A traditional Chinese medicine composition for resisting bacterial pneumonia and its application

Abstract

The application discloses a traditional Chinese medicine composition for resisting bacterial pneumonia and application thereof. The traditional Chinese medicine composition is composed of a platycodon grandiflorum extract, a leaf of loquat extract, a polygonatum odoratum extract, a mulberry leaf extract, a lily extract, a saussurea involucrata extract, a dendrobium officinale flower extract, a nenufar extract, a momordica grosvenori extract, a noni fruit powder and an acai berry powder. In addition, the application further provides a traditional Chinese medicine liquid preparation for resisting bacterial pneumonia. Gamma-cyclodextrin is used as a taste masking agent for the first time, which is used for covering the bitter and astringent tastes caused by the platycodon grandiflorum and the leaf of loquat. Meanwhile, the gamma-cyclodextrin has the functions of solubilization and suspension, and is beneficial to improving the bioavailability of platycodin. Experiments prove that the traditional Chinese medicine composition has the remarkable effect of resisting bacterial pneumonia and is safe and free of side effects. The application provides a new technical means for treating bacterial pneumonia.

Description

Technical Field

[0001] The present invention relates to a traditional Chinese medicine composition for resisting bacterial pneumonia and application thereof, and belongs to the field of biotechnology. Background Art

[0002] Bacterial pneumonia refers specifically to pneumonia caused by bacteria (usually Streptococcus pneumoniae or Staphylococcus aureus), and does not include pneumonia caused by pathogenic microorganisms such as mycoplasmas and chlamydia. Bacterial pneumonia accounts for 80% of all pathogen-related pneumonia in adults and is considered classic pneumonia, also known as typical pneumonia.

[0003] Traditional Chinese Medicine believes that bacterial pneumonia is often caused by exogenous wind-heat and phlegm-heat. The lungs control respiration and regulate the waterways. They are the upper source of water and are known to be purifying. Descending is considered normal, while ascending is considered adverse. When invaded by external pathogens, the lungs lose their normal descending function and their clearing and purifying functions are ineffective. Consequently, lung qi fails to descend and becomes adverse. Water is unable to be transported and transformed, stagnating in the lung meridians and forming phlegm. Alternatively, warm and hot pathogens can directly burn the lung fluid, refining it into phlegm. Phlegm and heat then intersect and block the airways, causing lung qi to become blocked. With lung qi blocked, heart blood circulation is impaired, the liver loses its ability to disperse and regulate, and the heart loses its nourishment, resulting in insufficient heart qi. Phlegm-heat becomes intense and persists. Consequently, heat and toxicity transform into fire, and the phlegm and fire fan into conflict, invading the heart and liver.

[0004] Since the advent of the antibiotic era, the prognosis of bacterial pneumonia has improved significantly, but the mortality rate has remained high since the 1960s. Currently, bacterial pneumonia has some new characteristics, including changes in the pathogen spectrum, especially a significant increase in the incidence of Gram-negative bacteria in hospitals. Although Streptococcus pneumoniae still dominates the pathogens of community-acquired pneumonia, its clinical manifestations are becoming more atypical. The rate of bacterial resistance has increased, and so-called "refractory" pneumonia is common, especially in children, the elderly, and immunosuppressed patients, with extremely high mortality rates. Improving etiological diagnosis, rationally using antibiotics, avoiding the emergence of drug-resistant bacteria, and improving supportive care are issues that urgently need to be emphasized and addressed in the clinical management of pneumonia. Summary of the Invention

[0005] In view of the problems existing in the current treatment of bacterial pneumonia, the purpose of the present invention is to provide a traditional Chinese medicine composition with anti-bacterial pneumonia efficacy and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] The invention discloses a traditional Chinese medicine composition for resisting bacterial pneumonia, which comprises platycodon grandiflorum extract, loquat leaf extract, polygonatum odoratum extract, mulberry leaf extract, lily extract, snow lotus extract, dendrobium officinale flower extract, grass jelly extract, momordica grosvenori extract, noni fruit powder and acai fruit powder.

