Traditional Chinese medicine composition as well as preparation method and application thereof

Abstract

The invention provides a traditional Chinese medicine composition and a preparation method and application thereof.The traditional Chinese medicine monomer composition in the traditional Chinese medicine composition comprises amygdalin-usnic acid-beta-sitosterol which is derived from traditional Chinese medicine bitter apricot kernel, pericarpium citri reticulatae and rhizoma pinelliae preparata in a compatible combination mode, all the components have a synergistic effect, and through apparent indexes, the traditional Chinese medicine composition can be used for preparing the traditional Chinese medicine composition. The traditional Chinese medicine monomer composition and the traditional Chinese medicine compatibility combination thereof are used for evaluating and investigating the effect of the traditional Chinese medicine monomer composition and the traditional Chinese medicine compatibility combination thereof on inhibiting acute bronchitis of mice according to indexes such as acute bronchitis, tissue pathological observation, inflammatory factors and sputum dilution, the acute bronchitis treatment effect is remarkable, the medical application of the traditional Chinese medicine monomer composition is expanded, the traditional Chinese medicine compatibility combination is optimized, and the traditional Chinese medicine composition has a good application prospect in treatment of bronchitis. Especially, a new treatment idea is provided for treatment of acute bronchitis.

Description

Technical Field

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

[0002] Acute bronchitis is an acute infection of the bronchial mucosa caused by various pathogens. It is a common respiratory disease, more common in infants and young children, often secondary to an upper respiratory tract infection, with a higher incidence in winter and spring. Clinically, it is characterized by cough and sputum production. Western medicine primarily treats acute bronchitis with symptomatic treatment, focusing on cough suppression, expectoration, and fever reduction. Commonly used antitussives include dextromethorphan. For sticky and difficult-to-expectorate sputum, bromhexine and ambroxol hydrochloride are used. Fever is treated with antipyretic and analgesic medications such as acetaminophen or ibuprofen.

[0003] Western medicines for acute bronchitis, such as dextromethorphan, effectively inhibit the medullary cough center, producing a significant antitussive effect. While their chemical structure is simple and their therapeutic effects are clear, they require targeted treatment, preventing a single drug from addressing multiple symptoms. Furthermore, these drugs are often associated with side effects, and long-term use can lead to drug resistance.

[0004] Traditional Chinese medicine starts from the root cause of the disease and emphasizes dialectical treatment. It has comprehensive therapeutic effects and few side effects. However, its composition is complex. Different prescriptions are composed of several to more than a dozen Chinese medicines. The choice of different combinations is crucial to the efficacy of the medicine. It has important clinical application value in the treatment of acute bronchitis.

[0005] Furthermore, a combination of TCM monomers, based on the clear composition and content of the TCM formula, has the advantages of clear mechanisms and controlled quality. Therefore, there is an urgent need to develop a TCM monomer combination for the treatment of acute bronchitis. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a traditional Chinese medicine composition.

[0007] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned traditional Chinese medicine composition.

[0008] Another technical problem to be solved by the present invention is to provide an application of the above-mentioned Chinese medicine composition.

[0009] The technical solution adopted in the present invention is:

[0010] A traditional Chinese medicine composition, the main active ingredients of which are bitter almonds, tangerine peel and dried pinellia tuber (bitter almond-tangerine peel-dried pinellia tuber composition, BDR), wherein the bitter almonds, tangerine peel and dried pinellia tuber are 1-10 parts by weight, 1-10 parts by weight, and 1-10 parts by weight.

[0011] Preferably, the above-mentioned traditional Chinese medicine composition comprises, by weight, 1 part of bitter almond, 1 part of dried tangerine peel, and 1 part of dried pinellia tuber.

[0012] Preferably, the above-mentioned traditional Chinese medicine composition, the main active ingredients are composed of amygdalin (a monomer component of bitter almonds), usnic acid (a monomer component of tangerine peel), and β-sitosterol (a monomer component of Pinellia ternata) (amygdalin-usnic acid-β-sitosterol monomer composition, AUS), and the weight ratio is 1-10 parts of amygdalin, 1-10 parts of usnic acid, and 1-10 parts of β-sitosterol.

[0013] Preferably, the above-mentioned traditional Chinese medicine composition comprises 3 parts of amygdalin, 3 parts of usnic acid and 4 parts of β-sitosterol in parts by weight.

[0014] The preparation method of the above-mentioned traditional Chinese medicine composition comprises the following steps: weighing bitter almonds, tangerine peel and pinellia tuber according to the formula amount, soaking them in pure water and then decocting them with water; after the water boils, evaporating and concentrating the decoction to obtain a concentrated solution.

[0015] Preferably, the preparation method of the above-mentioned traditional Chinese medicine composition is as follows: amygdalin, usnic acid and β-sitosterol are weighed according to the formula amount, the amygdalin is prepared into a traditional Chinese medicine monomer solution with ultrapure water, usnic acid and β-sitosterol are respectively prepared into traditional Chinese medicine monomer solutions with 0.5% Tween 80, and the three traditional Chinese medicine monomer solutions are mixed to obtain the product.

[0016] Use of the above-mentioned Chinese medicine composition in preparing medicine for preventing and / or treating bronchitis.

[0017] Preferably, in the above application, the bronchitis is acute bronchitis and / or chronic bronchitis.

[0018] Preferably, in the above application, the bronchitis is acute bronchitis.

