Fritillaria unibiacteata compatible prescription and application thereof

Abstract

The invention discloses a unibract fritillary bulb compatible prescription and application thereof, and belongs to the technical field of traditional Chinese medicines. The invention provides a traditional Chinese medicine composition for relieving cough, eliminating phlegm and / or resisting inflammation. The traditional Chinese medicine composition is prepared from 40g of fritillaria unibiacteata, 10-20g of folium eriobotryae, 20-40g of sea-buckthorn and 15-30g of poria cocos. The toxicity of the unibract fritillary bulb compatible prescription is researched by using a mouse acute toxicity experiment, and the prescription is non-toxic according to in-vitro and in-vivo related indexes. Then, a disease model is established through ammonia water cough inducing, tracheal phenol red excretion and xylene ear swelling inducing methods, cough relieving, phlegm eliminating and anti-inflammatory pharmacological experiments are conducted on nine compatibility groups designed through orthogonal experiments, and the optimal compatibility proportion is 40 g of fritillaria unibiacteata, 15 g of folium eriobotryae, 40 g of sea-buckthorn and 22.5 g of poria cocos. The invention provides a new medicine choice for relieving cough, eliminating phlegm or resisting inflammation.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a dark purple fritillaria compatibility prescription and application thereof. Background Art

[0002] "Laosou" is a disease name, which can be found in Volume 3 of "Emergency Prescriptions for the Elbow". It refers to a disease characterized by persistent cough, fatigue, shortness of breath, and speech weakness.

[0003] Bronchitis refers to a chronic, nonspecific inflammation of the trachea, bronchial mucosa, and surrounding tissues. The main cause of bronchitis is repeated viral and bacterial infection, which results in chronic, nonspecific inflammation of the bronchi. Drops in temperature, spasms and ischemia of small respiratory vessels, and decreased defense function are conducive to the onset of the disease; chronic irritation from smoke, dust, and polluted air can also cause the disease; smoking causes bronchospasm, mucosal mutations, decreased ciliary movement, and increased mucus secretion, which facilitates infection; allergic factors also play a role. Clinical symptoms include cough, recurrent coughing, sputum and wheezing, repeated infections, and a prolonged course of the disease. Clinically, antibiotics such as erythromycin and azithromycin, expectorants, antitussive drugs, and antiasthmatic drugs are used for treatment, but the disease recurs and the results are unsatisfactory. Commonly used antitussive and expectorant drugs in Chinese patent medicines include Kening Syrup, Zhike Ningsou Capsules, Compound Luo Han Guo Cough Granules, Kening Syrup, Xingbao / Lifei Tablets, Laiyang Pear Cough Granules, Chuanbei Cough Syrup, Compound Platycodon Syrup, Chuanbei Loquat Paste and Loquat Cough Syrup, etc. These drugs have the following defects: single ingredients, single efficacy, antitussive but not expectorant, expectorant but not cough suppressant; large dosage, inaccurate dosage, uncertain efficacy, etc.

[0004] Traditional Chinese Medicine (TCM) often uses compatibility as a primary method, with the use of medicinal materials and dosage being key considerations. The dosage of a TCM drug is closely linked to its efficacy, leading some to believe that "the secret of TCM lies in dosage." Too small a dose will not achieve the desired therapeutic effect, while too large a dose will not achieve the desired therapeutic effect and may even cause adverse effects. This invention uses data mining technology to determine the TCM formula and then explores the dosage to identify the optimal compatibility ratio, providing a new drug option for antitussive, expectorant, or anti-inflammatory treatments. Summary of the Invention

[0005] The purpose of the present invention is to provide a dark purple Fritillaria combination prescription and its application to solve the problems existing in the above-mentioned prior art. The present invention provides a traditional Chinese medicine composition with antitussive, expectorant and / or anti-inflammatory effects, providing a new drug option for antitussive, expectorant or anti-inflammatory effects.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a traditional Chinese medicine composition for relieving cough, eliminating phlegm and / or resisting inflammation. The composition comprises 40g of dark purple fritillaria, 10-20g of loquat leaves, 20-40g of sea buckthorn and 15-30g of poria.

[0008] Optionally, it includes 40 g of Fritillaria unibracteata, 15 g of Eriobotrya japonica leaves, 40 g of Hippohgae rhamnoides, and 22.5 g of Poria cocos.

[0009] The present invention also provides the application of the described traditional Chinese medicine composition in the preparation of a drug for relieving cough.

[0010] The present invention also provides the application of the described traditional Chinese medicine composition in the preparation of a drug for removing phlegm.

[0011] The present invention also provides the application of the described traditional Chinese medicine composition in the preparation of an anti-inflammatory drug.

[0012] The present invention also provides the application of the described traditional Chinese medicine composition in the preparation of a drug for treating acute tracheobronchitis.