[0008] Among them, preferably, the traditional Chinese medicine composition is composed of the following raw materials in the following parts by weight: 400-600 parts of platycodon grandiflorum extract, 400-600 parts of loquat leaf extract, 200-400 parts of polygonatum extract, 200-400 parts of mulberry leaf extract, 200-400 parts of lily extract, 80-120 parts of snow lotus extract, 80-120 parts of dendrobium officinale flower extract, 80-120 parts of grass jelly extract, 80-120 parts of momordica grosvenori extract, 80-120 parts of noni fruit powder and 80-120 parts of acai fruit powder.

[0009] Among them, preferably, the traditional Chinese medicine composition is composed of the following raw materials in the following parts by weight: 500 parts of platycodon grandiflorum extract, 500 parts of loquat leaf extract, 300 parts of polygonatum extract, 300 parts of mulberry leaf extract, 300 parts of lily extract, 100 parts of snow lotus extract, 100 parts of dendrobium officinale flower extract, 100 parts of grass jelly extract, 100 parts of momordica grosvenori extract, 100 parts of noni fruit powder and 100 parts of acai fruit powder.

[0010] Wherein, preferably, the snow lotus extract is prepared by the following method:

[0011] 1) Take dried snow lotus, crush it, add 10 times its weight of deionized water, heat and extract at 80°C for 25 minutes, filter, and retain the filtrate;

[0012] 2) Add 10 times the weight of deionized water to the filter residue, heat and extract at 80°C for 25 minutes, filter, and retain the filtrate;

[0013] 3) Repeat step 2) once;

[0014] 4) Combine the filtrates and set aside;

[0015] The Dendrobium officinale flower extract is prepared by the following method:

[0016] 1) taking dried Dendrobium officinale flowers, crushing them, adding 8 times the weight of deionized water, heating and extracting at 95° C. for 35 minutes, filtering, and retaining the filtrate;

[0017] 2) Add 8 times the weight of deionized water to the filter residue, heat and extract at 95°C for 35 minutes, filter, and retain the filtrate;

[0018] 3) Repeat step 2) once;

[0019] 4) Combine the filtrates and set aside;

[0020] The loquat leaf extract is prepared by the following method:

[0021] Take dried loquat leaves, crush them, and extract them twice with 95% w / w ethanol at a solid-liquid ratio of 1:10 g / ml under reflux, each time for 30 minutes, filter, and spray-dry the combined filtrates for later use;

[0022] The Platycodon grandiflorum extract is prepared by the following method:

[0023] Wash and dry fresh Platycodon grandiflorum, grind it in a grinder, and extract it twice with 75% ethanol w / w at a solid-liquid ratio of 1:9 g / ml under reflux, each time for 60 minutes, filter, and spray-dry the combined filtrates for later use;

[0024] The polygonatum extract, mulberry leaf extract, lily extract, grass jelly extract, monk fruit extract, noni fruit powder and acai fruit powder are all purchased from the market.

[0025] Furthermore, the present invention also proposes the use of the traditional Chinese medicine composition in the preparation of medicines for treating bacterial pneumonia.

[0026] Preferably, the medicine is a liquid preparation.

[0027] Furthermore, the present invention also provides a liquid Chinese medicine preparation for resisting bacterial pneumonia, wherein the liquid Chinese medicine preparation is prepared from the Chinese medicine composition according to the following method:

[0028] (1) all the extracts were prepared into a suspension with deionized water, and the suspension was mixed with the snow lotus extract and the Dendrobium officinale flower extract to obtain a raw material mixture;

[0029] (2) Debittering, flavor correction, and solubilization

[0030] Adding γ-cyclodextrin to the raw material mixed solution for flavor correction;

[0031] (3) Allocation

[0032] Add citric acid, potassium sorbate and sweetener for blending, and add deionized water in a quantitative amount to obtain a liquid preparation with good palatability and unique flavor;

[0033] (4) Filling and sterilization

[0034] The product is filled under aseptic conditions to obtain the finished product, which is then sterilized at 110°C for 20 minutes and cooled.

[0035] Preferably, the amount of γ-cyclodextrin used is 400-600 parts by weight, the amount of citric acid used is 20-30 parts by weight, the amount of sucralose used is 1-3 parts by weight, and the amount of potassium sorbate used is 5-15 parts by weight.

[0036] Preferably, the amount of gamma-cyclodextrin used is 500 parts by weight, the amount of citric acid used is 25 parts by weight, the amount of sucralose used is 2 parts by weight, and the amount of potassium sorbate used is 10 parts by weight.