[0019] The treatment of bronchitis mentioned in the above application refers to at least one of improving body weight, increasing cough latency, reducing the number of coughs within 2 minutes, improving lung tissue pathology, reducing inflammatory factor levels, and promoting serous gland secretion of serous fluid to dilute sputum (acting as an expectorant). Improving lung tissue pathology means reducing alveolar thickening, alveolar wall rupture, inflammatory cell infiltration, and edema and congestion in the bronchial mucosa; reducing inflammatory factor levels means inhibiting the increase in IL-6, IL-1β, TNF-α, and cAMP levels.

[0020] The beneficial effects of the present invention are:

[0021] The above-mentioned traditional Chinese medicine composition, the traditional Chinese medicine monomer composition includes amygdalin-usnic acid-β-sitoterol (AUS), which are respectively derived from the traditional Chinese medicine bitter almond-dried tangerine peel-Rhizoma pplnellinae preaparata (BDR) traditional Chinese medicine compatibility combination. The components act synergistically. The effects of the traditional Chinese medicine monomer composition and its traditional Chinese medicine compatibility combination on inhibiting acute bronchitis in mice were evaluated through apparent indicators, histopathological observation, inflammatory factors and sputum dilution. The results showed that the effect of treating acute bronchitis was significant, which expanded the medical use of the traditional Chinese medicine monomer composition, optimized the traditional Chinese medicine compatibility combination, and provided a new treatment idea for the treatment of bronchitis, especially the treatment of acute bronchitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 To establish an acute bronchitis model in mice and the effects of drug administration on the body weight of AUS and BDR mice.

[0023] Figure 2 The effects of a single Chinese herbal composition (AUS) and a combined Chinese herbal composition (BDR) on lung histopathology in mice with acute bronchitis are shown. Representative images of HE-stained lung tissue from each group are shown (magnification 200x, scale bar 100 μm; Con: normal control group; Mod: model group; AUS group: amygdalin-usnic acid-β-sitosterol group; BDR group: bitter almond-tangerine peel-cleared pinellia group; XJEC group: Xingju Erchen Decoction group). Solid arrows in the figures represent alveolar collapse; dashed arrows represent alveolar wall rupture and alveolar fusion. Note: #P<0.05 compared with the Con group; *P<0.05 compared with the Mod group. After smoke modeling and 7 days of oral gavage, compared with the Mod group, the AUS group showed less alveolar wall thickening and less alveolar wall rupture and fusion. The BDR group had an increased number of alveoli, less alveolar collapse, and regular alveolar morphology.

[0024] Figure 3 The effects of AUS and BDR on lung injury scores in mice with acute bronchitis (n=3).

[0025] Figure 4Figure 2 Effects of AUS and BDR on p65, p-CREB, and CREB protein expression in lung tissues of mice with acute bronchitis (n = 6). Note: Compared with the Con group, #p < 0.05; compared with the Mod group, *p < 0.05. After smoke induction, p65 expression was significantly increased in the Mod group compared with the Con group (#P < 0.05), while p-CREB and CREB expression were significantly decreased (#P < 0.05). After AUS and BDR intervention, p65 protein expression was significantly decreased in the AUS and BDR groups compared with the Mod group (P < 0.05), while CREB expression was significantly increased (*P < 0.05). p-CREB protein expression was increased in the AUS group (P < 0.05), and p-CREB expression showed an increasing trend in the BDR group (P > 0.05). The results showed that AUS and BDR exerted anti-inflammatory effects by downregulating p65 protein, inhibiting the NF-κB signaling pathway, upregulating cAMP levels, activating the cAMP / PKA / CREB signaling pathway, and inhibiting the production of inflammatory mediators. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0027] Example 1

[0028] A traditional Chinese medicine composition (BDR), whose main effective ingredients are bitter almond, tangerine peel and clear pinellia (bitter almond-tangerine peel-clear pinellia composition, BDR), which comprises 1 part of bitter almond, 1 part of tangerine peel and 1 part of clear pinellia in parts by weight.

[0029] The preparation method of the above-mentioned traditional Chinese medicine composition comprises the following specific steps: weighing bitter almonds, tangerine peel and pinellia tuber according to the formula amount, soaking them in pure water for 30 minutes, decocting them with water, decocting them on low heat for 1 hour after the water boils, decocting them twice with water, combining the two decoctions, and concentrating them by evaporation using a rotary evaporator to prepare a concentrated solution.

[0030] Example 2

[0031] A traditional Chinese medicine composition (BDR) has main active ingredients consisting of bitter almond, tangerine peel and dried pinellia tuber (bitter almond-tangerine peel-dried pinellia tuber composition, BDR), which comprises 1 part of bitter almond, 10 parts of tangerine peel and 1 part of dried pinellia tuber in parts by weight.

[0032] The preparation method of the above-mentioned Chinese medicine composition is the same as that in Example 1.

[0033] Example 3

[0034] A traditional Chinese medicine composition (BDR) has main active ingredients consisting of bitter almonds, tangerine peel and dried pinellia tuber (bitter almond-tangerine peel-dried pinellia tuber composition, BDR), which comprises 10 parts of bitter almonds, 1 part of tangerine peel and 10 parts of dried pinellia tuber in parts by weight.

[0035] The preparation method of the above-mentioned Chinese medicine composition is the same as that in Example 1.