[0013] The present invention also provides a drug for relieving cough, removing phlegm and / or having anti-inflammatory effect, comprising the described traditional Chinese medicine composition.

[0014] The present invention also provides a drug for treating acute tracheobronchitis, comprising the described traditional Chinese medicine composition.

[0015] Optionally, it further includes pharmaceutically acceptable excipients.

[0016] Optionally, the administration method of the drug includes oral administration or parenteral administration.

[0017] The present invention discloses the following technical effects:

[0018] The present invention uses the acute toxicity experiment of mice to study the toxicity of the compatibility formula of Fritillaria unibracteata. According to relevant in vitro and in vivo indicators, it can be known that this formula has no toxicity. Then, disease models are established by the methods of ammonia water-induced cough, tracheal phenol red excretion, and xylene-induced ear swelling. Pharmacodynamic experiments of relieving cough, removing phlegm and anti-inflammatory are carried out on nine groups of compatibility groups designed by orthogonal test, and the best compatibility ratio is obtained as 40 g of Fritillaria unibracteata, 15 g of Eriobotrya japonica leaves, 40 g of Hippohgae rhamnoides, and 22.5 g of Poria cocos. Fritillaria unibracteata clears heat and resolves phlegm and is the monarch drug; Eriobotrya japonica leaves clears the lung and relieves cough, and reduces adverse qi and stops vomiting, and can be the minister drug; Poria cocos promotes diuresis and eliminates dampness, and can be the assistant drug; Hippohgae rhamnoides relieves cough and removes phlegm, and can be the messenger drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1 It is the result diagram of H&E staining of mouse tissue pathology in Example 1, and the scale bar is 100 μm. Detailed implementation manners

[0021] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0022] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0024] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0025] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0026] Through data mining methods, the present invention analyzed and summarized the clinical application characteristics such as the compatibility rules of drug formulas containing Fritillaria unibracteata, and obtained the compatibility formula of Fritillaria unibracteata - Eriobotrya japonica - Poria cocos - Hippohgae rhamnoides. Furthermore, through network pharmacology, the components and targets of this formula were predicted, and its core potential chemical components, targets, and related pathways were found. Further, through the acute toxicity experiment of mice, the acceptability of the preparation formula was determined, and the cough - relieving, expectorant, and anti - inflammatory activities of the efficacy group of the preparation formula were verified by animal models, and the best proportion of the compatibility formula was obtained as 40 g of Fritillaria unibracteata, 15 g of Eriobotrya japonica, 40 g of Hippohgae rhamnoides, and 22.5 g of Poria cocos.

[0027] Example 1 Research on acute toxicity experiment of mice with the compatibility formula of Fritillaria unibracteata

[0028] The acute toxicity experiment is one of the important methods to evaluate the safety of clinical medications. According to the literature records, the oral toxicity of Fritillaria cirrhosa is relatively small; the water extract of Eriobotrya japonica leaves belongs to the non-toxic level and has no genetic toxicity; the toxicity of Hippohgae rhamnoides is extremely low; Poria cocos belongs to the food and medicine homologous food in the "Notice on Issuing the Administrative Regulations on Substances That Are Both Foods and Chinese Medicinal Materials in Traditional Use" issued by the National Health Commission. Referring to the "National Food Safety Standard Acute Oral Toxicity Test (GB / 15193.3-2014)", the limited-dose method was used to conduct the acute toxicity experiment on mice. By observing the general state of the mice, measuring the blood biochemical indexes of the mice, measuring the organ indexes, and examining the changes in the organ tissues of the mice, the toxicity of the compatibility formula of Fritillaria unibracteata was explored, so as to conduct animal pharmacodynamic experiments subsequently and further explore the optimal proportion of the compatibility formula of Fritillaria unibracteata for its efficacy.

[0029] 1. Sample preparation

[0030] Referring to the "National Food Safety Standard - Acute Oral Toxicity Test (GB / 15193.3-2014)", in the limited-dose range, 10 g / kg body weight of each medicinal material was determined. According to the traditional Chinese medicine decocting method, first add 12 times the amount of water and soak for 30 minutes, then decoct with strong fire until boiling, and continue to decoct with gentle fire for 60 minutes, and pour out the first decoction; add 10 times the amount of water, continue to decoct, and pour out the second decoction in the same way as the first time; add 10 times the amount of water, continue to decoct, and pour out the third decoction in the same way as the second time. The three decoctions were combined together and concentrated under reduced pressure into 25.614 g of extract by a rotary evaporator, and the extract yield was 32%.

