A composition containing synephrine, hesperetin, naringenin and nobiletin components and its use

Abstract

The application discloses a composition containing synephrine, hesperetin, naringenin and nobiletin components and application thereof, and belongs to the technical field of traditional Chinese medicine.The active ingredients of the composition are synephrine, hesperetin, naringenin and nobiletin.The composition prepared by the application can significantly reduce pathological damage of lung tissue, inhibit collagen deposition and expression of fibrosis-related proteins, and play a role through regulation of a miRNA-mediated mitochondrial autophagy pathway, has a curative effect equivalent to that of a clinical first-line drug, and is safer, has a significant treatment effect on idiopathic pulmonary fibrosis, can be extended to secondary pulmonary fibrosis caused by various reasons such as virus infection, radiotherapy, drugs and connective tissue diseases, and can be made into various dosage forms such as oral administration, injection and atomization inhalation according to clinical requirements, and is suitable for clinical medication requirements of patients with different courses and different conditions, and has extremely high clinical transformation and industrialization values.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a composition containing synephrine, hesperidin, naringenin and nosenoside and its application. Background Technology

[0002] Pulmonary fibrosis is a chronic, irreversible, and fatal interstitial lung disease that primarily affects middle-aged and elderly individuals. It is characterized by subpleural and basal fibrosis and honeycomb-like changes, as well as collagen and extracellular matrix (ECM) deposition around fibrotic lesions, ultimately leading to altered lung tissue structure, loss of ventilation and diffusion capacity, and death. Idiopathic pulmonary fibrosis (IPF) is the most common type of pulmonary fibrosis, with a median survival of only 2–4 years, making it the most serious non-lung lung disease.

[0003] Currently used pirfenidone and nintedanib can only slow the rate of lung function decline in patients with mild to moderate pulmonary fibrosis and reduce the risk of acute exacerbations, but they also have significant adverse reactions and are expensive. N-acetylcysteine ​​can only improve the survival rate of patients with the TOLLIP TT genotype. Deeply exploring the essence of traditional Chinese medicine, fully leveraging its unique advantages, and actively searching for effective Chinese herbal ingredients for treating pulmonary fibrosis is not only an urgent task to save patients' lives but also an important direction for future treatment of pulmonary fibrosis.

[0004] Pulmonary fibrosis falls under the categories of "lung obstruction" and "lung atrophy" in traditional Chinese medicine. Its pathogenesis is a condition of deficiency in the root and excess in the branch; the root deficiency is due to deficiency of the lung, spleen, and kidney, while the branch excess is due to phlegm, heat, and blood stasis obstructing the lung collaterals. Professor Hou Jie, a pioneer in the field of pulmonary fibrosis in my country, believes that treatment should address both the root and branch, focusing on tonifying qi and strengthening the spleen, while simultaneously resolving phlegm and removing blood stasis. Based on clinical experience, he formulated a basic formula for pulmonary fibrosis – "Huqi Yin" (Tiger-Astragalus Decoction). This formula consists of seven herbs: Polygonum cuspidatum, Astragalus membranaceus, Salvia miltiorrhiza, Ligusticum chuanxiong, Angelica sinensis, Citrus reticulata peel, and Glycyrrhiza uralensis. Together, they tonify qi and replenish deficiency, resolve phlegm, and invigorate blood circulation. Preliminary clinical studies have found that "Huqi Yin" has a significantly higher effective rate in treating pulmonary fibrosis than the control group, and also shows advantages in terms of adverse reaction rate and health economics. Simultaneously, experimental studies have also found that "Huqi Yin" can effectively inhibit the progression of bleomycin (BLM)-induced pulmonary fibrosis in rats and reduce collagen deposition. Further analysis of the formula revealed that Citrus reticulata peel has the most significant anti-pulmonary fibrosis effect.