[0036] Example 4

[0037] A traditional Chinese medicine composition (AUS), whose main active ingredients are amygdalin (a monomer component of bitter almonds), usnic acid (a monomer component of dried tangerine peel), and β-sitosterol (a monomer component of dried pinellia tuber) (amygdalin-usnic acid-β-sitosterol monomer composition, AUS), and the main active ingredients are 3 parts of amygdalin, 3 parts of usnic acid, and 4 parts of β-sitosterol, according to their weight parts.

[0038] The preparation method of the above-mentioned traditional Chinese medicine composition has the following specific preparation steps: weighing amygdalin, usnic acid, and β-sitosterol according to the formula amount, preparing amygdalin with ultrapure water to form a traditional Chinese medicine monomer solution, and preparing usnic acid and β-sitosterol with 0.5% Tween 80 to form traditional Chinese medicine monomer solutions respectively, and mixing the three monomer solutions prepared above to obtain a traditional Chinese medicine monomer composition solution.

[0039] Example 5

[0040] A traditional Chinese medicine composition (AUS), whose main active ingredients are amygdalin (a monomer component of bitter almonds), usnic acid (a monomer component of dried tangerine peel), and β-sitosterol (a monomer component of dried pinellia tuber) (amygdalin-usnic acid-β-sitosterol monomer composition, AUS), and the main active ingredients are 10 parts of amygdalin, 1 part of usnic acid, and 1 part of β-sitosterol.

[0041] The preparation method of the above-mentioned Chinese medicine composition is the same as that of Example 4.

[0042] Example 6

[0043] A traditional Chinese medicine composition (AUS), whose main active ingredients are amygdalin (a monomer component of bitter almonds), usnic acid (a monomer component of dried tangerine peel), and β-sitosterol (a monomer component of dried pinellia tuber) (amygdalin-usnic acid-β-sitosterol monomer composition, AUS), and the main active ingredients are 1 part of amygdalin, 10 parts of usnic acid, and 10 parts of β-sitosterol.

[0044] The preparation method of the above-mentioned Chinese medicine composition is the same as that of Example 4.

[0045] Component analysis:

[0046] Amygdalin is the most abundant active ingredient in bitter almonds and is also the main active ingredient that exerts its cough and asthma-relieving effects; tangerine peel has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm, and its ingredients include usnic acid; β-sitosterol is the main component of qingbanxia and is the main ingredient that exerts its effects of drying dampness and resolving phlegm, relieving adverse reactions and stopping vomiting, and eliminating lumps and dispersing nodules; bitter almonds (containing amygdalin) have a synergistic effect through the "lung's ability to descend" and tangerine peel's (containing usnic acid) "resolving phlegm and saliva, harmonizing the spleen and stopping coughs, and relieving the five types of stranguria" and qingbanxia's (containing β-sitosterol) "removing wind and relieving spasms". Through the optimized combination of the dosage of each ingredient, it has a significant effect in treating bronchitis, providing a safer and more targeted treatment option for respiratory diseases such as allergic asthma, acute bronchitis, and chronic bronchitis.

[0047] Experimental Example 1

[0048] 1.1 Experimental Materials

[0049] 1.1.1 Experimental Reagents

[0050] Table 1-1 Main reagents for the experiment

[0051] Table 1-1

[0052]

[0053] 1.1.2 Experimental instruments

[0054] Table 1-2 Main experimental instruments

[0055] Table 1-2

[0056]

[0057] 1.2 Solution preparation

[0058] (1) Preparation of amygdalin-usnic acid-β-sitosterol solution (AUS): 5 mg of amygdalin powder was added to 1 mL of ultrapure water to prepare a 5 mg / mL amygdalin solution; 5 mg of usnic acid powder was added to 1 mL of 0.5% Tween 80 solution to prepare a 5 mg / mL usnic acid solution; 5 mg of β-sitosterol powder was added to 1 mL of 0.5% Tween 80 solution to prepare a 5 mg / mL β-sitosterol solution. 0.75 mL of the above 5 mg / mL amygdalin solution, 0.75 mL of β-sitosterol solution, and 1 mL of usnic acid solution were taken and diluted to 5 mL with normal saline to obtain amygdalin-usnic acid-β-sitosterol combination (AUS) solution. The oral doses were 15 mg / kg, 20 mg / kg, and 15 mg / kg, respectively, and stored in a refrigerator at 4°C.

[0059] (2) Preparation of bitter almond-tangerine peel-clear pinellia (BDR) solution: 24 g of bitter almond, 24 g of tangerine peel, and 24 g of clear pinellia were weighed on a scale, then soaked in pure water for 30 min and decocted twice. After each boiling, the water was reduced to low heat and simmered for 1 h. The two decoctions were combined and concentrated using a rotary evaporator to obtain a 45 mL concentrate with a concentration of 1.60 g / mL. Before administration, 8.75 mL of the bitter almond-tangerine peel-clear pinellia concentrate was added to 14.95 mL of 0.9% saline to obtain a bitter almond-tangerine peel-clear pinellia solution with a concentration of 0.936 g / mL. The solution was shaken repeatedly before use and administered to mice by gavage. The BDR group received a dose of 18.72 g / kg.

