Composition of paeoniflorin and isoliquiritigenin and use thereof

Abstract

The present invention relates to a composition of paeoniflorin and isoliquiritigenin and the use thereof. The composition of the present invention involves rich raw materials, has the effects of resisting inflammation, resisting oxidation, inhibiting collagen deposition and the like, can be effectively used for treating diffuse pulmonary interstitial fibrosis, and has relatively high economic and social benefits.

Description

A composition of paeoniflorin and isoliquiritigenin and its application Technical Field

[0001] The invention belongs to the technical field of drugs for treating diffuse pulmonary interstitial fibrosis, and relates to a composition of paeoniflorin and isoliquiritigenin and an application thereof. Background Art

[0002] Diffuse pulmonary fibrosis (DPF) is a group of diffuse lung diseases that affect the pulmonary interstitium and alveolar spaces, leading to loss of alveolar-capillary functional units. It carries high rates of disability and mortality, a heavy socioeconomic burden, and poses a serious threat to public health. The etiology of this disease is complex and may be related to dust, hazardous fumes, infections, and medications. Clinical manifestations include progressively worsening dyspnea, restrictive ventilation with decreased diffusion capacity, and ultimately respiratory failure. Idiopathic pulmonary fibrosis (IPF) is the most representative of these diseases.

[0003] IPF is the most common interstitial lung disease clinically, affecting more men than women and more often in the elderly. Its prognosis is poor, and its incidence is increasing annually. The prevalence of IPF in Italy, the United States, and the United Kingdom is 7.5-9.3 per 100,000, 14-42.7 per 100,000, and 100-25 per 100,000, respectively. The average survival of IPF patients after diagnosis is 2.5-3.5 years, and survival rates decline significantly over time, reaching a 3-year survival rate of 50% and a 5-year survival rate of only 20%. IPF is characterized by diffuse alveolitis, disorganized alveolar architecture, and interstitial thickening. Its primary pathological mechanisms are inflammation, oxidative stress, apoptosis, and epithelial-mesenchymal transition. Its pathological features are characterized by the accumulation of numerous fibroblasts and the deposition of extracellular matrix, accompanied by inflammation and damage, leading to structural changes and functional loss of normal lung tissue. Accumulation primarily occurs in the interstitial space, alveoli, and bronchioles, ultimately leading to respiratory failure. Transforming growth factor (TGF)-β1 and interleukin (IL)-13 are important profibrotic factors that can promote fibrosis formation. Hydroxyproline (HYP) is the main component of collagen tissue, and its expression can reflect collagen deposition and the degree of pulmonary fibrosis.

[0004] In 2015, the American Thoracic Society (ATS), European Respiratory Society (ERS), Japanese Respiratory Society (JRS), and Latin American Thoracic Association (ALAT) revised their 2011 IPF guidelines, stating that pirfenidone is a recommended treatment. However, its adverse reactions are extensive, and even when treatment benefit is assessed using forced vital capacity (FVC), some patients may still be unable to tolerate certain adverse reactions. Furthermore, the high cost of pirfenidone treatment has made it difficult to promote clinically.

[0005] Therefore, it is necessary to further strengthen the research and development of drugs for the treatment of diffuse pulmonary fibrosis and improve the level and ability of clinical prevention and treatment.

[0006] Summary of the Invention

[0007] The inventors' team has developed a traditional Chinese medicine formula for the treatment of diffuse pulmonary fibrosis. The formula consists of ginsenoside Re, icariin, nobiletin, fritillaryin A, paeoniflorin, and isoliquiritigenin. A patent application for this formula has been filed and granted under patent number CN109394776B. Building on these findings, the inventors further discovered that a traditional Chinese medicine composition consisting of paeoniflorin and isoliquiritigenin can achieve even better therapeutic effects, leading to the completion of the present invention.

[0008] One aspect of the present invention provides a pharmaceutical composition consisting of paeoniflorin and isoliquiritigenin.

[0009] Paeoniflorin (CAS: 23180-57-6, molecular formula: C 23 H 28 O 11 , molecular weight: 480.47g / mol) has the following chemical structure:

[0010] Isoliquiritigenin (CAS: 961-29-5, molecular formula: C 15 H 12 The chemical structure of O4 (molecular weight: 256.25 g / mol) is as follows:

[0011] In the pharmaceutical composition according to the present invention, the ratio of paeoniflorin to isoliquiritigenin is not particularly limited, as long as a significant synergistic effect of the two can be achieved.

[0012] In some embodiments, in the pharmaceutical composition according to the present invention, the weight ratio of paeoniflorin to isoliquiritigenin can be 10:90 to 90:10, for example, 20:80 to 80:20, 30:70 to 70:30, 40:60 to 60:40, 40:60 to 55:45, or 40:60 to 50:50.

[0013] In some embodiments, in the pharmaceutical composition according to the present invention, the weight percentage of paeoniflorin based on the total weight of the pharmaceutical composition can be more than 10%, more than 20%, more than 25%, more than 30%, more than 35%, or more than 40%, and less than 90%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, or less than 50%, for example, 40% to 50%.

[0014] In some embodiments, in the pharmaceutical composition according to the present invention, the purity of the paeoniflorin and isoliquiritigenin is not less than 98% as determined by high performance liquid chromatography. High performance liquid chromatography is a conventional determination method and a well-known technique, so its description is omitted here.

