H1N1 virus;

[0021] Fig. 9 This is a schematic diagram of the lung index results of mice infected with the H1N1 virus;

[0022] Fig.10 The changes of peripheral blood lymphocyte subsets in mice after infection with H1N1 virus;

[0023] Fig.11The expression of inflammatory factors in mice after infection with H1N1 virus;

[0024] Fig.12 This is the influenza virus load in the lung tissue of mice infected with the H1N1 virus. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0026] In the first aspect, an embodiment of the present invention provides an application of a pharmaceutical composition in the preparation of a medicine for preventing and treating colds. The pharmaceutical composition includes the following components, by mass fraction: 10-20 parts of bupleurum, 20-25 parts of artemisia annua, 10-20 parts of kudzu vine, 10-20 parts of bitter almonds, 10-20 parts of forsythia, 10-20 parts of mulberry leaves, 10-20 parts of scutellaria, 5-10 parts of cardamom, 10-20 parts of talc and 10-20 parts of poria.

[0027] Bupleurum has the effect of reconciling the exterior and interior, soothing the liver and raising yang. It is used for colds and fevers, alternating colds and fevers, malaria, liver depression and qi stagnation, chest and rib pain, rectal prolapse, uterine prolapse, and irregular menstruation.

[0028] Artemisia annua has the effects of clearing heat, cooling blood, reducing steaming, relieving summer heat, dispelling wind and relieving itching. It is used as an antipyretic for hot flashes due to yin deficiency, and can also stop night sweats, heatstroke, etc. However, this species does not contain "artemisinin" and has no antimalarial effect.

[0029] The root of Pueraria lobata is used as medicine, which can relieve muscle and fever, promote the production of body fluid and quench thirst, dredge meridians, and detoxify alcohol. It can also be used to treat fever and headache caused by external factors, lower blood lipids, lower blood sugar, relieve fever, and protect cardiovascular system. Pueraria lobata is a green food that can be used as both medicine and food. It has a high starch content and contains inorganic elements such as iron and calcium and essential amino acids for the human body.

[0030] Bitter almond, also known as almond. Its main functions include lowering qi, relieving cough and asthma, moistening intestines and relieving constipation. It is used for cough and asthma, chest fullness and phlegm, blood deficiency and dry body fluid, dry intestines and constipation.

[0031] Forsythia suspensa has the effects of clearing away heat, detoxifying, reducing swelling and dispersing nodules. It is one of the traditional Chinese medicines commonly used in clinical practice in China. It is often used to treat acute wind-heat colds, lymph node tuberculosis and other diseases.

[0032] Mulberry leaves have the effects of dispersing wind-heat, clearing the lungs and moistening dryness, calming liver yang, and clearing the liver and improving eyesight.

[0033] The root of Scutellaria baicalensis is used as medicine, which tastes bitter and is cold in nature. It has the effects of clearing away heat and dampness, purging fire and detoxifying, stopping bleeding, and calming the fetus. It is mainly used to treat febrile diseases, upper respiratory tract infections, lung heat cough, damp-heat jaundice, pneumonia, dysentery, hemoptysis, red eyes, fetal movement disorders, hypertension, carbuncles, furuncles, etc.

[0034] Cardamom has the effects of removing dampness and promoting qi, warming the middle and stopping vomiting, and stimulating appetite and digestion.

[0035] Talc is a diuretic and anti-dampness agent, clears away heat and relieves summer heat, removes dampness and heals sores. It is used for hot stranguria, stranguria with stones, hot and painful urination, thirst due to summer heat and dampness, and diarrhea due to dampness and heat. It is also used for external treatment of eczema, wet sores, and prickly heat. When spread on the surface of inflamed or damaged tissues, it can have a protective effect. When taken internally, it can protect the inflamed gastrointestinal mucosa and exert antiemetic and antidiarrheal effects, and it can also prevent the absorption of poisons in the gastrointestinal tract.

[0036] Poria cocos has the effects of promoting diuresis and eliminating dampness, strengthening the spleen, and calming the mind and nerves. It is often decocted into juice and taken, or it can be boiled in water, cooked into porridge or stewed into soup.

[0037] In an optional embodiment of the present invention, the pharmaceutical composition includes the following components by mass fraction: 15 parts of bupleurum, 22.5 parts of artemisia annua, 15 parts of kudzu root, 15 parts of bitter apricot kernel, 15 parts of forsythia, 15 parts of mulberry leaf, 15 parts of scutellaria, 7.5 parts of cardamom, 15 parts of talc and 15 parts of poria. In other optional embodiments of the present invention, the raw materials can be reasonably adjusted within the dosage range according to actual needs.

[0038] It should be noted that the medicine prepared from the pharmaceutical composition of the present invention has the effects of clearing away heat and dampness, promoting lung function and relieving cough.

[0039] In an optional embodiment of the present invention, the dosage form of the drug is selected from any one of tablets, capsules, pellets and granules. The drug is administered orally; further, the drug also includes pharmaceutically acceptable excipients.

[0040] It should be noted that the selection and use of pharmaceutical excipients need to comply with pharmacopoeia standards and relevant regulatory requirements to ensure their quality and safety.

[0041] Pharmaceutically acceptable excipients include fillers, such as lactose, starch and dextrin; binders, such as methyl cellulose, hydroxypropyl cellulose, etc.; lubricants, such as magnesium stearate; sustained-release agents, such as ethyl cellulose; in actual applications, the excipients can be reasonably adjusted according to the actual dosage form.

[0042] In an optional embodiment of the present invention, the dosage form of the pharmaceutical composition is granules, and the excipients selected in the preparation process include lactose and dextrin in a mass ratio of (1.5-2.5):1.

[0043] In an optional embodiment of the present invention, the preparation of the drug includes: mixing all raw materials according to a ratio, adding water and boiling for multiple times to obtain multiple extracts; mixing all the obtained extracts, concentrating them to obtain an extract; adding pharmaceutically acceptable excipients to obtain the drug.

[0044] In an optional embodiment of the present invention, the pharmaceutical composition is decocted twice, and its preparation process is as follows: all raw materials are mixed according to the ratio, water is added at a solid-liquid ratio of 1:10, and the mixture is first soaked for 30 minutes and then decocted for 2 hours to obtain a first extract and a first medicinal residue; wherein, water 10 times the amount of the raw materials is added to the first medicinal residue, and the mixture is decocted again to obtain a second extract and a second medicinal residue; the first extract and the second extract are mixed and concentrated; during the concentration process, the temperature of the mixed extract is 75°C-85°C, the vacuum degree is -0.090MPa to -0.1MPa, and the mixture is concentrated to a relative density of 1.05-1.15g / cm 3 At this time, an extract is obtained, and the temperature of the extract is 60°C-70°C; then, pharmaceutically acceptable excipients, namely lactose and dextrin in a mass ratio of 2:1, are added, and granulation is performed by extrusion in one step. The obtained drug is a granular preparation, which is then packaged to obtain a finished drug.