[0031] 2. Grouping and administration of experimental animals

[0032] Twenty male and twenty female Kunming mice were taken. Every 10 mice of the same sex were randomly divided into 1 group. The experiment was carried out after the mice had adapted to the environment in the laboratory for one week. The mice were fasted for 12 hours without water restriction before administration. With the maximum gavage volume of 0.4 mL / 10 g, the administration group was gavaged with the maximum soluble mass concentration of 1 g / mL that could be aspirated by a No. 10 gavage needle, and the blank group was gavaged with an equal amount of normal saline. The state of the mice was observed within 2 hours after gavage. If there was death, the mice were dissected immediately. The diet was restored 4 hours after the last administration, and the mice were continuously observed for 14 days.

[0033] 3. Determination of blood biochemical indexes of mice

[0034] On the 15th day after administration, the mice were fasted for 12 hours, blood was taken from the eye socket, placed at room temperature for 30 minutes, centrifuged at 3000 r / min for 15 minutes, and then the supernatant was carefully aspirated and stored at -20°C for later use. The contents of GPT / ALT, GOT / AST, CRE, AKP, Urea, TBIL, and ALB in the serum of the mice were measured using a biochemical factor kit.

[0035] 4. Dissection and determination of organ indexes

[0036] After blood collection, the mice were sacrificed by cervical dislocation, dissected, and the hearts, livers, spleens, lungs, kidneys, and stomachs of the mice were taken. Observe the color and morphology of each tissue for abnormalities, quickly wash with refrigerated PBS, remove excess tissues such as fat, weigh, and calculate the organ index of the mice. Organ index (%) = [wet weight of organ (g) / body weight (g)]×100%.

[0037] 5. Histopathological examination

[0038] Refer to the national standard of the People's Republic of China "GB / 15193.24-2014 Technical Requirements for Pathological Examination in Toxicological Evaluation of Food Safety" for tissue sectioning. Soak the taken animal tissues with 4% paraformaldehyde and fix for more than 24 hours at room temperature. After fixation, put the paraffin sections with a thickness of 4μm into xylene solution for dewaxing for 20 minutes, change to fresh xylene solution and continue dewaxing for 20 minutes. After taking out, dehydrate with gradient alcohol, dehydrate with absolute ethanol for 5 minutes, change to fresh absolute ethanol solution and continue dehydrating for 5 minutes, change to 75% alcohol and dehydrate for 5 minutes, and then rinse with distilled water. Stain the processed sections with hematoxylin staining solution for 3 - 5 minutes, rinse with distilled water, use differentiating solution for differentiation, then rinse with distilled water, finally use bluing solution for bluing, and rinse with running water. Then put the processed sections into 95% alcohol for dehydration for 5 minutes, 85% alcohol for dehydration for 5 minutes, and stain with eosin staining solution for 5 minutes. Finally, put the processed sections into absolute ethanol for dehydration for 5 minutes, repeat 3 times, then make them transparent with xylene solution for 5 minutes, repeat once, and finally mount with neutral gum and observe and analyze the sections with a microscope.

[0039] 6. Data processing method

[0040] All experimental data were statistically analyzed using IBM SPSS Statistics 25.0 statistical software, with expressed. One-way analysis of variance (ANOVN) was used for comparison between groups; the significant difference level was expressed as P<0.05, P<0.01, and the extremely significant difference level was expressed as P﹤0.001.

[0041] 7. Experimental results

[0042] 7.1 Observation of general state of mice

[0043] 14 days after administration, the experimental group of mice showed drowsy symptoms 1 / 2 hour after intragastric administration and gradually improved to normal 1 hour later.

[0044] During the observation period, no deaths occurred in the experimental group and the blank group of mice, as shown in Table 1. The breathing of both groups of mice was normal, the general condition of the mice was good, their diet and drinking water were basically normal, and their body weights increased normally. There was no significant difference in the body weights of the mice in the experimental group compared with those in the blank group (P > 0.05, Table 2). No obvious abnormal behavioral activities or spontaneous activities were observed, and no general manifestations of poisoning were observed.

[0045] Table 1 Mortality in the acute toxicity experiment of mice

[0046]

[0047]

[0048] Table 2 Changes in body weight in the acute toxicity experiment of mice

[0049]

[0050] 7.2 Determination of blood biochemical indexes of mice

[0051] Fourteen days after administration, there was no significant difference in the biochemical factors of GOT / AST, CRE, AKP, Urea, TBIL, and ALB between the experimental group and the blank group of mice (P > 0.05). There was no significant difference in the GPT / ALT biochemical factor between the experimental group and the blank group of mice of the same sex (P > 0.05).

[0052] Table 3 Changes in blood biochemical factors of mice

[0053]

[0054] 7.3 Anatomical and organ index determination

[0055] Fourteen days after administration, during the dissection process, it could be observed that the colors, appearances, and sizes of the organs of the mice were normal, and no phenomena of congestion, hemorrhage, or edema occurred. According to the results of the organ indexes (Table 4), there were no significant changes in the hearts, livers, spleens, and lungs of the experimental group compared with those of the blank group (P > 0.05). There was no significant difference in the kidney organ index between the experimental group and the blank group of mice of the same sex (P > 0.05). There was a significant difference in the stomach organ index between the male mice in the experimental group and the male mice in the blank group (P < 0.05).