[0015] As demonstrated in the Examples, the pharmaceutical composition according to the present invention can be used to prevent or treat diffuse pulmonary fibrosis, and can achieve better therapeutic effects compared with the traditional Chinese medicine formula of CN109394776B.

[0016] Another aspect of the present invention provides use of the above-mentioned pharmaceutical composition for preparing a medicament for preventing or treating diffuse pulmonary fibrosis.

[0017] In another aspect, the present invention provides a drug for preventing or treating diffuse pulmonary fibrosis, comprising the above-mentioned pharmaceutical composition.

[0018] Particularly, the above-mentioned medicine does not contain ginsenoside Re, icariin, nobiletin, and fritillin A.

[0019] Ginsenoside Re (CAS: 489-32-7, molecular formula: C 48 H 82 O 18 , molecular weight: 947.154 g / mol) has the following chemical structure:

[0020] Icariin (CAS: 489-32-7, molecular formula: C 33 H 40 O 15 , molecular weight: 676.65g / mol) has the following chemical structure:

[0021] Nobiletin (CAS: 478-01-3, molecular formula: C 21 H 22 The chemical structure of O8 (molecular weight: 402.39 g / mol) is as follows:

[0022] Peimine (CAS: 23496-41-5, molecular formula: C 27 H 45 The chemical structure of NO3 (molecular weight: 431.65 g / mol) is as follows:

[0023] In another aspect, the present invention provides a method for preventing or treating diffuse pulmonary fibrosis, which comprises administering the pharmaceutical composition or the medicament according to the present invention to a subject in need thereof.

[0024] The subject mentioned here can be a human or an animal, such as a mammal, such as a human, ape, monkey, pig, horse, cow, sheep, dog, cat, lion, tiger, etc., but is not limited thereto.

[0025] The drug of the present invention may be in various dosage forms conventional in the art, such as solid, semisolid or liquid forms, and may be aqueous solutions, non-aqueous solutions, suspensions, lozenges, capsules, tablets, granules, pills, powders and inhalants. The drug may be administered by injection, oral administration or inhalation. The injection may include intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injections.

[0026] The drug may include the pharmaceutical composition of the present invention in any amount, as long as the desired preventive or therapeutic benefit can be obtained. For example, based on mass percentage, the drug may include 0.01 to 99.99%, such as 1% to 90%, of the pharmaceutical composition of the present invention, and 99.99 to 0.01%, such as 99% to 10%, of a pharmaceutically acceptable excipient.

[0027] The pharmaceutically acceptable conventional excipients may be selected from excipients, fillers, diluents, preservatives, colorants, flavoring agents, flavoring agents, sustained-release agents, coating agents, penetration enhancers, adhesives, wetting agents, disintegrants, surfactants, lubricants, solubilizers, fragrances, additives, buffers, soothing agents, etc., but are not limited thereto.

[0028] In addition, the medicament of the present invention may further comprise other active ingredients as required for treatment or complications, etc. The other active ingredients may be active ingredients for preventing or treating diffuse pulmonary fibrosis, or active ingredients of drugs for treating other diseases, disorders, or symptoms.

[0029] The dosage of the pharmaceutical composition and the drug according to the present invention can be selected according to the instructions for use, the patient's age, sex and other conditions and the severity of the disease.

[0030] The present invention has been described in detail above, but the above embodiments are merely illustrative in nature and are not intended to limit the present invention. In addition, the present invention is not limited by any theory described in the above prior art or invention summary or the following examples.

[0031] Unless otherwise expressly stated, numerical ranges throughout this application include any subranges therein and any numerical values ​​in increments of the smallest subunit of a given value therein. Unless otherwise expressly stated, numerical values ​​throughout this application represent approximate measures or limits of the range of embodiments that include minor deviations from the given value and have approximately the stated value as well as the stated exact value. Except for the working examples provided at the end of the detailed description, all numerical values ​​for parameters (e.g., quantities or conditions) in this application (including the appended claims) should be understood in all cases as being modified by the term "approximately", regardless of whether "approximately" actually appears before the value. "Approximately" means that the stated value allows for slight imprecision (some approach to exactness in the value; approximately or reasonably close to the value; approximately). If the imprecision provided by "approximately" is not understood in this ordinary sense in the art, "approximately" as used herein at least represents the variation that can be produced by ordinary methods of measuring and using these parameters. For example, "approximately" can include variations of less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1% or less than or equal to 0.5%. Beneficial effects

[0032] During the research and development, the inventors have obtained the same or similar results through repeated experiments. The method is effective and feasible, the raw materials are abundant, and it has good practical application value.

[0033] The present invention's composition for treating diffuse pulmonary fibrosis is extracted and screened from peony and liquorice, and has anti-inflammatory, antioxidant, and collagen deposition-inhibiting effects, making it effective for treating diffuse pulmonary fibrosis. The present invention has repeatedly studied the efficacy of the compositions obtained in various examples in treating rats with diffuse pulmonary fibrosis, and achieved satisfactory therapeutic results.