[0045] It should be noted that the number of times the drug composition is decocted is related to its actual dosage, and can be reasonably adjusted according to the actual dosage. The purpose of multiple decoctions is to dissolve the active ingredients in the drug in water as much as possible for easy absorption. Decoction can decompose or transform some toxic components in the drug and adjust its medicinal properties; in addition, decoction can also remove impurities and bad odors on the surface of the drug, improving the purity and use effect of the drug.

[0046] In other optional embodiments of the present invention, the added auxiliary materials can be adjusted to prepare the drug into any of the following dosage forms: tablets, capsules, pellets. The decoction extract can also be directly consumed without concentration, that is, prepared into a decoction form.

[0047] In other optional embodiments of the present invention, the drug is packed in 8g per bag, three times a day. The packing amount is reasonably adjusted according to the actual dosage form, such as 16g, 24g, etc. The packing amount and the number of times the drug is taken are different depending on the dosage form.

[0048] It should be noted that the embodiment of the present invention adopts the method of high-temperature extrusion of the extract to obtain the granules. The obtained extract has better efficacy, and after high-temperature extrusion, the particles of the obtained granules are more moderate, and the granules can be obtained without further drying.

[0049] In an optional embodiment of the present invention, the cold is caused by at least one of the following pathogens: influenza virus, bacteria or fungi.

[0050] The influenza virus is any one of influenza A virus selected from H1N1, H2N2, H5N1, H9N2, H13N6, H16N3 and H3N2; further, the influenza virus is influenza A virus H1N1.

[0051] Bacteria include endotoxins. The toxic component of endotoxins is mainly lipid A, which is heat-resistant and stable, with weak antigenicity. It can cause fever, microcirculation disorders, endotoxin shock and endotoxemia in the body.

[0052] Fungi include yeast, which can cause inflammatory responses in the body, release some inflammatory mediators, and further act on the body temperature regulation center, causing increased body temperature.

[0053] In an optional embodiment of the present invention, preventing and treating a cold comprises preventing and treating at least one of the following symptoms: fever, cough, body aches, runny nose, shortness of breath, pneumonia, and bronchitis.

[0054] In an optional embodiment of the present invention, the prevention and treatment of colds includes at least one of the following application methods: (1) antipyretic; (2) analgesic; (3) antiviral; (4) increasing the life extension rate of the subject; (5) increasing the weight of the subject.

[0055] In an optional embodiment of the present invention, antipyretic includes reducing the content of inflammatory cytokines in serum; further, inflammatory cytokines include interleukin 1β and tumor necrosis factor α, providing a pharmacological basis for the clinical application of the pharmaceutical composition.

[0056] In an optional embodiment of the present invention, the antiviral effect includes at least one of the following application modes: (1) effectively inhibiting viral replication at the gene level and protein level; (2) inhibiting the release of viral particles; (3) reducing the viral load in lung tissue; (4) alleviating pulmonary congestion, alveolar thickening and obvious inflammatory cell infiltration caused by viral infection.

[0057] Specifically, the virus replication is effectively inhibited at the gene level and protein level. In an optional embodiment of the present invention, the expression of PB2 and NB proteins is mainly inhibited. Among them, PB2 is an important protein in the influenza A virus genome and is part of the viral polymerase complex. NP is another important protein in the influenza A virus genome and is part of the ribonucleoprotein complex. The less the expression of PB2 and NB proteins, the better the inhibitory effect.

[0058] In a second aspect, an embodiment of the present invention provides a use of the aforementioned pharmaceutical composition in the preparation of a drug for fighting colds.

[0059] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0060] Example 1

[0061] This embodiment provides a pharmaceutical composition, and the method for preparing the pharmaceutical composition comprises the following steps:

[0062] (1) Mix the raw materials in proportion, add water at a solid-liquid ratio of 1:10, soak for 30 minutes, and then boil for 2 hours to obtain a first extract and a first medicinal residue, wherein the first extract is retained.

[0063] The pharmaceutical composition includes the following components by mass fraction: 15 parts of bupleurum, 22.5 parts of artemisia annua, 15 parts of kudzu vine, 15 parts of bitter apricot kernel, 15 parts of forsythia, 15 parts of mulberry leaf, 15 parts of scutellaria baicalensis, 7.5 parts of cardamom, 15 parts of talc and 15 parts of poria.

[0064] (2) adding 10 times the amount of water as the raw material to the first medicinal residue and boiling again to obtain a second extract and a second medicinal residue, wherein the second extract is retained.

[0065] (3) The first extract and the second extract are mixed and concentrated; during the concentration process, the temperature of the mixed extract is 80° C., the vacuum degree is -0.090 MPa, and the relative density is concentrated to 1.10 g / cm 3 At this time, the extract was obtained and the temperature of the extract was 65°C.

[0066] (4) Lactose and dextrin were added in a mass ratio of 2:1, and granulation was performed in one step by high-temperature extrusion. The obtained drug was a granular preparation, which was packaged in batches, with each bag containing 8 g. The finished product was in a dry paste powder state and was recorded as Qingxing granules.

[0067] Experimental Example 1

[0068] This experimental example uses animal experiments to test the efficacy of the drug prepared in Example 1, and the specific method is as follows:

[0069] 1.1 Experimental Materials

[0070] 1.1.1 Test and reference substances

[0071] Qingxing granules prepared in Example 1; Ganmaoling granules: China Resources Sanjiu Pharmaceutical Co., Ltd., specification: 10 g×9 bags / box, batch number: 2011098H.

[0072] 1.1.2 Experimental reagents

[0073] Lipopolysaccharide: SIGMA, specification: 1g / bottle, batch number: WXBD1402V;

[0074] Highly active dry yeast: Angel Yeast Co., Ltd., specification: 5g / bag, batch number: 20210203;

[0075] Rabbit interleukin 1β (1L-1β) ELISA kit: Jiangsu ELISA Industrial Co., Ltd., batch number: 202105;

[0076] Rabbit tumor necrosis factor α (TNF-a) ELISA kit: Jiangsu ELISA Industrial Co., Ltd., batch number: 202105;

[0077] Glacial acetic acid: Tianjin Damao Chemical Reagent Factory, specification: 500 mL / bottle, batch number: 20201201;

[0078] Sodium chloride injection: Meizhou High-tech Industrial Park, Guangdong, batch number: H20070703.