[0056] Table 4 Changes in organ indexes of mice

[0057]

[0058] 7.4 Histopathological examination

[0059] The results of H&E staining of each tissue pathology are shown in Figure 1It can be seen that the epicardial structure of the heart tissue is clear; the myocardial fibers are evenly colored, the cell boundaries are obvious, the direction is consistent, the myocardial cell striations are clear, light and dark alternating, and there is no abnormality in the interstitium; no obvious inflammatory cell infiltration is observed.

[0060] In the liver tissue, the central vein is located in the center of the hepatic lobule, surrounded by radially distributed hepatocytes and sinusoids. Many hepatocytes show hydropic degeneration, are enlarged, and have loose and lightly stained cytoplasm; no obvious inflammatory cell infiltration is observed.

[0061] The spleen tissue consists of two parts: red pulp and white pulp. The white pulp includes the lymphoid sheath around the central artery, splenic nodules and marginal zone. There is no obvious change in the number and size of the two. The red pulp is mainly distributed in a large area under the capsule, around the trabeculae and outside the marginal zone of the white pulp. It is composed of splenic cords and splenic sinusoids, is evenly distributed, and is clearly demarcated from the white pulp. No obvious abnormalities were found.

[0062] The lung tissue is essentially composed of the numerous alveoli at the ends of the bronchial branches at all levels, with no obvious abnormalities in the structure of the bronchioles. The alveolar walls are mildly thickened in multiple foci. There are small areas of hemorrhage, and a small number of red blood cells can be seen in the bronchioles and alveoli. No obvious inflammatory cell infiltration is observed.

[0063] The glomeruli are evenly distributed on the cortex of the renal tissue, with uniform cell number and matrix. The renal tubular epithelial cells are round and plump, with neat and regular brush borders, and no obvious abnormalities in the medulla. The connective tissue between the urinary tubules is the renal interstitium, which does not show significant proliferation. No obvious inflammatory cell infiltration is observed.

[0064] Based on the above results, it can be seen that the mice's organs did not show any pathological signs.

[0065] Example 2 Study on the antitussive, expectorant and anti-inflammatory effects of a dark purple Fritillaria compatibility prescription

[0066] Traditional Chinese Medicine (TCM) often uses compatibility as a primary method of medication, with the use of medicinal materials and dosage being key considerations. The dosage of a TCM herb is closely linked to its efficacy, leading some to believe that "the secret of TCM lies in dosage." Too small a dose will not achieve the desired therapeutic effect, while too large a dose will not achieve the desired therapeutic effect and may even cause adverse effects. In this example, data mining techniques were used to determine the TCM formula to be: Dark Purple Fritillaria - Loquat Leaves - Poria - Seabuckthorn. After determining the formula, the dosage was investigated.

[0067] According to the literature, orthogonal design, direct experimental design, orthogonal and uniform joint design, and other methods are often used to explore the dosage of traditional Chinese medicine prescriptions. Orthogonal design utilizes orthogonal tables to comprehensively compare efficacy results and screen for the optimal dosage at each level. This method saves resources and time while ensuring the accuracy of the results. This method is consistent with the characteristics of drug interactions within traditional Chinese medicine prescriptions and plays an important role in the construction of animal models, the extraction of Chinese medicinal materials and compound prescriptions, and the determination of dosage combinations for traditional Chinese medicine prescriptions. This study used orthogonal experimental design to obtain nine dosage combinations and then explored the antitussive, expectorant, and anti-inflammatory efficacy of these nine combinations. Further analysis was performed using a comprehensive weighted score based on the efficacy results.

[0068] In this example, an orthogonal experimental design was used to determine the compatibility ratio of the four drugs. A mouse ammonia-induced cough test, a mouse tracheal phenol red excretion test, and a xylene-induced mouse ear swelling test were performed to analyze the cough latency, number of coughs, tracheal phenol red content, corrected phenol red content, swelling degree, and swelling inhibition rate for each compatibility ratio to determine the optimal ratio for the dark purple Fritillaria combination prescription for the treatment of acute tracheobronchitis.

[0069] 1. Orthogonal experimental design

[0070] Table 5 Equivalent dose ratios between humans and animals based on body surface area

[0071]

[0072] The dosage for mice (g / kg) = 0.0026 × the daily dosage for humans (g) ÷ the weight of the mouse (kg).