[0037] Preferably, in 30 ml / bottle of the finished product, there are 500 mg of platycodon grandiflorum extract, 500 mg of loquat leaf extract, 300 mg of polygonatum odoratum extract, 300 mg of mulberry leaf extract, 300 mg of lily extract, 100 mg of saussurea involucrata extract, 100 mg of dendrobium officinale flower extract, 100 mg of mesona chinensis extract, 100 mg of momordica grosvenori extract, 100 mg of acai berry powder, 100 mg of gamma-cyclodextrin, 25 mg of citric acid, 2 mg of sucralose, and 10 mg of potassium sorbate.

[0038] Compared with the prior art, the present application has the following advantages:

[0039] 1. The oral liquid preparation using traditional Chinese medicinal materials as raw materials has rapid effect and high bioavailability, and is particularly suitable for children, the elderly, or people who have difficulty swallowing solid preparations. However, due to the inherent bad taste of traditional Chinese medicinal materials, such as bitterness, astringency, and pungency, people who take the traditional Chinese medicinal oral liquid preparation are not easy to accept, especially the bitterness, which is the most difficult to accept, and the compliance is extremely poor. At the same time, due to the dispersity of traditional Chinese medicinal oral liquid preparations (such as oral liquids and granules) being much greater than that of solid preparations, the dissolution, dissolution, and diffusion are completed in an instant, and once the taste components enter the oral cavity, they can be easily identified, thereby producing an unbearable taste, especially the bitter traditional Chinese medicinal components, resulting in poor compliance of the population. In order to improve the bitter taste, peculiar smell, and other bad tastes of traditional Chinese medicinal oral liquid preparations, and improve the compliance and long-term adherence of the population, common methods include adding sugar, adding aromatic substances, reducing the amount of active ingredients, and changing the dosage form, but these methods often compromise between good taste and health, and cannot synergistically improve the two. Cyclodextrin is a molecule with special properties, which can not only improve the bioavailability of raw materials, but also mask the unpleasant taste and smell of products without changing the nutritional value of the products. For example, beta-cyclodextrin, which is often used in the food and drug industries, has good taste-masking effect on many bitter traditional Chinese medicines, has low production cost, and is widely used in industry, but has low drug loading capacity, and has requirements for the structure and size of the drug molecules, and is difficult to complex with drugs with strong hydrophilicity or complex components. The present application finds that beta-cyclodextrin has poor bitter taste-masking effect on traditional Chinese medicinal oral liquid preparations containing loquat leaf and platycodon grandiflorum active ingredients. The present application first uses gamma-cyclodextrin as a taste-masking agent to mask the bitter and astringent taste caused by platycodon grandiflorum and loquat leaf. At the same time, while masking the bitter taste, it also has the effects of solubilization and suspension, making the active ingredients with poor solubility and easy to precipitate more easily mixed with the liquid, reducing the generation of precipitates. Further research finds that gamma-cyclodextrin is beneficial to improving the bioavailability of platycodon grandiflorum saponins.

[0040] 2. The present invention proposes a traditional Chinese medicine composition for treating bacterial pneumonia. The traditional Chinese medicine composition is composed of platycodon grandiflorum extract, loquat leaf extract, polygonatum odoratum extract, mulberry leaf extract, lily extract, snow lotus extract, dendrobium officinale flower extract, grass jelly extract, momordica grosvenori extract, noni fruit powder and acai fruit powder. Experiments have shown that the traditional Chinese medicine composition has significant anti-bacterial pneumonia efficacy and is safe and has no side effects. The present invention provides a new technical means for the treatment of anti-bacterial pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a typical graph of the number of zebrafish neutrophils after sample treatment;

[0042] Note: The dotted box is the zebrafish swim bladder, the analysis site, and the green fluorescent particles are neutrophils;

[0043] Figure 2 is the number of zebrafish neutrophils after sample treatment;

[0044] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001;

[0045] Figure 3 为tgfβ-1基因相对表达量;

[0046] Compared with the model control group, **p<0.01, ***p<0.001;

[0047] Figure 4 为斑马鱼鱼鳔组织结构示意图;

[0048] Note: The gray dotted box is the zebrafish swim bladder;

[0049] Figure 5 This is a typical diagram of the zebrafish swim bladder tissue structure after sample processing;

[0050] 注:黑色箭头指向炎性细胞。 DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0052] Example 1 Preparation of a Chinese medicine composition for antibacterial pneumonia

[0053] The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 500 parts of platycodon grandiflorum extract, 500 parts of loquat leaf extract, 300 parts of polygonatum odoratum extract, 300 parts of mulberry leaf extract, 300 parts of lily extract, 100 parts of snow lotus extract, 100 parts of dendrobium officinale flower extract, 100 parts of grass jelly extract, 100 parts of momordica grosvenori extract, 100 parts of noni fruit powder and 100 parts of acai fruit powder.