[0005] Dried tangerine peel (Chenpi) is pungent and bitter in taste, warm in nature, and enters the spleen and lung meridians. It has the functions of regulating qi and strengthening the spleen, drying dampness and resolving phlegm. After years of research, it has been found that the alkaline extract of dried tangerine peel (CAE) has the most significant anti-pulmonary fibrosis effect. CAE is one of many extracts of dried tangerine peel. Quality control and substance identification have revealed that its main active components are flavonoids and alkaloids, represented by hesperidin, naringenin, nodosumiole, hesperidin, neohesperidin, naringin, rutin, and synephrine. Further research on the main active components of CAE revealed that synephrine, hesperidin, naringenin, and nodosumiole have similar effects to CAE. The dried tangerine peel active complex (CASC) obtained by mixing these four substances in a certain proportion also showed good anti-pulmonary fibrosis effects. However, current technology has not disclosed a technical scheme for using a composition of synephrine, hesperidin, naringenin, and nodosumiole in a fixed ratio for the treatment of pulmonary fibrosis, nor has it provided a standardized preparation method and a clear mechanism of action for this composition. Therefore, developing a composition of active monomers from dried tangerine peel with clear ingredients, controllable quality, scientific compatibility, and definite efficacy is of great value and practical significance in meeting the clinical treatment needs of pulmonary fibrosis. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a composition containing synephrine, hesperidin, naringenin and nobiletin, which exhibits a significant synergistic effect in the treatment of pulmonary fibrosis, and the preparation method and pharmaceutical use of the composition are also clarified. Another technical problem to be solved by this invention is to provide the application of the composition in the preparation of drugs for the treatment of pulmonary fibrosis.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A pharmaceutical composition comprising synephrine, hesperidin, naringenin and nosenoside.

[0009] Furthermore, the mass ratio of synephrine, hesperidin, naringenin and nodosumetin is 3:5:5:2.

[0010] Further, it includes excipients selected from one or more of fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrices.

[0011] Furthermore, the pharmaceutical composition is an oral preparation selected from any one of capsules, tablets, and granules.

[0012] Further, synephrine, hesperidin, naringenin, and novo-tangeretin are passed through an 80-100 mesh sieve and mixed evenly according to the specified ratio to obtain the drug composition.

[0013] Furthermore, the mixing speed is 15~25 r / min, and the mixing time is 20~40 min.

[0014] Furthermore, the use of the pharmaceutical composition in the preparation of a medicament for the prevention or treatment of pulmonary fibrosis.

[0015] Furthermore, the pulmonary fibrosis includes any one of idiopathic pulmonary fibrosis, bleomycin-induced pulmonary fibrosis, post-viral infection pulmonary fibrosis, radiotherapy-associated pulmonary fibrosis, drug-induced pulmonary fibrosis, and connective tissue disease-associated pulmonary fibrosis.

[0016] Furthermore, the pharmaceutical composition exerts its anti-pulmonary fibrosis effect by regulating the miRNA-mediated mitophagy pathway.

[0017] Furthermore, the drug is administered orally, and the daily dose for adults is 75-300 mg / kg based on the active composition.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) The composition of the present invention overcomes the shortcomings of complex components and difficult quality standardization of traditional Chinese medicine compound and crude extract. It consists of only 4 natural active monomers derived from tangerine peel. The chemical structure of each component is clear and the content can be accurately determined. The preparation process is simple and stable and can be mass-produced. It fully meets the technical requirements of modern chemical drug / natural drug new drug research and development, and greatly reduces the difficulty of drug development.

[0020] (2) Based on previous formulation studies, single-component efficacy screening, and in vitro and in vivo gradient dose verification, this invention has determined the optimal ratio of the four components. The components work synergistically to exert anti-pulmonary fibrosis effects through multiple targets and pathways. In vivo animal experiments have confirmed that the composition of this invention can dose-dependently improve the weight loss of pulmonary fibrosis model mice, significantly reduce pathological damage to lung tissue, inflammatory cell infiltration and collagen deposition, improve fibrotic imaging features such as ground-glass opacities and honeycomb changes in the lungs, and significantly downregulate the expression of fibrosis marker proteins α-SMA and Vimentin. The efficacy of the high-dose group is comparable to that of the first-line clinical drug pirfenidone, and some indicators are better than those of the positive control group.

[0021] (3) This invention clarifies for the first time the core mechanism of action of the composition through "regulating key miRNAs - activating mitophagy - protecting alveolar type II epithelial cells - inhibiting fibrosis progression", filling the gap in the research on the mechanism of anti-pulmonary fibrosis of tangerine peel active monomer composition. Unlike existing drugs that only target the downstream links of fibrosis, the composition of this invention directly targets the core initiating link of pulmonary fibrosis pathogenesis - the imbalance of mitochondrial homeostasis in alveolar type II epithelial cells. By activating mitophagy to clear damaged mitochondria, it maintains the normal structure and function of alveolar epithelial cells, blocking fibroblast activation and abnormal collagen deposition from the root. It can not only delay the progression of the disease, but also has the potential value of reversing early fibrosis.