[0079] 1.1.3 Experimental instruments

[0080] Electronic balance: Ohaus Instruments (Changzhou) Co., Ltd., model: AX2202ZH, sensitivity: 0.01g;

[0081] Electronic balance: Changshu Shuangjie Testing Instrument Factory, model: JJ2000, sensitivity: 0.1g;

[0082] Electronic balance: Changshu Shuangjie Testing Instrument Factory, model: TC30KL, sensitivity: 1g;

[0083] Analytical balance: Ohaus Instruments (Changzhou) Co., Ltd., model: CP224C, sensitivity: 0.1 mg;

[0084] Electronic thermometer: Omron (Dalian) Co., Ltd., model: MC-347;

[0085] Intelligent hot plate instrument: Chengdu Taimeng Technology Co., Ltd., model: RB-200;

[0086] Labeling instrument: Thermo Fisher Scientific Inc., model: Multiskan FC.

[0087] 1.1.4 Experimental animals

[0088] SD rats, SPF grade, provided by Guangdong Medical Laboratory Animal Center, Laboratory Animal Production License No.: SCXK (Yue) 2018-0002, Laboratory Animal Quality Certificate No.: No.44007200090528. Facility Certificate No.: No.00268118. Used for rat fever experiments.

[0089] New Zealand rabbits, ordinary grade, were provided by Xinhua Experimental Animal Farm, Huadu District, Guangzhou, with the laboratory animal production license number: SCXK (Yue) 2019-0023, and the laboratory animal quality certificate number: No.44007600008691. Facility certificate number: No.00270990. They were used for the rabbit fever experiment.

[0090] KM mice, SPF grade, provided by Guangdong Medical Laboratory Animal Center, Laboratory Animal Production License No.: SCXK (Guangdong) 2018-0002, Laboratory Animal Quality Certificate No.: No.44007200089099, No.44007200089100. Facility Certificate No.: No.00264442, 00264443. They were used for mouse hot plate test and mouse writhing test, respectively.

[0091] 1.1.5 Observation of the adaptability of experimental animals

[0092] Adaptive feeding process: All animals were observed for adaptive feeding once a day from the day of receipt; the observation contents included: whether the mental state was good, whether the behavior was normal, whether the coat was smooth, whether there was loose stool, whether there was any abnormality in the eyes, whether there was any abnormal secretion and ulceration in the perineum, and whether there was any death. Animals that passed the adaptive feeding were included in the formal experiment.

[0093] 1.1.6 Experimental animal feeding management and environmental conditions

[0094] The experimental animals were housed in Room D5 and G3 of the Experimental Animal Laboratory of the Safety Assessment Center and were allowed to eat and drink freely.

[0095] During the experiment, the temperature of the experimental animal room was 20℃-25℃, the relative humidity was 40%-70% (recorded by WS-1 temperature and humidity recorder), and the lighting was 12h / 12h alternating light and dark.

[0096] 1.1.7 Dosage design and grouping

[0097] The daily dosage of Qingxing Granules for adults is 48g. Based on an adult body weight of 60kg, the proposed dosage of Qingxing Granules for adults is 0.8g of crude drug·kg -1 This experiment set up three dose groups: low, medium, and high, which are equivalent to 1, 2, and 4 times the intended clinical dose, respectively.

[0098] Among them, the equivalent dose of rats was calculated based on the body weight conversion factor of 6.25, and the three doses were 5g crude drug·kg -1 ·d -1 、10g herbal medicine kg -1 ·d -1 、20g herbal medicine kg -1 ·d -1 .

[0099] The equivalent doses for rabbits were calculated based on the body weight conversion factor of 2.30. The three doses were 1.84 g crude drug kg -1 ·d -1 、3.68g herbal medicine kg -1 ·d -1 、7.36g herbal medicine kg -1 ·d -1 .

[0100] The equivalent doses for mice were calculated using a body weight conversion factor of 9.01. The three doses were 7.21 g crude drug·kg -1 ·d -1 、14.42g herbal medicine kg -1 ·d -1 、28.84g herbal medicine kg -1 ·d -1 .

[0101] The dose of the positive control drug Ganmaoling granules was equivalent to that of Qingxing granules, which was equivalent to twice the clinical dose of Ganmaoling granules. The rat equivalent was 6.25 g·kg -1 ·d -1 The equivalent dose for rabbits is 2.30 g·kg -1 ·d -1 The equivalent dose for mice is 9.01 g·kg -1 ·d -1 The groups and dosages are shown in Table 1.

[0102] Table 1 Grouping and dosage

[0103]

[0104] 1.1.8 Statistical analysis

[0105] All data are expressed as "mean ± standard deviation "Indicated that the experimental data were statistically analyzed using SPSS17.0 statistical analysis software, and the differences between groups were compared using one-way analysis of variance (One-Way ANOVA), and the LSD test was used for pairwise comparisons between groups. P<0.05 was considered statistically significant.

[0106] 1.2 Experimental methods and results

[0107] 1.2.1 Effect of Qingxing Granules on Dry Yeast-Induced Fever in Rats

[0108] 1.2.1.1 Experimental methods

[0109] SD rats, SPF grade, half male and half female, weighing 180g-220g. After adaptive feeding, the rectal temperature was measured once a day with a thermometer for 7 consecutive days, and the animals with rectal temperature values ​​between 36.3℃-38.3℃ and fluctuating no more than 0.3℃ were selected for experiments. They were randomly divided into normal control group, model control group, low-, medium- and high-dose groups of Qingxing granules, and Ganmaoling granules group, with 8 rats in each group. Each group was gavaged at a volume of 10mL / kg, and the normal control group was given an equal volume of normal saline for 1 week. Fasting but not water control for 12h before the last administration. After administration, except for the normal control group, 20% yeast suspension (10mL / kg body weight) was injected subcutaneously into the back of the other rats, and the normal control group was given an equal volume of normal saline. Then the rectal temperature of the rats was measured at 1h, 2h, 4h, 6h, 8h, 10h and 12h, and the difference between the rectal temperature and the basal rectal temperature (△T) at different time points was compared as an indicator of body temperature change. [1-3] .

[0110] 1.2.1.2 Experimental results

[0111] Compared with the normal control group, the body temperature of the rats in the model control group increased significantly 6h-2h after modeling (P<0.01). Compared with the model control group, the low dose of Qingxing Granules can significantly reduce the body temperature of rats 6h-8h after modeling (P<0.05), the medium and high doses of Qingxing Granules can significantly reduce the body temperature of rats 6h-10h after modeling (P<0.05 or P<0.01), and Ganmaoling Granules can significantly reduce the body temperature of rats 6h-12h after modeling (P<0.05 or P<0.01). The above shows that Qingxing Granules have a certain antipyretic effect on fever model rats. The relevant results are shown in Figure 1 , the corresponding data are shown in Table 2.

[0112] Table 2 Effects of Qingxing Granules on body temperature of rats ( n=8)

[0113]

[0114] Note: Comparison between model group and normal control group △ P<0.05, △△ P<0.01; comparison between drug-treated group and model control group * P<0.05, ** p<0.01.