[0034] The invention has abundant raw materials and scientific and advanced methods, is effectively used for treating diffuse pulmonary interstitial fibrosis, can be developed into a new drug for treating diffuse pulmonary interstitial fibrosis, and has high economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1: H&E staining of lung tissue of rats in the model group in Example 1;

[0036] Figure 2 H&E staining of lung tissue of rats in the blank group in Example 1;

[0037] FIG3 is an H&E staining image of lung tissue of rats in the pirfenidone group in Example 1;

[0038] Figure 4 is an H&E staining image of lung tissue of rats in group 1 of Formula 1 in Example 1;

[0039] Figure 5 H&E staining of lung tissue of rats in Formulation 2 group in Example 1;

[0040] Figure 6 H&E staining of lung tissue of rats in group 3 of formula in Example 1;

[0041] Figure 7 H&E staining of lung tissue of rats in group 4 of formula in Example 1;

[0042] Figure 8 H&E staining of lung tissue of rats in group 5 of formula in Example 1;

[0043] Figure 9 H&E staining of lung tissue of rats in Group 6 of Formula 1;

[0044] Figure 10 H&E staining of lung tissue of rats in group 7 of formula in Example 1;

[0045] Figure 11 H&E staining of lung tissue of rats in group 8 of formula in Example 1;

[0046] Figure 12 H&E staining of lung tissue of rats in group 9 of Formula 9 in Example 1;

[0047] Figure 13 H&E staining of lung tissue of rats in group 10 of formula in Example 1;

[0048] Figure 14 H&E staining of lung tissue of rats in Formulation 11 group in Example 1;

[0049] Figure 15 H&E staining of lung tissue of rats in group 12 of formula in Example 1;

[0050] Figure 16 H&E staining of lung tissue of rats in Formulation 13 group in Example 1;

[0051] Figure 17 H&E staining of lung tissue of rats in group 14 of Formula 1 in Example 1;

[0052] Figure 18 is a Masson staining image of lung tissue of rats in the model group in Example 1;

[0053] Figure 19 shows Masson staining of lung tissue of rats in the blank group in Example 1;

[0054] Figure 20 is a Masson staining image of lung tissue of rats in the pirfenidone group in Example 1;

[0055] Figure 21 is a Masson staining image of lung tissue of rats in the Formula 1 group in Example 1;

[0056] Figure 22 is a Masson staining image of lung tissue of rats in Formulation 2 group in Example 1;

[0057] Figure 23 is a Masson staining image of lung tissue of rats in Group 3 of Formula 1;

[0058] Figure 24 is a Masson staining image of lung tissue of rats in group 4 of Formula 1;

[0059] Figure 25 is a Masson staining image of lung tissue of rats in group 5 of Formula 5 in Example 1;

[0060] Figure 26 is a Masson staining image of lung tissue of rats in Group 6 of Formula 1;

[0061] Figure 27 is a Masson staining image of lung tissue of rats in group 7 of Formula 7 in Example 1;

[0062] Figure 28 is a Masson staining image of lung tissue of rats in group 8 of Formula 8 in Example 1;

[0063] Figure 29 is a Masson staining image of lung tissue of rats in group 9 of Formula 9 in Example 1;

[0064] Figure 30 is a Masson staining image of lung tissue of rats in group 10 of Formula 1 in Example 1;

[0065] Figure 31 is a Masson staining image of lung tissue of rats in the Formulation 11 group in Example 1;

[0066] Figure 32 is a Masson staining image of lung tissue of rats in group 12 of Formula 1 in Example 1;

[0067] Figure 33 is a Masson staining image of lung tissue of rats in group 13 of formula in Example 1;

[0068] Figure 34 is a Masson staining image of lung tissue of rats in group 14 of Formula 1 in Example 1;

[0069] Figure 35 shows CoL I staining of lung tissue of rats in the model group in Example 1;

[0070] Figure 36 shows CoL I staining of lung tissue of rats in the blank group in Example 1;

[0071] Figure 37 shows CoL I staining of lung tissues of rats in the pirfenidone group in Example 1;

[0072] Figure 38 shows CoL I staining of lung tissue of rats in the Formulation 1 group in Example 1;

[0073] Figure 39 shows CoL I staining of lung tissue of rats in group 2 of formula in Example 1;

[0074] Figure 40 shows CoL I staining of lung tissue of rats in group 3 of formula in Example 1;

[0075] Figure 41: CoL I staining of lung tissue of rats in group 4 of Formula 4 in Example 1;

[0076] Figure 42 shows CoL I staining of lung tissue of rats in group 5 of formula in Example 1;

[0077] Figure 43: CoL I staining of lung tissue of rats in Formulation 6 group in Example 1;

[0078] Figure 44 shows CoL I staining of lung tissue of rats in group 7 of formula in Example 1;

[0079] Figure 45 is a CoL I staining image of lung tissue of rats in group 8 of formula in Example 1;

[0080] Figure 46 shows CoL I staining of lung tissue of rats in group 9 of formula in Example 1;

[0081] Figure 47: CoL I staining of lung tissue of rats in group 10 of formula 1 in Example 1;

[0082] Figure 48 shows CoL I staining of lung tissue of rats in the Formulation 11 group in Example 1;

[0083] Figure 49 shows CoL I staining of lung tissue of rats in group 12 of formula 1 in Example 1;

[0084] Figure 50 shows CoL I staining of lung tissue of rats in group 13 of formula 1 in Example 1;

[0085] Figure 51: CoL I staining of lung tissue of rats in group 14 of formula 1 in Example 1;

[0086] Figure 52 shows α-SMA staining of lung tissue in rats in the model group in Example 1;