[0022] (4) The four active ingredients in the composition of this invention are all derived from tangerine peel, a traditional Chinese medicinal material that is both food and medicine. It has a history of clinical application in my country for thousands of years and has extremely low toxicity and good safety. Compared with chemical drugs such as pirfenidone and nintedanib, it can significantly reduce the risk of adverse reactions such as gastrointestinal reactions and liver and kidney damage, and patients can tolerate it better. At the same time, the raw materials are readily available and the preparation cost is low. Compared with imported chemical drugs, it has significant health economic advantages and can greatly reduce the treatment burden on patients.

[0023] (5) The composition of the present invention not only has a significant therapeutic effect on idiopathic pulmonary fibrosis, but can also be extended to secondary pulmonary fibrosis caused by various reasons such as viral infection, radiotherapy, drugs, and connective tissue diseases. At the same time, it can be formulated into various dosage forms such as oral, injection, and nebulized inhalation according to clinical needs, to meet the clinical medication needs of patients with different disease courses and conditions, providing a new, safe and effective drug option for the clinical treatment of pulmonary fibrosis, and has extremely high clinical translation and industrialization value. Attached Figure Description

[0024] Figure 1 This is a graph showing the effect of CASC on mouse body weight in this application;

[0025] Figure 2 This is a diagram illustrating the effect of CASC on lung tissue pathology in this application.

[0026] Figure 3 This is a diagram showing the effect of CASC on lung imaging in this application;

[0027] Figure 4 This is a diagram showing the expression of Vimentin and α-SMA proteins in lung tissue of this application. Detailed Implementation

[0028] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0029] Synephrine (CAS: 94-07-5), hesperidin (CAS: 520-33-2), naringerin (CAS: 480-41-1), and novo-tangeretin (CAS: 478-01-3) monomers used in the following examples were all purchased from Chengdu Pury Technology Development Co., Ltd., with a purity ≥98% (HPLC); experimental animals, reagents, and instruments were all conventional commercial products in the field.

[0030] The following embodiments of the method for detecting content uniformity include the following steps:

[0031] 1. Instruments and Reagents

[0032] An ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS / MS) analysis system (mainly including ultra-high performance liquid chromatography (UPLC, SHIMADZU Nexera X2) and tandem mass spectrometry (MS / MS, AppliedBiosystems 4500 Q TRAP), equipped with an electrospray ionization (ESI) interface); a digitally controlled ultrasonic cleaner (250W / 40kHz, ShuMei); an ultrapure water system (Millipore); synephrine, hesperidin, naringenin, and nobiletin reference standards (Chengdu Pury Technology Development Co., Ltd., purity ≥98%, HPLC); acetonitrile and phosphoric acid were chromatographically pure, methanol was analytically pure, and the experimental water was ultrapure water.

[0033] 2. Chromatographic conditions

[0034] UPLC conditions: Column: Agilent SB-C18 1.8µm, 2.1mm 100 mm; Mobile phase: Phase A is ultrapure water (with 0.1% formic acid added), Phase B is acetonitrile (with 0.1% formic acid added); Elution gradient: 0.00 min, Phase B ratio is 5%, within 9.00 min, Phase B ratio linearly increases to 95% and maintains at 95% for 1 min, 10.00-11.10 min, Phase B ratio decreases to 5%, and equilibrates to 5% for 14 min; Flow rate: 0.35 mL / min; Column temperature: 40℃; Injection volume: 4 μL;

[0035] ESI conditions: Software: Analyst 1.6.3 (AB Sciex); Ion modes: Positive ion mode (ESI+) and Negative ion mode (ESI-); Ion source: Turbine spray; Source temperature: 550℃; Ion spray voltage: 5500V (ESI+) / -4500V (ESI-); Ion source gas I: 50psi; Ion source gas II: 60psi; Curtain gas: 25.0psi; Collision-induced ionization parameter: High;

[0036] MS / MS conditions: Triple Quadrupole (QQQ) mode: calibrated with 10 μmol / L polypropylene glycol solution; Linear ion trap mode: calibrated with 100 μmol / L polypropylene glycol solution; QQQ scan mode: Multiple Reaction Monitoring (MRM); Collision gas (nitrogen): Medium; Declustering and collision voltages for each MRM ion pair were optimized; A specific set of MRM ion pairs was monitored in each epoch based on the eluted material.