[0115] 1.2.2 Effect of Qingxing Granules on Endotoxin-Induced Fever in Rabbits

[0116] 1.2.2.1 Experimental methods

[0117] New Zealand rabbits, ordinary grade, male, weighing 1.5kg-2.5kg. After adaptive feeding, the rectal temperature of the rabbits was measured once a day for 1 week, and those with a rectal temperature between 38.5℃-39.5℃ and a fluctuation of no more than 0.5℃ were selected for the experiment. They were randomly divided into a normal control group, a model control group, a low-, medium-, and high-dose Qingxing granule group, and a Ganmaoling granule group, with 8 rabbits in each group. Each group was gavaged at a volume of 10 / kg, and the normal control group was given an equal volume of normal saline for 1 week. Before the last dose, lipopolysaccharide (LPS) was diluted to 2 mg / L with pyrogen-free normal saline. [4-5] Except for the normal control group, all other groups were injected with LPS at 1 mL / kg through the auricular vein of the left ear of the rabbits, and the normal control group was injected with pyrogen-free saline at 1 mL / kg through the auricular vein. 0.5 h after LPS injection, the rabbits were gavaged. The rectal temperature of the rabbits was recorded at 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h and 6 h after modeling, and the difference between the rectal temperature and the basal rectal temperature (△T) at different time points was compared as an indicator of body temperature change. 3 h after modeling, blood was collected from the auricular vein of the rabbits, and the levels of endogenous pyrogen interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in the serum of the rabbits were determined according to the instructions of the kit.

[0118] 1.2.2.2 Experimental results

[0119] Compared with the normal control group, the body temperature of rabbits in the model control group increased significantly 1h-6h after modeling (P<0.01). Compared with the model control group, low-dose Qingxing granules can significantly reduce the body temperature of rabbits 1.5h after modeling (P<0.05), medium-dose Qingxing granules can most significantly reduce the body temperature of rabbits 1.5h-2h and 5h-6h after modeling (P<0.05), high-dose Qingxing granules can significantly reduce the body temperature of rabbits 1.5h-3h and 6h after modeling (P<0.05), Ganmaoling granules can significantly reduce the body temperature of rabbits 1h-6h after modeling (P<0.05 or P<0.01). Results are shown in Figure 2 , the corresponding data are shown in Table 3.

[0120] Table 3 Effect of Qingxing Granules on Rabbit Body Temperature ( n=8)

[0121]

[0122] Note: Comparison between model group and normal control group △ P<0.05, △△ P<0.01; comparison between drug-treated group and model control group * P<0.05, ** p<0.01.

[0123] Compared with the normal control group, the levels of interleukin-1β and tumor necrosis factor-α in the serum of rabbits in the model control group were significantly increased 3 hours after modeling (P<0.05 or P<0.01). Compared with the model control group, low and medium doses of Qingxing granules can significantly reduce the levels of tumor necrosis factor-α in the serum of rabbits 3 hours after modeling (P<0.01), high doses of Qingxing granules can significantly reduce the levels of interleukin-1β and tumor necrosis factor-α in the serum of rabbits 3 hours after modeling (P<0.05), and Ganmaoling granules can significantly reduce the levels of interleukin-1β and tumor necrosis factor-α in the serum of rabbits 3 hours after modeling (P<0.05). The above shows that Qingxing granules can reduce the level of inflammatory cytokines in the serum of rabbits with fever model and have a certain antipyretic effect on rabbits with fever model. The results are shown in Table 4.

[0124] Table 4 Effects of Qingxing Granules on serum interleukin-1β and tumor necrosis factor-α in rabbits ( n=8)

[0125]

[0126] Note: Comparison between model group and normal control group △ P<0.05, △△ P<0.01; comparison between drug-treated group and model control group * P<0.05, ** p<0.01.

[0127] 1.2.3 Effect of Qingxing Granules on Hot Plate-Induced Pain Response in Mice

[0128] 1.2.3.1 Experimental methods

[0129] KM mice, SPF grade, female, weighing 18g-22g. After adaptive feeding, the mice were placed on a (55±0.5℃) intelligent hot plate instrument, and the time (s) required for the mice to lick their hind paws from the time they were placed on the hot plate was observed and recorded. 60 mice with a latency period of pain response of 5s-30s were selected and randomly divided into a blank control group, a low-, medium-, and high-dose Qingxing granule group, and a Ganmaoling granule group, with 12 mice in each group. Each group was intragastrically administered at a volume of 20mL / kg, and the blank control group was given an equal volume of normal saline once a day for 1 week. The pain threshold of each mouse was measured according to the above method 30min, 60min, 90min, and 120min after the last administration. If there is still no reaction after 60s, the mouse should be taken out to avoid burns, and its pain threshold is calculated based on 60s. [6-7] .

[0130] 1.2.3.2 Experimental results

[0131] Compared with the blank control group, the low, medium and high doses of Qingxing granules can significantly prolong the pain threshold of mice after 60 minutes of administration (P<0.05), and Ganmaoling granules can significantly prolong the pain threshold of mice after 30 minutes of administration (P<0.05). The above shows that Qingxing granules have a certain analgesic effect on pain model mice, and the results are shown in Table 5.

[0132] Table 5 Effects of Qingxing Granules on Pain Threshold of Mice ( n=12)

[0133]

[0134]

[0135] Note: Compared with the blank control group * P<0.05, ** p<0.01.

[0136] 1.2.4 Effect of Qingxing Granules on Acetic Acid-Induced Pain Response in Mice

[0137] 1.2.4.1 Experimental methods

[0138] KM mice, SPF grade, half male and half female, weighing 18g-22g. After adaptive feeding, they were randomly divided into a blank control group, a low-, medium-, and high-dose Qingxing granule group, and a Ganmaoling granule group, with 12 mice in each group. Each drug group was given oral administration at a volume of 20mL / kg, once a day, for 1 week. The blank control group was given an equal volume of normal saline. 60min after the last administration, 0.6% acetic acid 0.2mL / mouse was intraperitoneally injected to induce pain in each group of mice, and the time of occurrence of the twisting reaction of each mouse and the number of twisting reactions within 15min were immediately observed and recorded. [7] .

[0139] 1.2.4.2 Experimental results

[0140] Compared with the blank control group, the medium and high doses of Qingxing Granules and Ganmaoling Granules can significantly prolong the latency period of mice writhing (P<0.05 or P<0.01) and reduce the number of mice writhing (P<0.05 or P<0.01). The above shows that Qingxing Granules have a certain analgesic effect on pain model mice, and the results are shown in Table 6.

[0141] Table 6 Effect of Qingxing Granule on acetic acid-induced writhing reaction in mice ( n=12)

[0142]

[0143] Note: Compared with the blank control group * P<0.05, ** p<0.01.