[0087] Figure 53 shows α-SMA staining of lung tissue of rats in the blank group in Example 1;

[0088] Figure 54 shows α-SMA staining of lung tissues of rats in the pirfenidone group in Example 1;

[0089] Figure 55 shows α-SMA staining of lung tissue of rats in the Formulation 1 group in Example 1;

[0090] Figure 56 shows α-SMA staining of lung tissue of rats in Formulation 2 group in Example 1;

[0091] Figure 57 shows α-SMA staining of lung tissue of rats in group 3 of formula in Example 1;

[0092] Figure 58 shows α-SMA staining of lung tissue of rats in group 4 of Formula 4 in Example 1;

[0093] Figure 59 shows α-SMA staining of lung tissue in rats in group 5 of Formula 5 in Example 1;

[0094] Figure 60 shows α-SMA staining of lung tissue of rats in group 6 of Formula 6 in Example 1;

[0095] Figure 61 α-SMA staining of lung tissue of rats in Formulation 7 group in Example 1;

[0096] Figure 62 shows α-SMA staining of lung tissue in rats in group 8 of Formula 8 in Example 1;

[0097] Figure 63 shows α-SMA staining of lung tissue in rats in group 9 of Formula 9 in Example 1;

[0098] Figure 64 shows α-SMA staining of lung tissue in rats in group 10 of formula 1 in Example 1;

[0099] Figure 65 shows α-SMA staining of lung tissue of rats in the Formulation 11 group in Example 1;

[0100] Figure 66 shows α-SMA staining of lung tissue in rats in group 12 of Formula 1 in Example 1;

[0101] Figure 67 α-SMA staining of lung tissue of rats in Formulation 13 group in Example 1;

[0102] Figure 68 shows α-SMA staining of lung tissue in rats in group 14 of Formula 1 in Example 1;

[0103] Figure 69 shows H&E staining of lung tissue of rats in the model group in Example 2;

[0104] Figure 70 shows H&E staining of lung tissue of rats in the blank group in Example 2;

[0105] Figure 71 shows H&E staining of lung tissue of rats in the pirfenidone group in Example 2;

[0106] Figure 72 H&E staining of lung tissue of rats in the isoliquiritigenin group in Example 2;

[0107] Figure 73 H&E staining of lung tissue of rats in the paeoniflorin group in Example 2;

[0108] Figure 74 shows H&E staining of lung tissue of rats in the combination group in Example 2;

[0109] Figure 75 shows Masson staining of lung tissue of rats in the model group in Example 2;

[0110] Figure 76 shows Masson staining of lung tissue of rats in the blank group in Example 2;

[0111] Figure 77 shows Masson staining of lung tissues of rats in the pirfenidone group in Example 2;

[0112] Figure 78 is a Masson staining image of lung tissue of rats in the isoliquiritigenin group in Example 2;

[0113] Figure 79 shows Masson staining of lung tissue of rats in the paeoniflorin group in Example 2;

[0114] Figure 80 is a Masson staining image of lung tissue of rats in the combination group in Example 2. DETAILED DESCRIPTION

[0115] The specific embodiments of the present invention are further described in detail below with reference to the examples. However, the following examples are provided only for easier understanding of the present invention, and the scope of the present invention is not limited thereto.

[0116] Example 1 Screening of pharmaceutical composition formulations

[0117] 1 Experimental Materials

[0118] 1.1 Experimental Animals

[0119] A total of 204 SPF Sprague-Dawley rats, half male and half female (age: 6-8 weeks, weight: 200 ± 10 g; animal license number: 37009200018733), were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (SCXK[Lu]20140007).

[0120] 1.2 Experimental drugs

[0121] 1.2.1 Bleomycin hydrochloride for injection (production batch number: s1214-12, 15U / bottle, Hisun Ruihui Pharmaceutical Co., Ltd.) was dissolved in normal saline to a concentration of 30 mg / ml.

[0122] 1.2.2 Pirfenidone capsules (Aisiri, 100 mg × 54 capsules / bottle, Beijing Contini Pharmaceutical Co., Ltd.), positive control drug, were prepared with distilled water to a concentration of 33.3 mg / ml.

[0123] 1.2.3 Icariin, isoliquiritigenin, fritillin A, nobiletin, paeoniflorin, and ginsenosides were purchased from Chengdu Munster Biotechnology Co., Ltd. The purity of each monomer was determined by high-performance liquid chromatography (HPLC) and was greater than 98%. Immediately before use, each monomer was dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) to prepare a suspension.

[0124] 1.3 Experimental Reagents

[0125] 1.3.1 Immunohistochemistry (IHC)-related reagents

[0126] SABC ready-to-use rabbit IgG immunohistochemistry kit (SA1022), DAB color development kit (AR1022), and Mayor's hematoxylin (AR0005) were provided by Wuhan Boster Biotechnology Co., Ltd.

[0127] The primary antibodies rabbit anti-rat Anti-Collagen I (COL I) Antibody (Cat. No. AF7001) and Anti-alpha-SMA (α-SMA) Antibody (Cat. No. AF1032) were provided by Affinity Biopharmaceuticals, Inc., USA.

[0128] α-SMA (14395-1-AP) polyclonal antibody was provided by Wuhan Tri-Tian Biotechnology Co., Ltd.

[0129] 1.3.2 Hematoxylin-eosin (H&E) staining and Masson's Trichrome staining reagents

[0130] Paraformaldehyde (P804536) was provided by Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China).