[0073] Referring to the maximum human-acceptable dose recorded in the Chinese Pharmacopoeia (2020 Edition, Part I), which includes 10g of Fritillaria thunbergii, 10g of loquat leaf, 15g of Poria cocos, and 10g of Hippophae rhamnoides, the mice can tolerate 1.3g of Fritillaria thunbergii, 1.3g of loquat leaf, 1.95g of Poria cocos, and 1.3g of Hippophae rhamnoides, according to Table 5. Because most doses in clinical prescriptions exceed those specified in the Chinese Pharmacopoeia, we multiplied these doses by 2 and 4 times to obtain a four-factor, three-level orthogonal experimental design for the pilot experiment. Through preliminary experiments, dark purple Fritillaria (2.6g / kg, 3.9g / kg, 5.2g / kg), loquat leaves (1.3g / kg, 1.95g / kg, 2.6g / kg), Poria (1.95g / kg, 2.925g / kg, 3.9g / kg) and sea buckthorn (2.6g / kg, 3.9g / kg, 5.2g / kg) were determined, and a four-factor three-level orthogonal experiment was established (g / kg: the weight of the original medicinal material received by mice per kilogram of body weight), as shown in Table 6.

[0074] Table 6 Level graphs of four Chinese medicinal materials

[0075]

[0076] Using IBM SPSS Statistics 25.0 statistical software, select Data in the toolbar, select Generate in Orthogonal Design, input the factor names, and define the factor levels to create a new data set. Output the orthogonal experimental design table for four factors and three levels, which are 1: A1B%C1D1, 2: A1B2C2D2, 3: A1B3C3D3, 4: A2B1C2D3, 5: A2B2C3D1, 6: A2B3C1D2, 7: A3B1C3D2, 8: A3B2C1D3, 9: A3B3C2D1, a total of nine matching groups, as shown in Table 7.

[0077] Table 7 Orthogonal experimental design table for matching

[0078]

[0079] Select Fritillaria unibracteata, Hippohgae rhamnoides, Poria cocos, and Eriobotrya japonica. According to the results of the orthogonal experimental design in Table 7, the total weight of each result is expanded ten times for decoction. First, add 12 times the amount of water and soak for 30 minutes, then decoct with strong fire until boiling, and continue to decoct with slow fire for 60 minutes. Pour out the first decoction; add 10 times the amount of water and continue to decoct, the same as the first time, pour out the second decoction; add 10 times the amount of water and continue to decoct, the same as the second time, pour out the third decoction. Combine the three decoctions and use a rotary evaporator to concentrate them under reduced pressure into an extract. The extract yield is shown in Table 8. Calculate the amount of crude drug required according to the body weight of each mouse being 20 g, and then calculate the content of the extract required for each mouse per day through the extract yield. Refer to Wei Wei's "Pharmacological Experiment Methodology, Fourth Edition" to determine the gavage volume of mice as 0.1 mL / 10 g, and finally obtain the extract concentration when gavage for the matching groups for drug administration.

[0080] Table 8 Decoction extract yield

[0081]

[0082] 2. Experimental methods

[0083] 2.1 Grouping of experimental animals

[0084] (1) Cough suppression experiment s

[0085] Select 120 male mice about 4 weeks old and weighing 18 g - 22 g, randomly divide them into groups of 10 each, and set up a blank group: gavage with normal saline but not induced to cough with ammonia water; a model group: gavage with normal saline and induced to cough with ammonia water; a positive group: gavage with pentoxyverine citrate solution and induced to cough with ammonia water; matching groups 1, 2, 3, 4, 5, 6, 7, 8, 9: gavage with the corresponding extract solution and induced to cough with ammonia water.

[0086] (2) Expectorant experiment

[0087] Select 120 male mice about 4 weeks old and weighing 18 g - 22 g, randomly divide them into groups of 10 each, and set up the following groups: blank group: intragastric administration with normal saline without injecting phenol red solution into the abdomen; model group: intragastric administration with normal saline and injecting phenol red solution into the abdomen; positive group: intragastric administration with ambroxol hydrochloride solution and injecting phenol red solution into the abdomen; compatibility groups 1, 2, 3, 4, 5, 6, 7, 8, 9: intragastric administration with the corresponding extract solution and injecting phenol red solution into the abdomen.

[0088] (3) Anti-inflammatory experiment

[0089] Select 120 male mice about 4 weeks old and weighing 18 g - 22 g, randomly divide them into groups of 10 each, and set up the following groups: blank group: intragastric administration with normal saline without applying xylene solution to the right ear; model group: intragastric administration with normal saline and applying xylene solution to the right ear; positive group: intragastric administration with dexamethasone acetate solution and applying xylene solution to the right ear; compatibility groups 1, 2, 3, 4, 5, 6, 7, 8, 9: intragastric administration with the corresponding extract solution and applying xylene solution to the right ear.