[0144] Experimental Example 2

[0145] This experimental example tests the efficacy of the drug prepared in Example 1 against influenza A (H1N1) virus in vitro and in vivo, and the specific method is as follows:

[0146] 2.1 Experimental Materials

[0147] 2.1.1 Experimental cells and viruses

[0148] Canine kidney epithelial cell line (MDCK cells), human non-small cell lung cancer cell line (A549 cells); influenza virus A / WSN / 33 (H1N1), A / PR / 8 (H1N1), provided by Guangdong Key Laboratory of New Drug Screening, Southern Medical University.

[0149] 2.1.2 Experimental animals

[0150] SPF grade, 3-4 weeks old, BALB / c mice, half male and half female, a total of 90. Purchased from Guangzhou Southern Medical University Laboratory Animal Technology Development Co., Ltd., Animal Production License No.: SCXK (Guangdong) 2021-0041, Animal Certificate No.: N0.44002100034785, for death protection experiments.

[0151] SPF grade, 3-4 weeks old, BALB / c mice, half male and half female, a total of 60. Purchased from Guangzhou Southern Medical University Experimental Animal Technology Development Co., Ltd., animal production license number: SCXK (Guangdong) 2021-0041, animal certificate number: N0.44002100034784, for in vivo efficacy testing.

[0152] 2.1.3 Experimental drugs

[0153] Qingxing Granules: Engineering Center of Guangzhou University of Chinese Medicine Science and Technology Industrial Park Co., Ltd., batch number: 201221 (in order to facilitate administration and drug preparation, the experiment used Qingxing Granule dry paste powder, 1g dry paste powder = 5.7823g crude drug).

[0154] Lianhua Qingwen Capsules: Yiling Pharmaceutical, national medicine approval number 220040063, batch number: B2102286.

[0155] Oseltamivir phosphate capsules: Roche, national medicine approval number 20140121, batch number: M1075.

[0156] 2.1.4 Main reagents for the experiment

[0157] Cell RNA extraction kit: Fuji Biotechnology, catalog number: RE03113, batch number: R220701;

[0158] Animal RNA extraction kit: Fuji Biotechnology, catalog number: B03023, batch number: R210301;

[0159] RIPA lysis buffer: BD, catalog number: BD0032, batch number: AC39211;

[0160] Tissue fixative: Sevier, catalog number: GllOl, batch number: CR2112157;

[0161] PBS solution: Fude Biotechnology, product number: FD7032, batch number: 20220920;

[0162] Reverse transcriptase: Takara, catalog number: RR0362-1, batch number: ALE1704A;

[0163] Fluorescence quantitative PCR enzyme: Avid Biotechnology, catalog number: QE006-2, batch number: 20220604;

[0164] PVDF membrane: Roche, product number: 03010040001, batch number: 62344100;

[0165] GAPDH primary antibody: Fude Biotechnology, catalog number: FD0068, batch number: 20220310;

[0166] IAV NP primary antibody: GeneTex, catalog number: GTX636282, batch number: 42662;

[0167] IAV PB2 primary antibody: GeneTex, catalog number: GTX125925, batch number: 43187;

[0168] APC-CD4 primary antibody: BD, catalog number: 553051, batch number: 9134756;

[0169] Percy-cy5.5-CD8 primary antibody: BD, catalog number: 551162, batch number: 9098866;

[0170] Mouse HRP secondary antibody: Fude Biotechnology, catalog number: FDM007, batch number: 20211004;

[0171] Rabbit HRP secondary antibody: FDR Biotechnology, catalog number: FDR007, batch number: 20220829.

[0172] 2.1.5 Main experimental instruments

[0173] Grinding machine: Servicebio, model: KZ-111;

[0174] Electrophoresis equipment: BioRad, model: POWERPACHC;

[0175] Real-time fluorescence quantification instrument: Roche, model: LightCycler480;

[0176] Inverted microscope: NIKON, model: T1-SAM;

[0177] Chemiluminescence instrument: PROTEINSIMPLE, model: FLUORCHEME.

[0178] 2.1.6 Statistical analysis

[0179] All data are expressed as "mean ± standard deviation" "Indicated that the experimental data were statistically analyzed using SPSS17.0 statistical analysis software, and the paired t-test was used between the two groups, and P<0.05 was considered statistically significant.

[0180] 2.2 Experimental methods and results

[0181] 2.2.1 Qingxing Granule Cytotoxicity Assay (MTT Method) [8]

[0182] 2.2.1.1 Experimental methods

[0183] Take target cells (MDCK cells) in good logarithmic growth phase and suspend them to 1×10 5 / mL, 100μL / mL cell suspension was plated in a 96-well plate and cultured overnight in a cell culture incubator at 37°C and 5% CO2. The next day, the culture medium was discarded, the cells were washed with PBS, and 100μL / well gradient dilutions of the drug to be tested, Qingxing Granules (two-fold dilution of blank DMEM culture medium) were added, and 3 replicate wells were set up for each group. After the experimental plate was placed in the incubator and cultured for 48 hours, the supernatant was discarded, and the cells were washed with PBS, and 200μL / well of MTT working solution (0.5mg / mL) was added. After continuing to culture for 4 hours, the supernatant was carefully removed, and 150μL / well of DMSO was added. After shaking at room temperature for 10 minutes, the absorbance value was measured at OD=570nn on a full-wavelength fluorescence microplate reader, and the drug CC was calculated. 50 .

[0184] 2.2.1.2 Experimental results

[0185] The toxicity of Qingxing granules to MDCK cells was determined by MTT method. The results showed that Qingxing granules had a significant effect on CC of MDCK cells. 50 When the drug concentration was less than 5.78 mg / mL, the cell viability was >80%, so this concentration was used as the highest concentration for subsequent experiments. The relevant results are shown in Figure 3 .

[0186] 2.2.2 Determination of viral inhibition index under different administration modes[9]

[0187] The following four different administration modes were used to test the anti-influenza A virus effect of Qingxing Granules. 5 / well was inoculated into a 6-well plate and cultured overnight. The next day, the virus solution was diluted to MOI = 0.05 using serum-free 1640 medium.

[0188] Western blot technology was used to detect the expression of viral nucleoprotein (NP) and basic protein 2 (PB2) in four modes, and the viral inhibitory effect of Qingxing Granules in different administration modes was analyzed. The less the expression of NP and PB2, the better the inhibitory effect.

[0189] 2.2.2.1 Cell pretreatment (Prior to infection)

[0190] A drug solution containing 5.78 mg / mL Qingxing granules was prepared with blank 1640 medium, added to the corresponding cell wells and incubated in an incubator for 30 minutes, the drug-containing supernatant was discarded, and the cells were washed once with PBS. The virus solution was added and incubated for 1 hour, the virus solution was discarded, and the cells were washed once with PBS. Blank 1640 medium was added and continued to be incubated in an incubator for 24 hours to extract the protein.