[0060] (3) Preparation of Xingju Erchen Decoction (XJEC): Weigh 12g each of dried tangerine peel, bitter almond, and dried pinellia tuber on a scale, including 10g of platycodon, 5g of liquorice, and 20g of poria. Soak in pure water for 30min, decoct twice with water, boil the water, and then simmer for 1h. Combine the two decoctions and concentrate them using a rotary evaporator to prepare a concentrate with a concentration of 1.775g / mL and a total volume of approximately 40mL. Before administration, add 7.77mL of Xingju Erchen Decoction concentrate to 14.95mL of 0.9% saline to obtain a Xingju Erchen Decoction solution with a concentration of 0.923g / mL. Shake well before use and administer to mice by gavage. The XJEC group received a dose of 18.46g / kg.

[0061] (4) 1 mol / L NaOH solution: Dissolve 0.2 g of NaOH powder in 5 mL of water to obtain a 1 mol / L NaOH solution.

[0062] (5) 0.005 g / mL phenol red solution: Take 0.5 g of phenol red and add 5 mL of 1 mol / L NaOH to dissolve it to obtain a 0.005 g / mL phenol red solution.

[0063] (6) 50 mg / mL ammonium chloride solution: 0.5 g of ammonium chloride powder was added to 10 mL of ultrapure water to obtain a 50 mg / mL ammonium chloride solution.

[0064] (7) 5% sodium bicarbonate solution: 5% sodium bicarbonate solution can be obtained by adding 2.5 g of NaHCO3 powder to 47.5 mL of ultrapure water.

[0065] 1.3 Experimental animals

[0066] Four-week-old, SPF-grade Kunming (KM) mice weighing (20 ± 5) g, male, were purchased from Beijing Huafukang Biotechnology Co., Ltd. Mice were acclimated for 3 days at the Tianjin University of Traditional Chinese Medicine Animal Center, housed five mice per cage. The temperature was maintained at 22 ± 5°C, relative humidity was 50%–70%, and ventilation was good. The Tianjin University of Traditional Chinese Medicine Animal Center provided experimental feed (Huafukang life-sustaining feed for rats), bedding, and tap water. Drinking water was changed daily, and bedding was changed once or twice weekly. During the experiment, the mice had free access to food and water.

[0067] 1.4 Experimental methods

[0068] 1.4.1 Ammonia-induced cough elimination experiment before acute bronchitis model preparation

[0069] Culling experiment: KM mice were reared and acclimated for 3 days. One day before the formal experiment, male mice weighing approximately 25-30 g were placed in an inverted 2L beaker with a 250 g cotton ball containing 500 mL of concentrated ammonia solution at the bottom. A timer was started, and the cough latency and the number of coughs within 2 minutes were observed and recorded. Mice with a cough latency of less than 10 seconds, greater than 33 seconds, or fewer than 6 but more than 53 coughs within 2 minutes were excluded.

[0070] 1.4.2 Establishment of an acute bronchitis mouse model induced by cigarette smoke

[0071] Sixty KM mice that qualified after elimination testing were randomly divided into five groups: a normal control group (Con group) of 12 mice, a model group (Mod group), a laetrile-usnic acid-β-sitosterol group (AUS group), a bitter almond-tangerine peel-Pinellinum tuber group (BDR group), and an apricot kernel and orange Erchen decoction group (XJEC group), with 12 mice in each group. After observation, the acute bronchitis model was established. The acute bronchitis mouse model was established using cigarette fumigation using a Yu Yuyan C-100 cigarette smoke generator. The procedure was to administer 20 Honghe brand cigarettes for 60 minutes daily at 9:00 AM and 4:00 PM for one week. Mice in each model group were observed for signs of nasal scratching, squinting, tearing, lethargy, coughing, rapid breathing, clustering, abdominal breathing, and slowed mental response. Body weight (g) was recorded daily.

[0072] 1.4.3 Acute bronchitis model identification

[0073] After the model was established, two mice were randomly selected from the Con group and two from the Mod group, and their lung tissues were taken for HE staining to detect pathological morphology and identify whether the model was successfully established. Pathological changes in the trachea and lung tissues of the mice were observed. Light microscopy of the Con group mice showed that the alveoli in the lung sections had regular morphology, the alveolar walls were tightly connected, the alveolar cavities were of appropriate size and evenly distributed, and there was no inflammatory cell infiltration in the field of view. Light microscopy of the Mod group mice showed that the alveolar walls collapsed, broke and fused, the alveolar cavity area increased, the number of alveoli decreased, the alveoli thickened and the alveolar morphology was irregular, there were a small number of red blood cells in the alveolar cavity, inflammatory cell infiltration occurred, the bronchial mucosa was damaged, and the entire lung tissue was obviously edematous and congested.

[0074] 1.4.4 Study on the antitussive effects of AUS and BDR on mice with acute bronchitis

[0075] After the acute bronchitis mouse model was successfully established by smoke fumigation, the mice were randomly divided into the model group (Mod), AUS group, BDR group, and Xingju Erchen Decoction group (XJEC). At 9:00 am every day, each group was given different interventions. The AUS group (amygdalin: 15 mg / kg, usnic acid: 20 mg / kg, β-sitosterol: 15 mg / kg), the BDR group (18.72 g / kg), and the XJEC group (18.46 g / kg) were given the test drugs by gavage at a volume of 0.02 mL / g for 7 days. The normal group and the model group were gavaged with the same volume of normal saline. The body weight of the mice was recorded daily. The results are shown in Figure 1 .