[0090] 2.2 Model establishment and detection

[0091] (1) Antitussive experiment

[0092] After randomly grouping the mice, after one week of adapting to the laboratory environment, the experiment begins. Determine the intragastric volume of the mice to be 0.1 mL / 10 g. The blank group and the model group are intragastrically administered with normal saline, the positive group is intragastrically administered with pentoxyverine citrate solution, and the compatibility groups are intragastrically administered with the corresponding extract solution. Intragastric administration is carried out for 7 days, and the body weight changes and status of the mice are observed. 12 h before the last administration, the mice are fasted but not deprived of water. 0.5 h after the last administration, in a sealed cylindrical container with an inner diameter of 15 cm, a height of 15 cm, and a volume of 2.5 L, place a petri dish with an inner diameter of 35 mm at the bottom. Use a pipette to suck 1 mL of 25% analytical pure ammonia water into the small petri dish, put the mice into the cylindrical container and expose them to the ammonia water. After the ammonia water volatilizes for 20 s, start timing, record the cough latency of the mice (from the start of timing to the first occurrence of cough) and the number of coughs of the mice in 2 min, and calculate the cough inhibition rate. Typical cough actions: abdominal muscle contraction or chest retraction, and at the same time opening the mouth wide with a cough sound.

[0093] Cough inhibition rate (%) = (average cough number of the control group - average cough number of the administration group) ÷ average cough number of the control group × 100%.

[0094] (2) Expectorant experiment

[0095] After randomly grouping the mice, they were allowed to acclimatize to the laboratory environment for one week before starting the experiment. The gavage volume for the mice was determined to be 0.1 mL / 10 g. The blank group and the model group were gavaged with normal saline, the positive group was gavaged with ambroxol hydrochloride solution, and the compatibility group was gavaged with the corresponding extract solution. Gavage was performed for 7 days, and the body weight of the mice was recorded, and the health status of the mice was observed. 12 h before the last administration, the mice were fasted but allowed to drink water. Half an hour after the last administration, 0.5% phenol red solution (0.1 mL / 10 g) was injected intraperitoneally. After waiting for 30 minutes, the mice were sacrificed by cervical dislocation. The neck was dissected with a scalpel, the tissues around the trachea were dissected, and the trachea from the subthyroid cartilage to the tracheal bifurcation was removed. Note that the length of the trachea taken from each mouse was kept consistent. It was placed in a test tube containing 3 mL of normal saline, and 0.2 mL of 1 mol / L NaOH solution was added to the test tube. After soaking for 4 h, it was centrifuged at 3000 rpm / min for 15 min, and the OD value of the supernatant after centrifugation at a wavelength of 546 nm was measured, and the phenol red excretion and expectorant index of the mice were calculated.

[0096] Phenol red excretion (μg / mL) = OD value × 16.348;

[0097] Corrected phenol red excretion = phenol red excretion (μg / mL) ÷ body weight of the mouse (kg).

[0098] (3) Anti-inflammatory experiment

[0099] After randomly grouping the mice, they were allowed to acclimatize to the laboratory environment for one week before starting the experiment. The gavage volume for the mice was determined to be 0.1 mL / 10 g. The blank group and the model group were gavaged with normal saline, the positive group was gavaged with dexamethasone acetate solution, and the compatibility group was gavaged with the corresponding extract solution. Gavage was performed for 7 days, and the body weight change and status of the mice were observed. 12 h before the last administration, the mice were fasted but allowed to drink water. 0.5 h after the last administration, 20 μL of xylene was aspirated with a pipette and evenly coated on the upper and lower surfaces of the right ear of each mouse, and the left ear was not coated as a control. 0.5 h after coating, blood was collected by eye enucleation, and the mice were sacrificed by cervical dislocation. The collected blood was placed in a 1.8 mL clean EP centrifuge tube, allowed to stand for 30 min, centrifuged at 3000 r / min for 15 min, and the serum was taken to detect inflammatory factors such as IL-6, IL-1β, and TNF-α using an enzyme-linked kit. The two ears were cut along the auricle baseline, and ear pieces were punched at symmetric positions on the left and right ears using an 8 mm diameter punch. Ear tissues of the same size were taken, and the ear mass of each mouse was weighed with a precision electronic balance, and the ear swelling rate of the mice was calculated.

[0100] Degree of ear swelling (mg) = weight of the left ear - weight of the right ear;

[0101] Ear swelling inhibition rate (%) = (degree of swelling in the model group - degree of swelling in the administration group) ÷ degree of swelling in the model group × 100%.

[0102] 2.3 Data Processing Method

[0103] All experimental data were statistically analyzed using IBM SPSS Statistics 25.0 statistical software; expressed as ( ), one-way ANOVA (ANOVN) was used for between-group comparison; the significant difference level was expressed as P < 0.05, P < 0.01, and the extremely significant difference level was expressed as P < 0.001.