[0191] 2.2.2.2 Virus pretreatment (Pre-infection)

[0192] The diluted virus solution was prepared into a drug-containing virus solution containing 5.78 mg / mL, and the drug-virus mixture was placed in an incubator for 30 minutes. The above virus solution was added to the corresponding wells and incubated in the incubator for 1 hour. The virus solution was discarded, washed once with PBS, and added with spacer 1640 medium and continued to be placed in the incubator for 24 hours to extract the protein.

[0193] 2.2.2.3 Post infection

[0194] Add the diluted virus solution to the wells of the culture plate and incubate in an incubator for 1 hour. Discard the virus solution, wash once with PBS, add a drug solution with a concentration of 5.78 mg / mL prepared with blank 1640 culture medium, continue to culture for 24 hours, and extract the protein.

[0195] 2.2.2.4 Entire infection

[0196] The diluted virus solution was prepared into a drug-containing virus solution containing 5.78 mg / mL Qingxing granules and incubated in an incubator for 30 minutes. At the same time, a drug solution containing 5.78 mg / mL was prepared with blank 1640 culture medium and added to the corresponding cell wells for incubation for 30 minutes. The drug-containing supernatant was discarded, washed once with PBS, and the drug-containing virus solution was added to continue incubation. After 1 hour, the virus supernatant was discarded, washed once with PBS, and the blank 1640 culture medium containing the drug was added to continue culturing the cells for 24 hours to extract the protein.

[0197] 2.2.2.5 Experimental results

[0198] Using 5.78 mg / ml as the dosing concentration, the virus inhibition effect of Qingxing Granules under different dosing modes was determined. Four different intervention methods, including cell pretreatment, virus pretreatment, post-infection treatment and full-course dosing, were used to detect the expression of PB2 and NP virus antigens in A549 cells. The results showed that Qingxing Granules (5.78 mg / mL) can significantly downregulate the expression of PB2 and NP proteins in the post-infection treatment stage. The less NP and PB2 are expressed, the better the inhibitory effect is, suggesting that Qingxing Granules can significantly play an antiviral role after viral infection. Related results can be found in Figure 4 .

[0199] 2.2.3 Calculation of progeny virus titer by well plate experiment [9]

[0200] 2.2.3.1 Experimental methods

[0201] A549 cells were cultured at 6×10 6 Each well was inoculated in a 6-well plate, and 1 mL / well of virus solution with an infection recovery (i.e., multiplicity of infection, MOI) of 0.05 was added to the well, and the well was placed in an incubator for incubation for 1 hour. After discarding the virus solution, it was washed once with PBS, and 0.64 mg / mL, 1.90 mg / mL, and 5.78 mg / mL Qingxing granule solution were added to the corresponding cell wells, and continued to stand in the incubator for 24 hours. The supernatant in the 6-well plate was then collected, centrifuged at 4°C, 1000g for 10 minutes, and the supernatant was collected. The number of progeny virus particles in the supernatant (PFU / mL) was determined by the plaque method.

[0202] 2.2.3.2 Experimental results

[0203] Plaque formation test was used to detect the progeny virus load in mice. Figure 5 As shown, Qingxing Granules can significantly reduce the plaques infected by IAV, suggesting that Qingxing Granules can significantly reduce the viral load of influenza A IAV progeny.

[0204] 2.2.4 Expression of viral nucleoprotein (NP) at the RNA level[9]

[0205] 2.2.4.1 Experimental methods

[0206] The cell treatment method was referred to 2.2.3.1, and the virus supernatant was discarded to extract the cell RNA. RNA was extracted according to the operation steps of the RNA extraction kit, and the RT-qPCR method was used to detect the expression of NP in cells after Qingxing granules were treated. The design of each mRNA primer is shown in Table 7.

[0207] Table 7 mRNA primers

[0208]

[0209] 2.2.4.2 Experimental results

[0210] RT-qPCR was used to detect the gene of viral nucleoprotein NP. The diluted virus solution (MOI = 0.05) was incubated with A549 cells for 1 hour, and the virus solution was discarded and a blank 1640 medium containing a specific concentration of Qingxing granules was added. After continuing to culture for 24 hours, the RNA was extracted. Results (see Figure 6 ) showed that compared with the blank control group, Qingxing Granules could inhibit and reduce the NP gene expression in A549 cells, and the inhibitory effect increased with the increase of concentration, showing a concentration-dependent manner, and the difference was statistically significant.

[0211] 2.2.5 Influenza virus infection mouse modeling method

[10]

[0212] 2.2.5.1 Mouse LD 50 Determination

[0213] SPF grade, 3-4 weeks old, BALB / c mice, half male and half female, were adaptively fed for 1 week and randomly divided into 7 groups according to body weight. Each group of mice was infected with different concentrations of virus. The virus solution was diluted 10 times, and 50 μL of the diluted virus solution was dripped into the nasal cavity. After the virus challenge, the mice were fed for 14 days, and the weight changes and deaths of the mice were recorded every day. The LD was calculated according to the Reed-Muench method. 50 .

[0214] 2.2.5.2 Mouse viral pneumonia model

[0215] SPF grade, 3-4 weeks old, BALB / c mice, half male and half female, were adaptively fed for 1 week and randomly divided into 7 groups according to body weight: blank control group, model control group, low-dose Qingxing granules group, medium-dose Qingxing granules group, high-dose Qingxing granules group, Lianhua Qingwen group and oseltamivir group (see Table 8 for details). The virus solution was instilled into the nasal cavity (50 μL), and the blank control group was instilled with an equal amount of PBS (this time defined as the 0th day of virus infection). By observing the status of the mice in the virus control group, the mice in the virus control group showed signs of weight loss, anorexia, erect hair, and huddling on the 2nd to 3rd day after infection, and the influenza virus pneumonia mouse model was successfully constructed.

[0216] 2.2.6 Qingxing Granules Death Protection Test

[0217] 2.2.6.1 Experimental methods

[0218] One day before virus infection, experimental mice were given preventive oral administration: the Qingxing granule group was given the corresponding dose of Qingxing granule solution every day, 0.2 mL per mouse; the Lianhua Qingwen group and the oseltamivir group were given 0.2 mL of Lianhua Qingwen and oseltamivir phosphate per mouse every day; the blank control group and the model control group were given an equal volume of distilled water by oral administration; the administration was repeated 2-3 hours before virus infection and continued for 6 days after infection, for a total of 8 days. On the day of infection, 2 times the LD 50 The virus solution was dripped into the nasal cavity, and the status of mice in each group was observed every day, the weight of mice was weighed, and the time of death of mice was recorded. The process lasted until the 14th day after infection. The dosage is shown in Table 8.