[0054] Wherein, the snow lotus extract is prepared by the following method:

[0055] 1) Take dried snow lotus, crush it, add 10 times its weight of deionized water, heat and extract at 80°C for 25 minutes, filter, and retain the filtrate;

[0056] 2) Add 10 times the weight of deionized water to the filter residue, heat and extract at 80°C for 25 minutes, filter, and retain the filtrate;

[0057] 3) Repeat step 2) once;

[0058] 4) Combine the filtrates and set aside;

[0059] Wherein, the Dendrobium officinale flower extract is prepared by the following method:

[0060] 1) taking dried Dendrobium officinale flowers, crushing them, adding 8 times the weight of deionized water, heating and extracting at 95° C. for 35 minutes, filtering, and retaining the filtrate;

[0061] 2) Add 8 times the weight of deionized water to the filter residue, heat and extract at 95°C for 35 minutes, filter, and retain the filtrate;

[0062] 3) Repeat step 2) once;

[0063] 4) Combine the filtrates and set aside;

[0064] Wherein, the loquat leaf extract is prepared by the following method:

[0065] Take dried loquat leaves, crush them, and extract them twice with 95% w / w ethanol at a solid-liquid ratio of 1:10 g / ml under reflux, each time for 30 minutes, filter, and spray-dry the combined filtrates for later use;

[0066] Wherein, the Platycodon grandiflorum extract is prepared by the following method:

[0067] Wash and dry fresh Platycodon grandiflorum, grind it in a grinder, and extract it twice with 75% ethanol w / w at a solid-liquid ratio of 1:9 g / ml under reflux, each time for 60 minutes, filter, and spray-dry the combined filtrates for later use;

[0068] Among them, the polygonatum extract (batch number 230710), mulberry leaf extract (batch number 230710), lily extract (batch number 230711), grass jelly extract (batch number 230710), monk fruit extract (batch number 230710), noni fruit powder (batch number 230710) and acai fruit powder (batch number 230711) were all purchased from Xi'an Sanjiang Bioengineering Co., Ltd.

[0069] Example 2 Preparation of a Chinese medicine composition for antibacterial pneumonia

[0070] The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 400 parts of platycodon grandiflorum extract, 600 parts of loquat leaf extract, 200 parts of polygonatum odoratum extract, 400 parts of mulberry leaf extract, 200 parts of lily extract, 80 parts of snow lotus extract, 120 parts of dendrobium officinale flower extract, 120 parts of grass jelly extract, 80 parts of momordica grosvenori extract, 80 parts of noni fruit powder and 120 parts of acai fruit powder.

[0071] The preparation and purchase sources of the above raw materials are the same as in Example 1.

[0072] Example 3 Preparation of a Chinese medicine composition for antibacterial pneumonia

[0073] The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 600 parts of platycodon grandiflorum extract, 400 parts of loquat leaf extract, 400 parts of polygonatum odoratum extract, 200 parts of mulberry leaf extract, 400 parts of lily extract, 120 parts of snow lotus extract, 80 parts of dendrobium officinale flower extract, 80 parts of grass jelly extract, 120 parts of momordica grosvenori extract, 120 parts of noni fruit powder and 80 parts of acai fruit powder.

[0074] The preparation and purchase sources of the above raw materials are the same as in Example 1.

[0075] Example 4 Preparation of a Chinese herbal liquid preparation (Fei Rui Qing botanical beverage) for use in combating bacterial pneumonia I. Formulation

[0076] Brown glass bottle, gold cap, 30mL / bottle. The formula is shown in Table 1:

[0077] Table 1

[0078] raw material mg / bottle Platycodon grandiflorum extract 500 loquat leaf extract 500 Polygonatum odoratum extract 300 mulberry leaf extract 300 Lily Extract 300 Snow lotus culture 100 Dendrobium officinale 100 Grass Jelly Extract 100 Monk Fruit Extract 100 Noni fruit powder 100 Acai powder 100 γ-cyclodextrin 500 citric acid 25 Sucralose 2 Potassium sorbate 10

[0079] 二、制备方法

[0080] 1、提取

[0081] The preparation and purchase sources of the raw materials in Table 1 are the same as those in Example 1.