[0104] 3. Experimental Results

[0105] 3.1 Antitussive Experiment - Ammonia-Induced Cough Experiment in Mice

[0106] The effects of Fritillaria unibracteata complex with different compatibility ratios on ammonia-induced cough in mice. From the statistical results of the experimental data in Table 9, it can be seen that compared with the model group, in terms of cough latency, it can be known that each administration group can prolong the cough latency. Except for compatibility groups 2, 3, and 6 without significant significance (P > 0.05), other administration groups have extremely significant significance (P < 0.001). In terms of the number of coughs in 2 minutes, it can be obtained that each administration group can significantly reduce the number of coughs and has extremely significant significance (P < 0.001).

[0107] Sorted according to the antitussive effect from excellent to poor, the cough latency is: Y > 8 > 9 > 7 > 1 > 5 > 4 > 2 > 6 > 3 > M; the number of coughs is: 8 < Y < 9 < 4 < 2 < 6 < 3 < 7 < 1 < 5 < M.

[0108] Table 9 Ammonia-Induced Cough in Mice

[0109]

[0110] Note: Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.00.

[0111] 3.2 Expectorant Experiment - Tracheal Phenol Red Excretion Experiment in Mice

[0112] The effects of the new Fritillaria unibracteata complex with different compatibility ratios on the tracheal phenol red excretion volume in mice. From the statistical results of the experimental data in Table 10, it can be seen that compared with the model group, each administration group can increase the tracheal phenol red excretion volume in mice. In terms of the phenol red excretion volume, except for compatibility group 1 and compatibility group 6 with significant significance (P < 0.05), compatibility group 5 has significant significance for the expectorant effect (P < 0.01), and other administration groups have extremely significant significance for the expectorant effect (P < 0.001). In terms of the corrected phenol red excretion volume, compatibility group 6 has significant significance (P < 0.05), compatibility group 5 has significant significance for the expectorant effect (P < 0.01), and other administration groups have extremely significant significance for the expectorant effect (P < 0.001).

[0113] According to the expectorant effect from excellent to poor for sorting, the tracheal phenol red content can be obtained as: Y > 9 > 8 > 2 > 7 > 4 > 3 > 5 > 6 > 1 > M > K; the corrected phenol red content: Y > 9 > 3 > 7 > 4 > 8 > 1 > 2 > 5 > 6 > M > K.

[0114] Table 10 Excretion of phenol red in the trachea of mice

[0115]

[0116] Note: Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0117] 3.3 Anti-inflammatory experiment - Xylene-induced ear swelling experiment in mice

[0118] The effects of different compatibility ratios of the new Fritillaria unibracteata complex on xylene-induced ear swelling in mice. From the statistical results of the experimental data in Table 11, it can be seen that compared with the model group, each orthogonal test group can significantly reduce the ear swelling degree of mice caused by xylene. There is no significant difference in compatibility groups 1, 4, and 6 (P > 0.05), and compatibility groups 2, 3, 5, 7, 8, and 9 have significant differences (P < 0.05, P < 0.01).

[0119] Since inflammation plays an important role in the pathogenesis of acute tracheobronchitis, and some studies have shown that drugs can protect against acute tracheobronchitis by reducing the content of IL-6 and TNF-α and activating the PKA-NF-κB pathway, relevant inflammatory factors were detected. Compared with the model group, each orthogonal test group can significantly reduce the content of inflammatory factors in mice caused by xylene. From the TNF-α data, there is no significant difference in compatibility groups 1, 2, 4, 6, and 7 (P > 0.05), and compatibility groups 3, 5, 8, and 9 have significant differences (P < 0.05, P < 0.01). From the IL-6 data, compatibility group 4 has a significant difference (P < 0.01), and compatibility groups 1, 2, 3, 5, 6, 7, 8, and 9 have extremely significant differences (P < 0.001). From the IL-1β data, there is no significant difference in compatibility group 4 (P > 0.05), compatibility groups 3 and 5 have significant differences (P < 0.05, P < 0.01), and compatibility groups 1, 2, 6, 7, 8, and 9 have extremely significant differences (P < 0.001).

[0120] Sorting the anti-inflammatory effects from best to worst yields the following results: swelling degree: K<Y<8<9<2<3<7<5<4<1<6<M; swelling inhibition rate: K>Y>8>9>2>3>7>5>4>1>6>M. Sorting the levels of related anti-inflammatory factors from low to high yields: IL-6: 8<Y<9<7<K<6<2<1<5<3<4<M; IL-1β: 8<7<9<2<1<Y<6<3<5<K<4<M; and TNF-α: K<Y<8<9<3<5<2<4<7<1<6<M.

[0121] Table 11 Xylene-induced ear swelling in mice

[0122]

[0123] Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0124] Based on the above results, the effects of combination groups 8 and 9 were the best. Among them, dark purple Fritillaria cleared away heat and resolved phlegm and was the main drug, loquat leaves cleared the lungs and relieved cough, and relieved adverse reactions and stopped vomiting and was the minister drug, Poria promoted diuresis and eliminated dampness and was the adjuvant drug, and sea buckthorn relieved cough and eliminated phlegm and was the guiding drug.