[0219] Table 8 Experimental grouping and dosage design of death protection test

[0220]

[0221]

[0222] 2.2.6.2 Experimental results

[0223] Qingxing granules protect mice from death due to H1N1 virus infection. 50 The virus solution was 50 μL, and the mice were observed for 15 consecutive days. The mortality and weight changes were statistically analyzed. The results showed that Qingxing granules could not significantly improve the death protection rate. Compared with the model control group, the Qingxing granules medium dose group improved the life extension rate better than the Lianhua Qingwen group and the Qingxing granules low and high dose groups, reaching 20.63%. The results are shown in Tables 9 and Figure 7 .

[0224] Table 9 Death protection rate and life extension rate of mice infected with influenza

[0225]

[0226] Note: Death protection rate = [(mortality rate of model control group - mortality rate of treatment group) / mortality rate of model control group] × 100%; life extension rate = [(average survival days of treatment group - average survival days of model control group) / average survival days of model control group] × 100%.

[0227] 2.2.7 In vivo efficacy test of Qingxing granules

[0228] 2.2.7.1 Experimental methods

[0229] One day before virus infection, experimental mice were given preventive oral administration: the Qingxing granule group was given 0.2 mL of Qingxing granule solution of corresponding concentration by oral administration every day, the Lianhua Qingwen group and the oseltamivir group were given 0.2 mL of Lianhua Qingwen and 0.2 mL of oseltamivir phosphate by oral administration every day, and the blank control group and the model control group were given an equal volume of distilled water by oral administration; the administration was repeated 2-3 hours before virus infection and continued for 5 days after infection, for a total of 7 days. On the day of infection, LD was instilled into the nasal cavity 50 The virus solution was dripped into the nasal cavity, and the status of mice in each group was observed and weighed every day. The mice were dissected and sampled on the 6th day after infection, and the corresponding tests were performed. The dosage is shown in Table 10.

[0230] Table 10 Experimental grouping and dosage design of in vivo efficacy test

[0231]

[0232]

[0233] Detection indicators: At the end of the experiment, the lungs were weighed and the lung index and lung index inhibition rate were calculated; the lung consolidation was observed and pathological observation was performed; flow cytometry was used to detect T lymphocyte subsets (CD4 + T cells, CD8 + T cells) and RT-qPCR were used to detect the changes in mRNA of inflammatory factors (IFN-γ, IL-6, TNF-α, IL-10) in lung tissues; the virus titer in the lungs of each group of mice was detected. The design sequences of each primer are shown in Table 11.

[0234] Table 11 Primer design sequences

[0235]

[0236] 2.2.7.2 Qingxing Granules can inhibit lung inflammation in mice infected with influenza virus

[0237] Mouse lung tissue Figure 8As shown in the figure, the volume of lung tissue of mice in the virus group was slightly larger, the color was dark red, and most of the congestion and edema consolidation areas appeared. The area of ​​consolidation area of ​​lung tissue of mice in the high-dose Qingxing granules group was reduced. The results of HE staining of lung tissue pathological sections showed that the virus group developed interstitial pneumonia, characterized by a large number of alveolar atrophy, thickening of alveolar walls, and severe inflammatory infiltration around bronchus. The high-dose Qingxing granules group can inhibit the occurrence of interstitial pneumonia, the degree of alveolar atrophy is mild, and the congestion of alveolar walls and interstitial blood vessels is significantly improved.

[0238] 2.2.7.3 Effect of Qingxing Granules on Lung Index of Mice Infected with Influenza Virus

[0239] The lung index of influenza virus mice treated with low-dose and high-dose Qingxing Granule groups was significantly reduced (the lung index of the virus group was 20.19, the lung index of the low-dose Qingxing Granule group was 15.81, and the high-dose Qingxing Granule group was 15.80), and the inhibition rates of the low-dose Qingxing Granule group and the high-dose Qingxing Granule group were 31.79% and 31.89%, respectively, effectively inhibiting the edema and consolidation of lung tissue caused by the virus. The results are shown in Tables 12 and Fig. 9 .

[0240] Table 12 Effects of each drug group on mouse lung index ( n=12)

[0241]

[0242]

[0243] Note: t test analysis was used to compare the blank control group VS the model control group. ## P<0.01; model control group VS Qingxing granule low-dose group, Qingxing granule high-dose group * P<0.05; model control group VS oseltamivir group, ** P<0.01.

[0244] Lung index: lung weight (g) / body weight (kg); Lung index inhibition rate: [(model control group-drug treatment group) / (model control group-blank control group)]×100%.

[0245] 2.2.7.4 Effect of Qingxing Granules on the Changes of Peripheral Blood Lymphocyte Subsets in Mice Infected with Influenza Virus

[0246] 60 μL of mouse peripheral blood was collected to detect changes in lymphocyte subsets in mouse peripheral blood. Fig.10 As shown, the CD4 + T cells (see Fig.10A, the average value of the model control group was 17.8%, and the average values ​​of the low-, medium-, and high-dose Qingxing granules groups were 23.88%, 29.92%, and 25.3%, respectively. CD8 + T cells (see Fig.10 B. The mean value of the model control group was 6.8%, and the low-, medium-, and high-dose groups of Qingxing Granules were 8.6%, 9.38%, and 8.96%, respectively. All of them showed an upward trend, but the differences were not statistically significant, suggesting that Qingxing Granules did not exert its antiviral effect by regulating immune cells.

[0247] 2.2.7.5 Effect of Qingxing Granules on the Expression of Inflammatory Factors in Mice Infected with Influenza Virus

[0248] After influenza virus infection, it can continue to cause a strong inflammatory response and produce a variety of inflammatory factors. The lung tissues of infected mice were collected and cytokine mRNA was measured. The results showed that the expression of IL-10 and TNF-α mRNA in the lung tissues of mice in the Qingxing Granule high-dose group was significantly reduced (see Fig.11 , suggesting that high doses of Qingxing Granules can inhibit the inflammatory factors produced by infected mice and reduce lung damage.

[0249] 2.2.7.6 Qingxing Granules Can Significantly Reduce the Flu Virus Load in Mouse Lung Tissue

[0250] The virus load in the mouse lung tissue was detected by cytopathic method. The right lower two lobes of the mouse lung tissue were taken to detect the virus titer of the lung tissue. The lung tissue was ground and centrifuged to obtain the virus supernatant. The virus supernatant was diluted in a 10-fold gradient and added to MDCK cells. The lesion holes were observed and recorded. The results showed that Qingxing granules reduced the virus load in the mouse lung after intervention. Virus 10 6· 71 PFU / mL, Qingxing granule low dose group 10 4·79 PFU / mL, Qingxing granule high dose group 10 4·70 PFU / mL, results are shown in Fig.12 .