[0131] Paraffin wax (39601006) was provided by Leica Microsystems Trading Co., Ltd. (Illinois, USA).

[0132] Anhydrous ethanol (500 ml) and xylene (500 ml) were provided by Tianjin Zhiyuan Chemical Reagent Co., Ltd.

[0133] Hematoxylin staining solution (G1140), eosin staining solution (G1100), and neutral gum (G8590) were provided by Beijing Solebow Technology Co., Ltd.

[0134] Masson stain (G1006) was provided by Wuhan Savier Biotechnology Co., Ltd.

[0135] 1.4 Experimental Instruments

[0136] The experimental instruments are shown in Table 1.

[0137] Table 1

[0138] 2 Research Methods

[0139] 2.1 Model preparation

[0140] Rats were acclimatized to the environment for 7 days after purchase. They were housed in individual ventilated cages (IVC) and maintained at a temperature of (20-25)°C, relative humidity of (50±10)%, air exchange rate of 15 times / h, and ammonia concentration of ≤14 mg / m 3 , Noise level 60 ≤ db, maintain a 12-hour day and night cycle. Regularly check the purification and water and electricity operating systems to maintain a quiet environment.

[0141] A pulmonary fibrosis rat model was prepared by a one-time intratracheal instillation of bleomycin. After the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3.0 ml / kg), they were tilted back and fixed on a mouse board. A light source was shone on the throat, and the tongue was gently pulled to open the mouth. The glottis could be seen to open and close in an orderly manner with breathing. The indwelling needle was gently pushed into the trachea as the glottis opened and closed, and the needle core was pulled out. The swing of the cotton thread could be used to verify whether the tracheal intubation was successful. Bleomycin was injected into the trachea at a rate of 5 mg / kg, and 0.5 ml of air was continued to be injected to ensure the depth of the drug. The rats were stood upright and shaken from side to side to ensure that the drug was evenly distributed in the lungs. The anesthetized rats were placed on an electric blanket to maintain body temperature and placed in corresponding mouse cages after waking up. The rats were raised normally until the 28th day, and were free to drink pure water and sterile feed every day.

[0142] 2.2 Processing

[0143] 204 rats were randomly divided into blank group, model group, pirfenidone group (positive control) and each formula group (formula 1-14) (formula 1 is the Chinese medicine formula in CN109394776B), with a total of 17 groups, 12 rats in each group, 6 female rats / group and 6 male rats / group. The modeling day was set as day 0. From day 29 on, the blank group (normal rats without modeling) and the model group were given normal saline by gavage (2 ml / rat), and the pirfenidone group was given pirfenidone solution by gavage (108 mg kg -1 d -1 ), each formula-administered group was gavage-treated (dosages shown in Table 3) once a day for 14 consecutive days, and samples were collected after the 42nd day.

[0144] The drug dosage is calculated using the equivalent dose coefficient conversion formula, which is D 大鼠 =D 人 ×(HI 大鼠 / HI 人 )×(W 大鼠 / W 人 ) 2 / 3

[0145] Wherein, D is the dose, HI is the body shape index, and W is the body weight.

[0146] 2.3 Index detection

[0147] 2.3.1 General:

[0148] Observe the rats for death, bleeding around the eyes, mouth and nose, and daily observation of the rats' fur color, mental state, eating habits, and activity.

[0149] 2.3.2 Lung function:

[0150] Before sampling, the rats were anesthetized, tracheotomy was performed, and a tracheal tube was inserted into the rat trachea and fixed. TM The series PFT measurement system was used to detect the forced vital capacity (FVC), forced expiratory volume in 0.3s (FEV 0.3), vital capacity (VC), and dynamic lung compliance (Cydn) of rats.

[0151] 2.3.3 Lung tissue pathology:

[0152] The left lung was fixed with 10% formaldehyde for 72 h, embedded in paraffin, and sectioned at 4 μm for HE and Masson staining.

[0153] The pathological scores of alveolitis and pulmonary fibrosis were calculated according to the Szapiel method. The degree of alveolitis and pulmonary fibrosis were detected by HE staining and Masson staining, respectively.

[0154] The pathological changes of lung tissue were observed using an optical microscope. Six sections were selected from each group, and six fields of view were selected from each section. The degree of alveolitis and pulmonary fibrosis was scored according to the criteria shown in Table 2:

[0155] Table 2 Alveolitis and pulmonary fibrosis grading and scoring standards

[0156] 2.3.4 Expression of CoL I and α-SMA in lung tissue:

[0157] Immunohistochemistry was used for detection.

[0158] Lung tissue was sliced ​​at 4 μm and baked at 60°C for 3 h. Paraffin sections were sequentially dewaxed in xylene, dehydrated in gradient alcohol, and rinsed in distilled water for 2 min. Antigen retrieval was performed by boiling in a microwave oven with 0.01 M citrate buffer. 3% hydrogen peroxide was added to the sections to block endogenous peroxidase. The sections were blocked with 5% BSA blocking solution and incubated at 37°C for 30 min. Primary antibody (CoL) was added. The sections were incubated overnight at 4°C in a humidified chamber. HRP-conjugated goat anti-rabbit secondary antibody was added dropwise and incubated at 37°C for 20 min. SABC was added dropwise and incubated at 37°C for 30 min. Freshly prepared DAB color development solution was added dropwise and observed under a microscope. Color development was terminated when the sections turned brownish-yellow. The sections were counterstained with hematoxylin for 30 s and differentiated with 1% hydrochloric acid and alcohol for 3 s. The sections were then dehydrated in a gradient of alcohol. The sections were air-dried in a fume hood, mounted with neutral gum, and air-dried. Images were acquired under an Olympus-DP70 microscope (Image-Pro Plus 6.0, IPP 6.0). Six fields of view were randomly selected for each section, and the integrated optical density (IOD) of the images was calculated.