[0076] Thirty minutes after the last dose on the seventh day, the mice were placed in an inverted 1-liter glass beaker and subjected to an ammonia-induced cough test according to the method in 1.4.1. The cough latency and the number of coughs within 2 minutes were observed and recorded (violent abdominal contraction accompanied by coughing sounds, scratching the nose, or opening the mouth were used as the criteria). The cough suppressant effects of the AUS and BDR groups were evaluated by comparing the cough latency and the number of coughs in each group. The results are shown in Table 1.

[0077] 1.4.5 Sample Collection

[0078] 1.4.5.1 Collection of mouse serum

[0079] On the afternoon before the last administration, mice in each group were fasted but not watered for about 16 h. One hour after the last administration, 1% sodium pentobarbital was injected, and the left eye of the mice was removed to collect ocular venous blood. After standing at room temperature for 2 h, the blood was centrifuged at 2500 rpm for 20 min at room temperature to separate the serum and store it at -80°C.

[0080] 1.4.5.2 Collection of bronchoalveolar lavage fluid (BALF)

[0081] The mice were killed, the neck skin was cut open, the muscles around the trachea were separated, the trachea was exposed, and the tube was intubated about 2 mm below the thyroid cartilage. The tube was tied with surgical thread 2 mm below the intubation site to prevent fluid reflux. At the same time, the chest cavity was opened, and the right lung was tied with surgical thread near the right bronchus near the lung. 0.3 mL of normal saline solution was slowly injected into the left lung with a syringe and then withdrawn. The above process was repeated 3 times. The lavage fluids from the 3 times were combined and mixed evenly (a total of 0.50 mL, with a recovery rate of approximately 55.55%). The tube was centrifuged at 4°C, 3000 rpm for 10 min, and the supernatant was stored in a -80°C refrigerator.

[0082] 1.4.5.3 Collection and fixation of lung tissue

[0083] After carefully separating the surrounding mesentery of the ligated right lung, cut off the connection with the trachea below the ligature of the surgical thread. After gently washing the surface blood of the right lung in clean PBS buffer, place the right upper lobe in a centrifuge tube containing 4% PFA fixative for fixation. Wrap the remaining lung tissue in aluminum foil, place it in a 2 mL cryovial, and quickly store it in a -80°C refrigerator.

[0084] 1.4.6 HE staining

[0085] (1) Fixation of mouse lung tissue, its paraffin embedding and sectioning: The right upper lobe of the right lung of the mouse was placed in a centrifuge tube containing 4% PFA fixative for 24 to 48 hours, and cut into appropriate shapes and sections with surgical scissors. After dehydration with gradient ethanol (100% to 75%) and transparentization with xylene twice, each group of lung tissue was embedded in a paraffin embedding machine, cooled and fixed for 30 minutes, and then demolded and placed in a 4°C refrigerator overnight. After overnight, the paraffin block was slightly trimmed, and the mouse lung tissue was sliced ​​into 4 to 5 μm thickness on a microtome. After the slices were spread in water at a temperature of about 40°C, they were attached to adhesive slides and baked at 37°C until the water on the slides evaporated. The slices were then baked at 60°C for 2 hours.

[0086] (2) Mouse lung tissue staining: Mouse lung tissue sections were sequentially placed in xylene for dewaxing, gradient ethanol for rehydration, and ultrapure water for immersion. Hematoxylin was then dripped onto the lung tissue for staining for 4-5 minutes. After removing excess dye, the lung tissue was washed with water. Hydrochloric acid ethanol differentiation solution was dripped onto the lung tissue for 2-3 seconds. The lung tissue was then immersed in tap water for 3 minutes each. The lung tissue was stained with eosin for 8 minutes. The above steps made the nuclei of lung tissue cells blue-purple and the cytoplasm and extracellular matrix red. The sections were then quickly placed in gradient ethanol solution (75%-100%) for dehydration. The sections were then placed in xylene for transparency twice. Finally, neutral gum was added and the sections were sealed.

[0087] Mouse lung section observation: Pathological images of mouse lung sections in each group were observed under a Nikon optical microscope. Figure 2 .

[0088] Lung injury scores were also assessed based on the following four pathological features: 1) alveolar hemorrhage; 2) alveolar edema; 3) neutrophil infiltration / aggregation in the alveoli or vascular cavity; and 4) alveolar wall thickening or hyaline membrane formation. Figure 3 .

[0089] 1.4.7 ELISA assay for IL-6, IL-1β, TNF-α, and cAMP expression in mouse serum and bronchoalveolar lavage fluid (BALF)

[0090] (1) ELISA method to detect the content of IL-6, IL-1β and TNF-α in mouse bronchoalveolar lavage fluid

[0091] 1) Detect the levels of different indicators in mouse serum according to the ELISA kit instructions.

[0092] 2) Take out the reagents and microplates coated with specific antibodies 30 minutes in advance and equilibrate them to room temperature; thaw the serum or bronchoalveolar lavage fluid samples and standards and prepare them to the appropriate concentrations.

[0093] 3) Set up standard wells, blank wells, and sample wells. Add 100 μL of serially diluted standard to the standard wells.

[0094] 4) Slowly add 100 μL of the standard and sample diluent to the blank well, add 100 μL of the test sample to each experimental well, cover each well with a film to prevent evaporation of the liquid, and incubate in a 37°C incubator for 90 min.

[0095] 5) Drain all liquid from the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, cover the plate with film, and incubate at 37°C for 1 hour.

[0096] 6) After vertically shaking out the liquid in the wells, pat dry on clean absorbent paper. Add 350 μL of wash solution to each well, soak for 1 minute, then aspirate or shake off the liquid in the ELISA plate, pat dry, and repeat three times.