[0082] 2、混料

[0083] All the extracts are prepared into a suspension with deionized water, and the suspension is mixed with the snow lotus extract and the dendrobium officinale flower extract to obtain a raw material mixed solution.

[0084] 3、脱苦矫味、增溶

[0085] A certain proportion of γ-cyclodextrin is added to the raw material mixed solution to correct the taste, thereby changing the bad taste of the product to the greatest extent, so that the product retains the unique taste and smell of Chinese herbal medicine without any peculiar smell, and increases the solubility of the extract.

[0086] 4、调配

[0087] Add appropriate amounts of citric acid, potassium sorbate and sweetener for blending, and add a quantitative amount of water to obtain a liquid preparation with good palatability and unique flavor.

[0088] 5、灌装、灭菌

[0089] The product is filled under aseptic conditions, sterilized at 110°C for 20 minutes, and cooled.

[0090] 6、包装

[0091] 将成品装盒、装大箱。

[0092] This plant beverage has the unique fragrance of traditional Chinese medicine, no peculiar smell, a refreshing taste, and a sweet and sour taste. All quality indicators meet the requirements of relevant national standards and are in line with people's pursuit of health.

[0093] Example 5: Efficacy of a Chinese herbal liquid preparation (Fei Rui Qing botanical beverage) against bacterial pneumonia

[0094] 1.检测材料

[0095] 1.1.样品配制信息

[0096] The liquid Chinese medicine preparation for resisting bacterial pneumonia prepared in Example 4 (named Feiruiqing botanical beverage or "Feiruiqing" for short) was directly absorbed as a stock solution.

[0097] Positive control: dexamethasone acetate, white powder, batch number B1828095, Shanghai Aladdin Biochemical Technology Co., Ltd., solvent is DMSO.

[0098] 1.2.实验动物

[0099] Zebrafish were reared in aquaculture water at 28°C (water quality: 200 mg of instant sea salt was added to each liter of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3). They were bred and provided by our company's fish farming center. The experimental animal use license number is: SYXK(Zhejiang)2022-0004. The breeding and management of zebrafish complies with the requirements of the international AAALAC accreditation (certification number: 001458) and the IACUC ethics review number is: IACUC-2023-7487-01.

[0100] 1.3.仪器、耗材与试剂

[0101] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); motorized focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan); precision electronic balance (CP214, OHAUS, America); microinjector (IM-300, Narishige, Japan); needle puller (PC-10, Narishige, Japan); fully automatic sample rapid grinding instrument (JXFSTPRP-24L, Shanghai Jingxin Laboratory Equipment Technology Department, China); fully automatic nucleic acid extraction instrument (Auto-Pure32A, Hangzhou Aosheng Instrument Co., Ltd., China); conventional PCR amplifier (T100, BIO-RAD, Singapore); fluorescence quantitative PCR instrument (CFX Connect, BIO-RAD, Singapore); high-speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany); UV-visible spectrophotometer (Nanodrop2000, Thermo, USA); microplate mini centrifuge (BE-6100, Haimen Qilin Bell Instrument Manufacturing Co., Ltd., China); optical adhesive seal B (MSB1001, Bio-rad, USA); low-skirt 96-well plate (transparent) (HSP9601, Bio-rad, USA); microtome (KD2258, Jinhua Kedi Medical Equipment Co., Ltd., China); electronic constant temperature stainless steel water bath (HHS-2S, Shanghai Kanglu Instrument Equipment Co., Ltd., China); intelligent electric hot plate (400X280, Tianjin Leyue Nager Laboratory Instrument Sales Co., Ltd., China); 6-well plate (Zhejiang Belanbo Biotechnology Co., Ltd., China).