[0159] 2.4 Statistical Methods: IBM SPSS 22.0 statistical software was used for data analysis. Chi-square test was used for count data; repeated measurement data of rat body weight and lung function were analyzed by repeated measurement variance analysis; one-way ANOVA was used for comparison between groups. The least significant difference method was used for homogeneous variances, and Dunnett's T3 method was used for unequal variances. The significance level was set at α = 0.05. Data were expressed as mean. Means are ± standard error (SE).

[0160] 2.5 Comprehensive evaluation of R value:

[0161] The R value comprehensive evaluation method (copyright number: Guozuo Dengzi-2013-A-00096833) was used to comprehensively evaluate eight indicators of rat lung function (forced expiratory vital capacity (FVC), forced expiratory volume in 0.3 seconds (FEV0.3), deep inspiratory capacity (IC), dynamic lung compliance (Cydn), lung tissue pathology (HE staining, MASSON staining), lung tissue immunohistochemistry CoL I, α-SMA, etc.

[0162] Standardize the data and build R 综合 Value dataset (R 综合 =R-(-2)), after statistical analysis, the comprehensive correction effect R 综合 When R 综合 When R<1, it means that the comprehensive correction strength of the drug on such indicators exceeds the normal value. 综 合 =1 means that the drug has the best effect on the comprehensive correction intensity of such indicators. <R 综合 When R <2, it means that the drug has a certain corrective effect on such indicators. 综合 =2 means that the drug has no corrective effect on such indicators. 综合 When it is >2, it means that the drug has a reverse exacerbating effect on such indicators. 综合 The order of the comprehensive effect of drugs on indicators is obtained by the size of .

[0163] 3. Formulation screening

[0164] The inventors screened the Chinese medicine formula in CN109394776B as a basis and optimized the formula to test the therapeutic effect of the formula obtained by simplifying the formula or reducing the dosage.

[0165] The inventors first confirmed through preliminary optimization experiments that the formula obtained by not using or using less of some of the components in the traditional Chinese medicine formula in CN109394776B (for example, ginsenosides and / or fritillaryin A) still has the therapeutic effect of treating rats with diffuse pulmonary fibrosis, and used the R value comprehensive evaluation method to confirm that some formulas have better correction intensity effects.

[0166] Based on the above experiments, the inventors further combined the components of the Chinese herbal formula in CN109394776B to create a new formula, and then treated rats in groups according to the dosage. The specific formula and dosage are shown in Table 3. Among them, Formula 1 is the Chinese herbal formula in CN109394776B.

[0167] Table 3 Experimental groups and drug combinations

[0168] Each composition was provided and identified by the Drug Analysis Laboratory of the School of Pharmacy of Henan University of Traditional Chinese Medicine. Before use, the mixture was dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) to prepare a suspension.

[0169] Table 4 Grouping and treatment

[0170] 4 Experimental results

[0171] 4.1 General

[0172] The rats in the blank group moved freely, reacted quickly, had smooth and shiny fur, and ate and drank normally.

[0173] On the first day of modeling, rats in the model group showed a significant decrease in activity, fatigue, lethargy, and decreased food and water intake, but no mortality occurred. In the first week after modeling, bleeding and scabs were observed around the eyes, nose, mouth, and nails. Food and water intake recovered somewhat, but not to normal levels. In the second week after modeling, no bleeding was observed in the model group, and food and water intake returned to normal. Their fur became white and dull, and some rats developed sneezing. By the third and fourth weeks after modeling, the rats in the model group were able to move freely and their food and water intake remained normal.

[0174] After the 5th to 6th week of administration, the pirfenidone group and each formula group were able to improve the above symptoms to varying degrees.

[0175] 4.2 Lung function

[0176] The results of pulmonary function monitoring are shown in Table 5. Compared with the blank group, FVC, FEV0.3, IC, and Cydn in the model group were significantly decreased (P<0.01, P<0.05).

[0177] Compared with the model group, the FVC of formula 7, 8, and 11 groups were significantly increased (P<0.01, P<0.05), the FEV0.3 of formula 1, 2, 7, 8, and 11 groups were significantly increased (P<0.01, P<0.05), the IC of pirfenidone group and formula 1, 7, and 8 groups were significantly increased (P<0.01, P<0.05), and the Cydn of formula 5 and 13 groups were increased (P<0.05).

[0178] Compared with the pirfenidone group, FVC and FEV0.3 in formula 7 and 8 groups were increased (P<0.05).