[0097] 7) Add 100 μL of HRP conjugate working solution to each well, cover the plate with film, and incubate at 37°C for 30 minutes.

[0098] 8) Drain all liquid from the wells and wash the plate five times, following the same procedure as in step 3. Add 90 μL of substrate solution (TMB) to each well, cover the plate with film, and incubate at 37°C in the dark for approximately 15 minutes. Terminate the reaction by adding 50 μL of stop solution to each well. Immediately measure the optical density (OD) of each well at 450 nm using a microplate reader. Calculate the corresponding levels of the corresponding indicators in the bronchoalveolar lavage fluid of each group of mice using the standard curve. Results are shown in Table 2.

[0099] (2) ELISA method to detect the content of cAMP in mouse serum

[0100] 1) After equilibration at room temperature for 10 minutes, remove the desired strips from the aluminum foil bag. Seal the remaining strips in a ziplock bag and return them to 4°C.

[0101] 2) Sample addition: Add 50 μL of sample and standard to each well, add 50 μL of universal diluent to the blank well, and then add 50 μL of Biotin-antibody working solution to each well. Cover with sealing film and incubate at 37°C for 1 hour.

[0102] 3) Washing: Discard the liquid and add 300 μL of diluted 1x washing buffer to each well. Let it stand for 1 minute. Discard the washing buffer and pat dry on absorbent paper. Repeat this washing process 3 times.

[0103] 4) Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film, and incubate at 37°C for 30 minutes.

[0104] 5) Washing: Discard the liquid and wash the plate 5 times according to the washing method in step 3).

[0105] 6) Add substrate: Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 min.

[0106] 7) Add stop solution: Add 50 μL of stop solution to each well and immediately measure the OD value of each well at a wavelength of 450 nm. The results are shown in Table 2.

[0107] 1.4.8 Western blotting to detect the expression of p65 / NF-κB, cAMP / PKA / CREB proteins in lung tissues of mice with acute bronchitis by AUS and BDR

[0108] 1) Extraction of total protein from mouse lung tissue

[0109] ① Remove the mouse lung tissue from the -80℃ freezer. Use scissors to cut about 0.01g of lung tissue from each group. Place the lung tissue in a clean 1.5mL centrifuge tube and mince it with scissors. Wrap the remaining lung tissue with tin foil and store it in a -80℃ refrigerator.

[0110] ② Prepare lysis buffer at a ratio of RIPA lysis buffer: PMSF: phosphatase inhibitor = 100:1:1. Add 100 μL of lysis buffer to each centrifuge tube containing lung tissue.

[0111] ③ Homogenize the tissue on ice using a handheld homogenizer for approximately 45 seconds. Then, sonicate the tissue using a 30 Hz ultrasonic disruptor for six cycles, each cycle for 5 seconds, followed by a 5-second pause. After preparing the lung tissue homogenate, centrifuge it at 4°C, 12,000 rpm, for 10 minutes. After centrifugation, collect the supernatant, aliquot, and store at -80°C for long-term storage.

[0112] 2) Quantification of mouse lung tissue protein concentration by BCA method

[0113] ① Dilute the BSA standard: prepare BSA standards of different concentrations according to the BCA kit instructions (concentrations are: 0, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5 mg / mL), and add 20 μL of each concentration into a 96-well plate.

[0114] ② Dilute samples: Dilute each sample twenty-fold, take 20 μL of each sample and add it to a 96-well plate for use.

[0115] ③ Prepare BCA working solution: Mix reagents A and B in the kit at a ratio of 1:50 and prepare them before use.

[0116] ④ Sample addition: Add 200 μL of BCA working solution to all wells, shake thoroughly to mix, and cover the 96-well plate with a lid.

[0117] ⑤Incubation: Place the 96-well plate in a 37°C incubator and incubate for 30 minutes.

[0118] ⑥ Detection: Take out the 96-well plate and measure the OD value of each sample and BSA standard at 562nm using a microplate reader; make a standard curve and the linear correlation coefficient R 2 A minimum of 0.99 is recommended. Calculate the sample concentration based on the equation and the OD value of the protein sample to be tested, and then standardize the protein concentration for each sample. The sample protein concentration is 20 μg. Prepare 100 μL of each sample. First, add the protein sample to a centrifuge tube, then add 20 μL of 5× protein loading buffer. Finally, add 1x PBS buffer to make up to 100 μL. Vortex thoroughly to mix.

[0119] ⑦ Protein denaturation: Use a metal bath to boil at 100℃ for 10 minutes. After natural cooling, divide into packages and store at -20℃ until use.

[0120] 3) Glue making

[0121] Place clean Biosharp 1.0 mm glass plates on the gel preparation rack and assemble. First, fill with ultrapure water to test for leaks. Wait approximately 10 minutes until the liquid level has not dropped, indicating a successful leak test. Prepare the lower gel according to the instructions of the 12% one-step gel preparation kit: 1.35 mL of lower gel solution, 1.35 mL of lower gel buffer, and 30 μL of coagulant. Add the prepared lower gel solution between the two glass plates. To prevent air from affecting the solidification of the separation gel, add a seal layer of ultrapure water and wait approximately 20 minutes. Prepare the upper gel: 0.375 mL of upper gel solution, 0.375 mL of upper gel buffer, and 7.5 μL of coagulant. When a refraction line appears between the ultrapure water and the separation gel, discard the upper ultrapure water and blot dry with absorbent paper. Add the prepared upper gel solution and slowly insert the sample comb to avoid introducing bubbles. Once the upper gel is completely solidified, sample loading can be performed.