[0102] Lipopolysaccharide (LPS, batch number 0000108436, SIGMA, Israel); dimethyl sulfoxide (DMSO, batch number I2229063, Shanghai Aladdin Biochemical Technology Co., Ltd., China); sodium chloride injection (batch number S21091609, Hunan Kelun Pharmaceutical Co., Ltd., China); anhydrous ethanol (batch number 20230329, Sinopharm Chemical Reagent Co., Ltd., China); 4% tissue cell fixative (batch number 20230401, Beijing Solebold Technology Co., Ltd., China); xylene (batch number 20230424, Sinopharm Chemical Reagent Co., Ltd., China); PBS phosphate buffered saline (PBS). Buffer (catalog number BL601A, Biosharp, China); Mayer's hematoxylin staining solution (lot number 20220120, Shanghai Yihe Biotechnology Co., Ltd., China); Eosin staining solution (lot number 20220120, Shanghai Yihe Biotechnology Co., Ltd., China); neutral gum (lot number 330A021, Solarbio, China); high-efficiency section paraffin (melting point 54-56°C, lot number 20230322, Shanghai Huayong Paraffin Co., Ltd., China); high-efficiency section paraffin (melting point 62-64°C, lot number 20221116, Shanghai Huayong Paraffin Co., Ltd., China); ChamQ Universal SYBR qPCR Master Mix (Lot No. 027E2201CD, Vazyme, China); FastKing cDNA First-Strand Synthesis Kit (Genomic Depleted) (Lot No. X1213, Tiangen Biochemical Technology (Beijing) Co., Ltd., China); Universal RNA Extraction TL Kit C (Cat. No. TL2204001643C, Foshan Aowei Biotechnology Co., Ltd., China).

[0103] 2.检测方法

[0104] 2.1.最大检测浓度(MTC)测定

[0105] Transgenic neutrophil-producing green fluorescent zebrafish (MPX) at 5 days post-fertilization (dpf) were randomly selected and plated in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (concentrations shown in Table 2) were administered in water. A normal control group and a model control group were also established, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups received LPS intra-swim injection to establish a zebrafish bacterial pneumonia model. After treatment at 28°C for 4 hours, the mean temperature (MTC) of the sample in the model zebrafish was determined.

[0106] 2.2. Evaluation of efficacy against bacterial pneumonia (neutrophils)

[0107] Transgenic neutrophil-producing green fluorescent zebrafish (MPX) at 5 days post-fertilization (dpf) were randomly selected and plated in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water (concentrations shown in Table 3), along with a positive control of 100 μM dexamethasone acetate. A normal control group and a model control group were also established, with the volume per well being 3 mL. Except for the normal control group, all other experimental groups received LPS via swim bladder injection to establish a zebrafish bacterial pneumonia model. After 4 hours of treatment at 28°C, 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. Neutrophil counts in the swim bladder were analyzed, and the anti-bacterial pneumonia efficacy of the samples was evaluated using this indicator. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. P < 0.05 indicated statistical significance.

[0108] 2.3.抗细菌性肺炎功效评价(基因)

[0109] Wild-type AB zebrafish (5 dpf) were randomly selected and plated in a 6-well plate. Thirty zebrafish were treated in each well (experimental group). Water-soluble samples (concentrations shown in Table 3) and a positive control, dexamethasone acetate at a concentration of 100 μM, were administered. A normal control group and a model control group were also established. Each well contained 3 mL of culture medium. Three parallel experiments were performed. Except for the normal control group, all other experimental groups received LPS intra-swim injection to establish a zebrafish bacterial pneumonia model. After treatment at 28°C for 2 h, total RNA was extracted from each zebrafish using the Universal RNA Extraction TL Kit C. Total RNA concentration and purity were determined using a UV-visible spectrophotometer. 2.00 μg of total RNA from each zebrafish sample was synthesized into 20.0 μL of cDNA according to the instructions of the cDNA First-Strand Synthesis Kit. β-actin and tgfβ-1 gene expression was analyzed by q-PCR. β-actin was used as an internal control for gene expression, and the relative RNA expression of the tgfβ-1 gene was calculated. Statistical analysis was performed using SPSS 26.0 software. P < 0.05 indicated a statistically significant difference.

[0110] 2.4.抗细菌性肺炎功效评价(病理切片)

[0111] 5dpf wild-type AB strain zebrafish were randomly selected and placed in a 6-well plate. Thirty zebrafish were treated in each well (experimental group). Figure 1), a positive control of dexamethasone acetate at a concentration of 100 μM. A normal control group and a model control group were also established, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups received LPS injected into the swim bladder to establish a zebrafish bacterial pneumonia model. After treatment at 28°C for 4 hours, the zebrafish were fixed with 4% tissue cell fixative and subjected to a series of dehydration-embedding-sectioning-staining steps for histopathological analysis using H&E staining. Histopathological analysis of the swim bladder was used to evaluate the effects of the samples on the zebrafish swim bladder tissue structure.