[0179] Table 5 Pulmonary function related indicators ( ) Note 1: N = 10, compared with the blank group: *P < 0.05, **P < 0.01; compared with the model group: # P<0.05, ## P<0.01; compared with the pirfenidone group: △ P<0.05, △△ P<0.01

[0180] 4.3 Lung Tissue Pathology

[0181] In the control group, rats showed intact alveolar structure, with no thickening of the alveolar septa. In the model group, rats showed fractured and fused alveolar walls, with alveolar cavities of varying sizes visible. Alveolar structure was significantly damaged, with widened alveolar walls, extensive inflammatory cell infiltration, increased collagen deposition, and fibrotic scarring. The pirfenidone group and the various formulations improved these pathological lesions to varying degrees.

[0182] Scoring results based on H&E staining and Masson staining of rat lung tissue are shown in Table 6, H&E staining results are shown in Figures 1-17, and Masson staining results are shown in Figures 18-34. Compared with the blank group, the alveolitis and pulmonary fibrosis scores in the model group were significantly increased (P < 0.01). Compared with the model group, the alveolitis scores in Formulas 3 and 6 were significantly reduced (P < 0.01, P < 0.05); and the pulmonary fibrosis scores in Formulas 7, 8, 10, 13, and 14 were significantly reduced (P < 0.01, P < 0.05).

[0183] Table 6 Alveolitis and pulmonary fibrosis scores ( ) Note: Compared with the blank group: *P<0.05, **P<0.01; Compared with the model group: # P<0.05, ## P<0.01.

[0184] 4.4 Changes in CoL I and α-SMA protein expression in lung tissue

[0185] The results of CoL I and α-SMA staining in rat lung tissues are shown in Table 7. The results of CoL I staining are shown in Figures 35-51, and the results of α-SMA staining are shown in Figures 52-68. Compared with the blank group, the protein expression of CoL I and α-SMA in the lung tissues of rats in the model group was significantly increased (P < 0.01, P < 0.05). Compared with the model group, the expression of CoL I protein in the pirfenidone group and formulas 1, 2, 4, 7, 8, 9, 11, 12, and 14 was significantly decreased (P < 0.01, P < 0.05); the expression of α-SMA protein in formulas 2, 5, 6, and 7 was significantly decreased (P < 0.05).

[0186] Table 7 Changes of COL I and α-SMA in each group ( )

[0187] Same as Note 1. N = 6.

[0188] 4.6 Comprehensive evaluation of R value

[0189] The R-value comprehensive evaluation method was used to calculate eight indicators, including lung function (FVC, FEV0.3, IC, Cydn), lung tissue pathology (H&E staining, Masson staining), and collagen deposition (COL I, α-SMA). The results are shown in Table 8.

[0190] The results showed that all treatment groups had a significant corrective effect on PF rats (P<0.01). The corrective strength was as follows: Formulation 8 > Formulation 1 > Formulation 7 > Formulation 5 > Formulation 6 > Pirfenidone > Formulation 11 > Formulation 9 > Formulation 14 > Formulation 12 > Formulation 13 > Formulation 2 > Formulation 3 > Formulation 10 > Formulation 4.

[0191] Except for formula 3, 4, 10, and 13, all treatment groups showed significant improvement compared with the model group (P<0.01, P<0.05); formula 7 (peoniflorin) was superior to formula 3 (icariin), formula 4 (isoliquiritigenin), and formula 5 (nobiletin) (P<0.05); formula 8 (isoliquiritigenin + paeoniflorin) was superior to pirfenidone, formulas 2, 3, 4, and 9-14.

[0192] Table 8 R values ​​of total efficacy related indicators ( ) Note: Compared with the blank group: *P<0.05, **P<0.01; Compared with the model group: # P<0.05, ## P<0.01; compared with the pirfenidone group: △ P<0.05; compared with formula 1: ○ P<0.05, ○○P<0.01; compared with formula 2: □ P<0.05, □□ P<0.01; compared with formula 3: ■ P<0.05; compared with formula 4: ◇ P<0.05, ◇◇ P<0.01; compared with formula 8: ★★ P<0.01.

[0193] 5. Conclusion

[0194] Each formula can improve the lung function and pulmonary fibrosis manifestations of rats with pulmonary fibrosis to varying degrees, reduce inflammatory response and collagen deposition in lung tissue. Among them, formula 8 (isoliquiritigenin combined with paeoniflorin) has a more significant efficacy and is more economical.

[0195] Example 2: Evaluation of the efficacy of monomers of Formulation 8

[0196] To evaluate the difference in efficacy between isoliquiritigenin and paeoniflorin used alone and in combination, this study conducted a split study of formula 8, splitting formula 8 into isoliquiritigenin, paeoniflorin, and the combination of the two, all at 0.55 mg / kg / d.

[0197] 1 Experimental Materials

[0198] The experimental materials and research methods are the same as those in Example 1.

[0199] 2. Processing

[0200] Seventy-two rats were randomly divided into six groups: blank group, model group, isoliquiritigenin group, paeoniflorin group, combination group, and pirfenidone group, with 12 rats in each group (6 female rats per group and 6 male rats per group). The modeling day was designated as day 0. Starting from day 29, the blank group (normal rats without modeling) and the model group were gavaged with normal saline (2 ml per rat), the isoliquiritigenin group was given isoliquiritigenin (0.55 mg / kg / d), the paeoniflorin group was given paeoniflorin (0.55 mg / kg / d), and the combination group was given isoliquiritigenin + paeoniflorin (0.55 mg / kg / d), once daily for 14 days. The samples were collected after the 42nd day.