[0122] 4) Sample loading and electrophoresis

[0123] Install the electrophoresis instrument, fill the electrophoresis tank with freshly prepared 1× electrophoresis buffer, carefully pull out the sample comb vertically upward, and use a 20μL pipette tip to draw up the buffer and repeatedly and slowly rinse the electrophoresis channel to flush out the remaining gel in the loading channel to prevent it from hindering the loading. Add the above-mentioned quantified protein sample solution to the loading channel and add pre-stained protein markers on both sides of the sample solution. After the loading is completed, adjust the voltage to 150V and perform constant voltage electrophoresis (in an ice water bath) for about 50 minutes. The time to stop electrophoresis is determined according to the position of the pre-stained protein marker band on the SDS-PAGE gel.

[0124] 5) Transfer

[0125] After electrophoresis, place the methanol-activated PVDF membrane and adhesive strip in the equilibration solution. After equilibration for 1-2 minutes, transfer the membrane to a dry sponge in the interlayer of a rapid transfer apparatus. Use a roller dipped in the equilibration solution and carefully roll it in one direction to dislodge any air bubbles between the adhesive strip and the PVDF membrane, ensuring a tight fit. Finally, place a dry sponge over the adhesive strip and roll it several times with a roller dipped in the equilibration solution. Close the interlayer, select the appropriate program, and transfer the membrane for 15 minutes.

[0126] 6) Closed

[0127] Use tweezers to transfer the transferred PVDF membrane to an incubation chamber containing protein-free rapid blocking buffer. Block for 25 minutes. Discard the blocking buffer and briefly rinse once or twice with 1× TBST buffer to remove any residual rapid blocking buffer.

[0128] 7) Primary antibody incubation

[0129] Dilute the antibodies p65 (NF-κB subunit) (1:1000), CREB (1:2000), and p-CREB (1:2000) in 1× TBST buffer and place them in the corresponding PVDF membrane incubation box. Incubate in a 4°C refrigerator overnight (approximately 16-18 hours). Then, remove the PVDF membrane, recover the primary antibody, and wash the membrane five times with 1× TBST buffer at room temperature on a shaker for 1, 3, 5, 7, and 7 minutes.

[0130] 8) Secondary antibody incubation

[0131] Dilute goat anti-rabbit secondary antibody (1:10,000) with 5% skim milk powder and incubate on a shaker at room temperature for 1 hour. After incubation, wash the membrane five times with 1× TBST buffer at room temperature on a shaker for 1, 3, 5, 7, and 7 minutes.

[0132] 9) Development

[0133] Following the instructions in the ECL chemiluminescence kit, take 1 mL of each developer solution A and B at a 1:1 ratio, mix thoroughly, and store in a dark place. Remove the PVDF membrane and immerse it in the working solution. Stain for approximately 30 seconds, then remove it with tweezers and gently blot with absorbent paper to remove any excess liquid. Place the membrane in an ultrasensitive multifunctional imager and adjust the appropriate parameters for imaging.

[0134] 10) Image analysis

[0135] The grayscale value of the optical density of the imaged bands was analyzed using Image J software. Figure 4 .

[0136] 1.4.9 Effects of AUS and BDR on phenol red secretion in mice

[0137] The tracheal segment phenol red excretion test is a commonly used model for screening secretory expectorants. The principle is that expectorants stimulate bronchial secretion (including secretion from serous glands or tracheal goblet cells), which dilutes sputum. Following intraperitoneal injection, phenol red dye is secreted into respiratory mucus. Tracheal lavage is performed to collect mucus, and measuring phenol red concentration indirectly reflects serous secretion. By measuring changes in phenol red excretion, the expectorant effect of a drug can be quantitatively assessed.

[0138] 1.4.9.1 Experimental methods

[0139] Fifty KM mice weighing 20-25g (fasted for 16 hours prior to the experiment, but not water) were randomly divided into five groups of 10 mice each. These groups included: Con group (normal saline); AUS group (amygdalin: 15mg / kg, usnic acid: 20mg / kg, β-sitosterol: 15mg / kg); BDR group (18.72g / kg); XJEC group (18.46g / kg); and the positive drug group (50mg / mL ammonium chloride). All groups were administered via gavage at a volume of 0.02mL / g. Thirty minutes after administration, each group received an intraperitoneal injection of 5% phenol red in normal saline at a dose volume of 0.1mL / 10g. The mice were immediately killed by cervical dislocation 30 minutes after injection. The skin of the mouse neck was cut open, and the trachea and its surrounding muscle tissue were separated. The lower edge of the thyroid cartilage and the tracheal bifurcation were ligated with surgical thread to prevent the outflow of the tracheal contents. The trachea was cut with surgical scissors about 2 mm above and below the ligature. The surface blood was washed with PBS buffer, and the surface was slightly dried with absorbent paper. The ligature was untied, and the tracheal segment was placed in two centrifuge tubes containing 0.8 mL of sodium bicarbonate aqueous solution for washing. The washing solutions of the two centrifuge tubes were combined and centrifuged at 3000 rpm / min for 8 minutes. The supernatant was taken and its OD value was measured at a wavelength of 546 nm.