[0125] 3.4 Comprehensive Weighted Score

[0126] A comprehensive weighted scoring method was used to evaluate the antitussive, expectorant, and anti-inflammatory effects of the new dark purple Fritillaria compound in different compatibility ratios. According to the guidance of clinical doctors, the weight coefficients of the three efficacy indicators of antitussive, expectorant, and anti-inflammatory in acute tracheobronchitis were evenly distributed (cough incubation period, cough frequency, tracheal phenol red content, corrected phenol red content, tracheal swelling degree, and swelling inhibition rate each accounted for 1 / 6). Therefore, the comprehensive scoring formula is as follows:

[0127] OD=(A i / A max ±B min / B i ±C i / C max ±D i / Dmax ±E min / E i ±F i / F max )×1 / 6;

[0128] OD is the comprehensive score, A i is the cough incubation period of each group, A max is the maximum value of cough latency, B i is the number of coughs in each group, B min is the minimum number of coughs, C i is the phenol red content in the trachea of each group, Cmax is the maximum value of tracheal phenol red content, D i is the corrected phenol red content of each group, D max is the maximum value of corrected phenol red content, E i is the swelling degree of each group, E min is the minimum value of swelling degree, F i is the swelling inhibition rate of each group, F max is the maximum value of swelling degree. The intuitive analysis table of the comprehensive score is shown in Table 12.

[0129] According to the intuitive analysis table, the order of the factors affecting the comprehensive score is: A > B > D > C, and the optimal pharmacodynamic compatibility ratio is A3B2D3C2, that is, Fritillaria unibracteata 5.2 g / kg; Eriobotrya japonica 1.95 g / kg; Hippohgae rhamnoides 5.2 g / kg; Poria cocos 2.925 g / kg. According to Table 5, converting to the drug dosage applicable to humans, it is Fritillaria unibracteata 40 g; Eriobotrya japonica 15 g; Hippohgae rhamnoides 40 g; Poria cocos 22.5 g.

[0130] Table 12 Intuitive Analysis Table of Comprehensive Score

[0131]

[0132] Conclusion: The toxicity of the compatibility formula of Fritillaria unibracteata was studied by the acute toxicity experiment of mice. According to the relevant in vitro and in vivo indicators, it can be known that this formula has no toxicity. Then, disease models were established by the ammonia water-induced cough, tracheal phenol red excretion, and xylene-induced ear swelling methods. Antitussive, expectorant, and anti-inflammatory pharmacodynamic experiments were carried out on the nine compatibility groups designed by the orthogonal test, and the best compatibility ratio was obtained as Fritillaria unibracteata 40 g, Eriobotrya japonica 15 g, Hippohgae rhamnoides 40 g, and Poria cocos 22.5 g. Fritillaria unibracteata clears heat and resolves phlegm and is the monarch drug; Eriobotrya japonica clears the lungs and relieves cough, and reduces adverse qi and stops vomiting, and is the minister drug; Poria cocos promotes diuresis and percolates dampness and is the assistant drug; Hippohgae rhamnoides relieves cough and reduces phlegm and is the envoy drug.

[0133] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A traditional Chinese medicine composition for relieving cough, reducing phlegm and / or anti-inflammatory, characterized in that, It includes 40 g of Fritillaria unibracteata, 10 - 20 g of Eriobotrya japonica leaves, 20 - 40 g of Hippohgae rhamnoides and 15 - 30 g of Poria cocos.

2. The traditional Chinese medicine composition according to claim 1, wherein It includes 40 g of Fritillaria unibracteata, 15 g of Eriobotrya japonica leaves, 40 g of Hippohgae rhamnoides and 22.5 g of Poria cocos.

3. Use of the traditional Chinese medicine composition according to claim 1 in the preparation of a drug for relieving cough.

4. Use of the traditional Chinese medicine composition according to claim 1 in the preparation of a drug for reducing phlegm.

5. Use of the traditional Chinese medicine composition according to claim 1 in the preparation of a drug for anti - inflammation.

6. Use of the traditional Chinese medicine composition according to claim 1 in the preparation of a drug for treating acute tracheobronchitis.

7. A drug for relieving cough, reducing phlegm and / or having anti-inflammatory effects, characterized in that, It includes the traditional Chinese medicine composition according to claim 1.

8. A drug for treating acute tracheobronchitis, characterized in that, It includes the traditional Chinese medicine composition according to claim 1.

9. The medicament according to claim 7 or 8, characterized in that, It further includes pharmaceutically acceptable excipients.

10. The medicament according to claim 7 or 8, characterized in that, The administration mode of the drug includes oral administration or parenteral administration.

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