[0201] 3.Indicator detection

[0202] 3.1 General:

[0203] Observe the rats for death, bleeding around the eyes, mouth and nose, and daily observation of the rats' fur color, mental state, eating habits, and activity.

[0204] 3.2 Lung function:

[0205] Before sampling, the rats were anesthetized, tracheotomy was performed, and a tracheal tube was inserted into the rat trachea and fixed. TM The series PFT measurement system was used to detect the forced vital capacity (FVC), forced expiratory volume in 0.3s (FEV 0.3), vital capacity (VC), and dynamic lung compliance (Cydn) of rats.

[0206] 3.3 Lung tissue pathology:

[0207] The left lung was fixed with 10% formaldehyde for 72 h, embedded in paraffin, and sectioned at 4 μm for HE and Masson staining.

[0208] The pathological scores of alveolitis and pulmonary fibrosis were calculated according to the Szapiel method. The degree of alveolitis and pulmonary fibrosis were detected by HE staining and Masson staining, respectively.

[0209] Lung tissue pathological changes were observed using an optical microscope. Six sections were selected from each group, and six fields of view were selected from each section. The degree of alveolitis and pulmonary fibrosis was scored according to the criteria shown in Table 2 in Example 1:

[0210] 4. Results

[0211] 4.1 Lung function

[0212] The results of pulmonary function monitoring are shown in Table 9.

[0213] Compared with the blank group, the model group had significantly decreased FVC, FEV0.3, IC, and Cydn (P<0.01, P<0.05). Compared with the model group, the isoliquiritigenin group had increased FEV0.3, the paeoniflorin group had increased FVC and FEV0.3, and the combination group had significantly increased FVC, FEV0.3, IC, and Cydn, while the pirfenidone group had increased FEV0.3 and IC (P<0.01, P<0.05). The combination group was superior to the pirfenidone group in improving FVC, superior to the isoliquiritigenin group in improving FVC and FEV0.3, and superior to the paeoniflorin group in improving FEV0.3 (P<0.05).

[0214] Table 9 Changes in lung function in each group ( ) Note 1: N = 10, compared with the blank group: *P < 0.05, **P < 0.01; compared with the model group: # P<0.05, ## P<0.01; compared with the pirfenidone group: △ P<0.05, △△ P<0.01; compared with the isoliquiritigenin group, ◇ P<0.05, ◇◇ P<0.01; compared with the paeoniflorin group: ▲P<0.05, ▲▲ P<0.01.

[0215] 4.2 Lung tissue pathology

[0216] The scoring results based on the H&E staining and Masson staining images of rat lung tissue are shown in Table 10, the H&E staining results are shown in Figures 69-74, and the Masson staining results are shown in Figures 75-80.

[0217] Compared with the blank group, the model group showed significantly increased alveolitis and pulmonary fibrosis (P < 0.01). Compared with the model group, the isoliquiritigenin and paeoniflorin groups reduced the pulmonary fibrosis score (P < 0.05). Both the combination group and the pirfenidone group showed significant reductions in both the alveolitis and pulmonary fibrosis scores (P < 0.01, P < 0.05). The combination group was superior to isoliquiritigenin in reducing the alveolitis and pulmonary fibrosis scores, and superior to paeoniflorin in reducing the pulmonary fibrosis score (P < 0.05).

[0218] Table 10 Lung pathology scores ( )

[0219] Note: Same as Table 9.

[0220] 4. Conclusion

[0221] In summary, Formula 8 has a good therapeutic effect in the treatment of pulmonary fibrosis, can alleviate symptoms, improve lung function, improve the degree of alveolitis and pulmonary fibrosis in lung tissue, reduce collagen deposition in lung tissue, and can effectively alleviate the progression of pulmonary fibrosis. Formula 8 has the advantages of clear ingredients, small dosage form, and easy portability. Further evaluation of the effect of Formula 8 found that even if a single ingredient in Formula 8 is used in the same dosage as Formula 8, it still cannot achieve the same effect as Formula 8. Therefore, Formula 8 (isoliquiritigenin + paeoniflorin) is an innovation in the long-term treatment of pulmonary fibrosis and has high practical value.

[0222] The embodiments described above are merely preferred embodiments of the present invention and do not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it does not limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical content disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are within the scope of the technical solution of the present invention.

Claims

1. A pharmaceutical composition consisting of paeoniflorin and isoliquiritigenin.

2. The pharmaceutical composition according to claim 1, wherein The weight ratio of paeoniflorin to isoliquiritigenin is 10:90 to 90:10, for example, 20:80 to 80:20, 30:70 to 70:30, 40:60 to 60:40, 40:60 to 55:45, or 40:60 to 50:

50.

3. The pharmaceutical composition according to claim 1, wherein The weight percentage of paeoniflorin is greater than 10%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, or greater than 40%, and less than 90%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, or less than 50%, for example, 40% to 50%.

4. The pharmaceutical composition according to claim 1, wherein The purity of the paeoniflorin and isoliquiritigenin is not less than 98% as determined by high performance liquid chromatography.

5. Use of the pharmaceutical composition according to any one of claims 1 to 4 for preparing a medicament for preventing or treating diffuse pulmonary fibrosis.

6. A drug for preventing or treating diffuse pulmonary fibrosis, comprising the pharmaceutical composition according to any one of claims 1 to 4, but excluding ginsenoside Re, icariin, nobiletin, and fritillarin A.

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