[0140] Phenol red standard curve preparation: 1.95 mg of phenol red was weighed on an electronic analytical balance and dissolved in 3.9 mL of 5% NaHCO₃ to a phenol red concentration of 0.5 mg / mL. 0.1 mL of the 0.5 mg / mL phenol red solution was diluted with 3.9 mL of 5% NaHCO₃ to obtain a phenol red solution with a concentration of 12.5 μg / mL. This solution was then diluted to seven different concentrations using a 50 / 50 dilution method: 10 μg / mL, 5 μg / mL, 3 μg / mL, 1 μg / mL, 0.7 μg / mL, 0.3 μg / mL, and 0.1 μg / mL. The OD value was measured at 546 nm using a multifunctional microplate reader. A standard curve was prepared and the phenol red content (μg / mL) was calculated based on the standard curve. The phenol red content in the tracheal supernatant samples of each group of mice was also calculated. The results are shown in Table 3.

[0141] 1.5 Statistical analysis

[0142] GraphPad Prism 8.0 software was used for statistical analysis of the experimental data, SPSS software was used for data analysis, and one-way ANOVA was used to evaluate the differences in means among multiple groups. When P < 0.05, the results were considered to have significant differences, and when P < 0.01, the results were considered to have extremely significant differences.

[0143] Table 1

[0144]

[0145] Note: Compared with the Con group, # p<0.05; compared with the Mod group, * p<0.05.

[0146] Table 2

[0147]

[0148] Note: Compared with the Con group, # p<0.05; compared with the Mod group, * p<0.05.

[0149] Table 3

[0150]

[0151] Note: Compared with the Con group, # p<0.05.

[0152] Table 1 shows the therapeutic effects of the Chinese medicine monomer composition (AUS) and the Chinese medicine compatibility combination (BDR) on cough in mice with acute bronchitis induced by ammonia (n=10); Table 2 shows the changes in IL-6, IL-1β, TNF-α and cAMP levels in serum and bronchoalveolar lavage fluid (BALF) of mice with acute bronchitis treated with AUS and BDR Table 3 shows the effects of AUS and BDR on phenol red excretion in mice (n = 6). Expectorants can stimulate increased bronchial secretion (including serous gland secretion or tracheal goblet cell secretion) to dilute sputum. Increased phenol red excretion primarily reflects increased serous gland secretion, suggesting an enhanced expectorant effect.

[0153] In summary, the traditional Chinese medicine composition of bitter almond, tangerine peel, and dried pinellia tuber (BDR) and its monomer composition, amygdalin, usnic acid, and β-sitosterol solution (AUS), exhibit significant therapeutic effects in mice with acute bronchitis, significantly reducing cough frequency, prolonging cough latency, and improving lung tissue damage. Its mechanism of action is to inhibit PDE4B2 activity, upregulating cAMP levels and activating the cAMP / PKA signaling pathway; inhibiting the NF-κB signaling pathway and downregulating the expression of inflammatory factors such as IL-6, IL-1β, and TNF-α, thereby achieving anti-inflammatory effects. Furthermore, AUS promotes sputum secretion to dilute sputum. Studies have confirmed that AUS can treat acute bronchitis through its multiple anti-inflammatory, expectorant, and antitussive properties.

[0154] The above-described embodiments are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements to the technical solutions of the present invention made by ordinary engineers and technicians in this field should fall within the scope of protection of the present invention.

Claims

1. A Chinese medicine composition, characterized in that: The main active ingredients are composed of bitter almonds, tangerine peel and pinellia tuber, which are calculated by weight as 1-10 parts of bitter almonds, 1-10 parts of tangerine peel and 1-10 parts of pinellia tuber.

2. The Chinese medicine composition according to claim 1, characterized in that: Calculate 1 part of bitter almonds, 1 part of dried tangerine peel, and 1 part of dried pinellia tuber by weight.

3. The Chinese medicine composition according to claim 1, characterized in that: The main active ingredients are composed of amygdalin, usnic acid and beta-sitosterol, which are calculated by weight as 1-10 parts of amygdalin, 1-10 parts of usnic acid and 1-10 parts of beta-sitosterol.

4. The Chinese medicine composition according to claim 3, characterized in that: According to their weight parts, the ingredients include 3 parts of amygdalin, 3 parts of usnic acid, and 4 parts of β-sitosterol.

5. The method for preparing the Chinese medicine composition according to claim 1 or 2, characterized in that: Weigh bitter almonds, tangerine peel and clear pinellia according to the formula, soak them in pure water and then boil them with water. After the water boils, evaporate and concentrate the decoction to prepare a concentrated solution.

6. The method for preparing the Chinese medicine composition according to claim 3 or 4, characterized in that: Weigh amygdalin, usnic acid and β-sitosterol according to the formula amount, prepare the amygdalin into a Chinese medicine monomer solution with ultrapure water, prepare the usnic acid and β-sitosterol into Chinese medicine monomer solutions with 0.5% Tween 80 respectively, and mix the three Chinese medicine monomer solutions to obtain the product.

7. Use of the traditional Chinese medicine composition according to any one of claims 1 to 4 in the preparation of a medicament for preventing and / or treating bronchitis.

8. The use according to claim 7, characterized in that: The bronchitis is acute bronchitis and / or chronic bronchitis.

9. The use according to claim 7 or 8, characterized in that: The bronchitis is acute bronchitis.

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