[0112] 3.检测结果

[0113] 3.1.MTC测定

[0114] Under the experimental conditions, the MTC for Feiruiqing's antibacterial pneumonia efficacy was 50.0 μL / mL. See Table 2 for details.

[0115] Table 2 Results of the experimental study on the concentration of antibacterial pneumonia efficacy of samples (n=30)

[0116]

[0117] 3.2. Evaluation of efficacy against bacterial pneumonia (neutrophils)

[0118] Under the experimental conditions, Feiruiqing has the effect of resisting bacterial pneumonia, which is specifically manifested by reducing the number of neutrophils. See Table 3 for details. Figure 1 and Figure 2 .

[0119] Table 3 Results of the experimental evaluation of the efficacy of samples against bacterial pneumonia (neutrophils) (n=10)

[0120]

[0121] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0122] 3.3RNA提取结果及引物序列信息

[0123] At the end of the experiment, total RNA was extracted from zebrafish. RNA concentration and A260 / A280 ratio were measured using a UV-visible spectrophotometer (Table 4). The A260 / A280 ratios were between 1.8 and 2.2, indicating that the extracted zebrafish total RNA was of good quality and suitable for subsequent q-PCR experiments. Primer sequences are shown in Table 5.

[0124] 表4总RNA的浓度及A260 / A280比值(n=3)

[0125]

[0126]

[0127] 表5引物序列信息

[0128]

[0129] 3.4.抗细菌性肺炎功效评价(基因)

[0130] LPS is the main component of the outer membrane of Gram-negative bacteria and has been identified as a key risk factor for the pathogenesis of acute lung injury. Its use in animal models to induce acute lung injury has been widely reported. When LPS induces acute lung injury, inflammatory cells such as neutrophils are recruited and activated. After activation, inflammatory cells infiltrate the lung tissue and release inflammatory mediators. Transforming growth factor tgfβ-1 attracts monocytes and fibroblasts, further inducing monocytes to release different growth factors and inflammatory mediators, amplifying inflammation and inhibiting the repair of damaged tissues, inducing the differentiation of Treg and Th17 cells, and enhancing CD4 + The inflammatory function of T cells plays an important role in regulating the process of inflammation.

[0131] Under the experimental conditions, Feiruiqing can downregulate the relative expression of tgfβ-1 gene. Figure 3 .

[0132] Table 6 Sample anti-bacterial pneumonia efficacy evaluation test results (gene) (n=3)

[0133]

[0134]

[0135] 与模型对照组比较,**p<0.01,***p<0.001

[0136] 3.5.抗细菌性肺炎功效评价(病理切片)

[0137] The swim bladder tissue of zebrafish in the normal control group showed regular structure, intact cell morphology, and uniform cytoplasm. Obvious inflammatory infiltrates were observed in the swim bladders of zebrafish in the model control group, indicating successful model establishment. The inflammatory infiltrates were reduced in the positive control group, dexamethasone acetate, indicating that dexamethasone acetate has an ameliorative effect on LPS-induced bacterial pneumonia.

[0138] The swim bladder tissue of the fish in the Feiruiqing 12.5μL / mL concentration group was similar to that of the model control group, while the inflammatory infiltration of the swim bladder tissue of the fish in the 25.0μL / mL and 50.0μL / mL concentration groups was reduced, suggesting that Feiruiqing has the effect of resisting bacterial pneumonia ( Figure 4 ).

[0139] In conclusion, under the experimental conditions, Feiruiqing has the effect of resisting bacterial pneumonia, which is specifically manifested by the reduction of inflammatory infiltration in the swim bladder tissue. Figure 5 .

Claims

1. Use of a traditional Chinese medicine composition for resisting bacterial pneumonia in the preparation of a medicament for treating bacterial pneumonia, characterized in that: The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 400-600 parts of platycodon grandiflorum extract, 400-600 parts of loquat leaf extract, 200-400 parts of polygonatum odoratum extract, 200-400 parts of mulberry leaf extract, 200-400 parts of lily extract, 80-120 parts of snow lotus extract, 80-120 parts of dendrobium officinale flower extract, 80-120 parts of grass jelly extract, 80-120 parts of momordica grosvenori extract, 80-120 parts of noni fruit powder and 80-120 parts of acai fruit powder.