[0251] The results of the above viral pneumonia model mouse treatment experiment showed that Qingxing Granules can reduce the viral load in lung tissue, effectively improve mouse lung damage, and alleviate symptoms such as pulmonary congestion, alveolar thickening, and obvious inflammatory cell infiltration caused by viral infection. Its mechanism of action may be to reduce the production of inflammatory factors caused by the virus, thereby reducing the damage caused by the unbalanced inflammatory factor storm in the body, indicating that Qingxing Granules have a therapeutic effect on mice infected with influenza virus. The above research can provide a pharmacological basis for the clinical application of Qingxing Granules.

[0252] In summary, the pharmaceutical composition (Qingxing Granules) provided by the present invention has the following characteristics:

[0253] (1) Qingxing granules have a certain antipyretic effect on fever model rats;

[0254] (2) Qingxing granules can reduce the level of inflammatory cytokines in the serum of rabbits with fever model and have a certain antipyretic effect on rabbits with fever model;

[0255] (3) Qingxing granules can significantly prolong the pain threshold of mice 60 minutes after administration, and have a certain analgesic effect on pain model mice;

[0256] (4) Qingxing granules can significantly prolong the latency period of mice writhing and reduce the number of mice writhing, indicating that it has a certain analgesic effect on pain model mice;

[0257] (5) The results of in vitro experiments showed that after viral infection, Qingxing granules could effectively inhibit viral replication at the gene and protein levels, and also inhibit the release of viral particles;

[0258] (6) The results of the survival protection test showed that Qingxing granules could increase the life extension rate of influenza mice. The life extension rate of the medium-dose Qingxing granules group was 20.63%, but it had no significant effect on the survival rate of mice.

[0259] (7) The results of the viral pneumonia model mouse treatment experiment showed that Qingxing Granules can reduce the viral load in lung tissue, effectively improve lung damage in mice, and alleviate symptoms such as pulmonary congestion, alveolar thickening, and obvious inflammatory cell infiltration caused by viral infection. The above results can provide a pharmacological basis for the clinical application of Qingxing Granules.

[0260] Specific references are as follows:

[0261] [1] Wu Jialin, Lü Shaowa, Sun Yali, et al. Study on the antipyretic mechanism of Zihu decoction on different rat fever models [J]. Central South Pharmacy, 2018, 16(4): 492-495.

[0262] [2] An Xiang, Gao Jingfei, Sun Jieyu, et al. Study on the antipyretic effect of Chaicen Qingning Capsules and Ganmaoling Capsules on LPS and dry yeast rat fever model [J]. Chinese Journal of Pharmacology and Clinical Medicine, 2017, 33(5): 157-162.

[0263] [3] Chu Jin, Ma Xingyue, Qin Tingting, et al. Study on the antipyretic effect of compound Yinhua Jiedu granules on dry yeast-induced fever model in young mice [J]. Pharmacy and Clinical Research, 2020, 28(4): 246-250.

[0264] [4] Lü Shaowa, Wu Yinqi, Li Yingpeng, et al. Effects of different phases of Baihu decoction on serum TNF-α, IL-1β and IL-6 levels in rabbits with fever model [J]. International Journal of Pharmaceutical Research, 2020, 47(10): 870-875.

[0265] [5] Gong Ming, Yang Ruocong, Dai Yaolan, et al. Study on the antipyretic effect of Houttuynia cordata wall-broken slices on endotoxin-induced fever in rabbits [J]. World Chinese Medicine, 2020, 15(13): 1930-1933.

[0266] [6] Du Junxia, ​​Gong Binbin, Song Fei, et al. Study on the anti-inflammatory and analgesic effects of wolfberry polysaccharide [J]. Natural Product Research and Development, 2019, 31: 236-239.

[0267] [7] Sun Zhanpeng, Gao Changjiu, Zhou Xue, et al. Study on the analgesic effect of water and alcohol extracts of the Chinese herbal medicine Dongfengcai [J]. Modern Distance Education of Traditional Chinese Medicine, 2021, 19(4): 152-154.

[0268] [8] Li Jingyan. Study on the anti-Zika virus activity and mechanism of action of Salvia miltiorrhiza monomer compounds salvianolic acid and cryptotanshinone[D]; Southern Medical University, 2021.

[0269] [9] Ye Yilu. Tetrahydroquercetin derivative YX-48-26 targets PA_N to inhibit influenza A virus replication[D]; Southern Medical University, 2021.

[0270]

[10] Liu Hongtao. Study on the antiviral effect and mechanism of ABMADABMA against influenza virus and herpes zoster virus[D]; Jilin University, 2022.

[0271] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for preventing and treating colds, characterized in that: The pharmaceutical composition comprises the following components by weight: 10-20 parts of bupleurum, 20-25 parts of artemisia annua, 10-20 parts of kudzu root, 10-20 parts of bitter almond, 10-20 parts of forsythia, 10-20 parts of mulberry leaf, 10-20 parts of scutellaria, 5-10 parts of cardamom, 10-20 parts of talc and 10-20 parts of poria.

2. The use according to claim 1, characterized in that: The cold is caused by at least one of the following pathogens: influenza virus, bacteria or fungi.

3. The use according to claim 1, characterized in that: The prevention and treatment of colds includes prevention and treatment of at least one of the following symptoms: fever, cough, body aches, runny nose, shortness of breath, pneumonia and bronchitis.

4. The use according to claim 1, characterized in that: The cold prevention and treatment comprises at least one of the following application methods: (1) Antipyretic; (2) Analgesia; (3) Antiviral; (4) Improve the life extension rate of the subject; (5) Increase the body weight of the subject.

5. The use according to claim 4, characterized in that: The antipyretic effect includes reducing the content of inflammatory cytokines in serum.

6. The use according to claim 5, characterized in that: The inflammatory cytokines include interleukin 1β and tumor necrosis factor α.

7. The use according to claim 5, characterized in that: The anti-virus comprises at least one of the following application methods: (1) Effectively inhibit viral replication at the gene and protein levels; (2) Inhibit the release of viral particles; (3) reduce viral load in lung tissue; (4) Alleviate pulmonary congestion, alveolar thickening and obvious inflammatory cell infiltration caused by viral infection.

8. The use according to claim 1, characterized in that: The dosage form of the drug is selected from any one of tablets, capsules, pellets and granules; preferably granules; The drug is administered orally; preferably, the drug also includes pharmaceutically acceptable excipients.

9. The use according to claim 1, characterized in that: The preparation of the drug comprises: All raw materials are mixed according to the proportion, and water is added and then boiled for multiple times to obtain multiple extracts; All the obtained extracts are mixed and concentrated to obtain an extract; The medicine is prepared by adding pharmaceutically acceptable excipients.

10. Use of the pharmaceutical composition according to claim 1 in the preparation of medicines for fighting colds.

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