2. The use according to claim 1, characterized in that The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 500 parts of platycodon grandiflorum extract, 500 parts of loquat leaf extract, 300 parts of polygonatum odoratum extract, 300 parts of mulberry leaf extract, 300 parts of lily extract, 100 parts of snow lotus extract, 100 parts of dendrobium officinale flower extract, 100 parts of grass jelly extract, 100 parts of momordica grosvenori extract, 100 parts of noni fruit powder and 100 parts of acai fruit powder.

3. The use according to claim 1, characterized in that The snow lotus extract is prepared by the following method: 1) Take dried snow lotus root, crush it, add 10 times its weight of deionized water, heat and extract at 80°C for 25 minutes, filter, and retain the filtrate; 2) Add 10 times the weight of deionized water to the residue, heat and extract at 80°C for 25 minutes, filter, and retain the filtrate; 3) Repeat step 2) once; 4) Combine the filtrates and set aside; The Dendrobium officinale flower extract is prepared by the following method: 1) Take dried Dendrobium officinale flowers, crush them, add 8 times their weight of deionized water, heat and extract at 95°C for 35 minutes, filter, and retain the filtrate; 2) Add 8 times the weight of deionized water to the residue, heat and extract at 95°C for 35 minutes, filter, and retain the filtrate; 3) Repeat step 2) once; 4) Combine the filtrates and set aside; The loquat leaf extract is prepared by the following method: Dried loquat leaves were crushed and extracted twice with 95% w / w ethanol at a solid-liquid ratio of 1:10 g / ml under reflux for 30 minutes each time, filtered, and the combined filtrates were spray-dried for later use; The Platycodon grandiflorum extract is prepared by the following method: Wash and dry fresh Platycodon grandiflorum, grind it in a grinder, and extract it twice with 75% ethanol w / w at a solid-liquid ratio of 1:9 g / ml under reflux, each for 60 minutes, filter, and spray-dry the combined filtrates for later use; The polygonatum extract, mulberry leaf extract, lily extract, grass jelly extract, monk fruit extract, noni fruit powder and acai fruit powder are all purchased from the market.

4. The use according to claim 3, characterized in that The medicine is a liquid preparation.

5. The use according to claim 4, characterized in that The liquid preparation is prepared from the Chinese medicine composition according to claim 1 or 2 according to the following method: (1) All the extracts were prepared into a suspension with deionized water, and mixed with the snow lotus extract and the Dendrobium officinale flower extract to obtain a raw material mixture; (2) Debittering, flavor correction, and solubilization Adding γ-cyclodextrin to the raw material mixed solution for flavor correction; (3) Allocation Add citric acid, potassium sorbate and sweetener for blending, and add deionized water in a quantitative amount to obtain a liquid preparation with good palatability and unique flavor; (4) Filling and sterilization The product was filled under aseptic conditions to obtain the finished product, which was then sterilized at 110°C for 20 min and cooled.

6. The use according to claim 5, characterized in that The usage amount of the gamma-cyclodextrin is 400-600 parts by weight, the usage amount of citric acid is 20-30 parts by weight, the usage amount of sucralose is 1-3 parts by weight, and the usage amount of potassium sorbate is 5-15 parts by weight.

7. The use according to claim 6, characterized in that The usage amount of the γ-cyclodextrin is 500 parts by weight, the usage amount of citric acid is 25 parts by weight, the usage amount of sucralose is 2 parts by weight, and the usage amount of potassium sorbate is 10 parts by weight.

8. The use according to claim 5, characterized in that Each 30ml / bottle of the finished product is made of 500mg of platycodon grandiflorum extract, 500mg of loquat leaf extract, 300mg of polygonatum extract, 300mg of mulberry leaf extract, 300mg of lily extract, 100mg of snow lotus extract, 100mg of dendrobium officinale flower extract, 100mg of grass jelly extract, 100mg of momordica grosvenori extract, 100mg of noni fruit powder, 100mg of acai fruit powder, 500mg of γ-cyclodextrin, 25mg of citric acid, 2mg of sucralose, and 10mg of potassium sorbate.

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