[0037] 3. Preparation of mixed reference solution

[0038] Accurately weigh appropriate amounts of synephrine, hesperidin, naringenin, and nodosumetin reference standards, and prepare single-component stock solutions with methanol respectively; accurately measure appropriate amounts of each stock solution, dilute with methanol, and prepare a mixed reference solution containing 30 μg synephrine, 50 μg hesperidin, 50 μg naringenin, and 20 μg nodosumetin per 1 mL, and store at 4℃ protected from light for later use.

[0039] 4. Preparation of the test solution

[0040] Nine parallel sampling points were taken from seven locations at the discharge port of the three-dimensional motion mixer (upper, middle, lower, left, right, front, and rear) and two locations on the inner wall and center of the mixer cylinder. Each sample was accurately weighed to approximately 15 mg and placed in a 50 mL volumetric flask. 40 mL of methanol was added, and the sample was sonicated for 20 min to completely dissolve it. After cooling to room temperature, the sample was diluted to the mark with methanol and shaken well. The solution was then filtered through a 0.22 μm organic phase filter membrane, and the filtrate was collected to obtain a single test solution.

[0041] 5. Determination Method

[0042] Accurately pipette 10 μL of the mixed reference solution and each individual test solution into the UPLC-ESI-MS / MS analysis system and record the chromatograms. Calculate the measured content of synephrine, hesperidin, naringenin, and nobiletin in each test sample by peak area using the external standard method.

[0043] 6. Result Determination

[0044] The relative standard deviation (RSD) of the measured content of each active ingredient in the 9 test samples was calculated. The RSD of the contents of synephrine, hesperidin, naringenin and nodosumetin were all ≤3.0%, which means that the mixing uniformity meets the requirements.

[0045] Example 1

[0046] A method for preparing a composition containing synephrine, hesperidin, naringenin and nobiletin, comprising the following steps:

[0047] (1) Weigh 3 g of synephrine, 5 g of hesperidin, 5 g of naringin, and 2 g of neohesperidin by mass parts respectively, pass them through a 100-mesh sieve, and reserve for use.

[0048] (2) Place the above 4 raw materials in a three-dimensional motion mixer, mix them at a rotation speed of 20 r / min for 30 min under a dry environment at room temperature, detect the content uniformity after discharging, and when RSD≤3.0%, 15 g of the active composition (CASC) of the present invention can be obtained. After sealing, store it in the dark and dry at 4°C.

[0049] Example 2

[0050] Pharmacodynamic verification of the composition prepared in Example 1 on bleomycin-induced pulmonary fibrosis model mice

[0051] 2.1 Experimental materials

[0052] 1. Experimental animals: SPF-grade male C57BL / 6J mice, 6 weeks old, weighing 17 - 20 g, a total of 36 mice, provided by Jiangsu Qinglongshan Biotechnology Co., Ltd., animal license number: SCXK (Hu) 20190004; Adaptively raise them in the SPF-grade animal center of Nanjing University of Chinese Medicine for 1 week, temperature (22±2)°C, humidity (75±5)%, 12 h day and night alternation, free food and water intake.

[0053] 2. Experimental drugs and reagents: The composition of the present invention (prepared in Example 1); pirfenidone (PFD, Beijing Kangdini Pharmaceutical Co., Ltd.); bleomycin sulfate (Med Chem Express, USA); HE staining kit, Masson staining kit (Wuhan Sevier Biotechnology); Vimentin antibody, α-SMA antibody, GAPDH antibody (Wuhan Sanying Biotechnology Co., Ltd.).

[0054] 3. Experimental instruments: Quantum GX microCT small animal in vivo CT imaging system (Perkin Elmer, USA); QuantStudio7Flex western blot multi-functional imaging system (BioRAD, USA); NikonTi-E inverted microscope (Nikon, Japan); LeicaRM2235 automatic paraffin slicer (Leica, Germany).

[0055] 2.2 Experimental methods

[0056] 1. Animal grouping and modeling: Randomly divide 36 mice into 6 groups, with 6 mice in each group, namely: blank group (Control), model group (PF), CASC low-dose group (CASC-L, 75 mg·kg -1 ·d -1CASC medium-dose group (CASC-M, 150 mg·kg) -1 ·d -1 CASC high-dose group (CASC-H, 300 mg·kg) -1 ·d -1 ), positive control group (PFD, 300 mg·kg) -1 ·d -1 ).

[0057] Except for the control group, the mice in the other groups were used to construct a pulmonary fibrosis model by a single intratracheal bleomycin aerosol nebulization method: after anesthesia, the mice were tracheotomized and the nebulization needle was inserted into the trachea. During the inspiratory phase, bleomycin solution (5U / kg) was nebulized into the trachea; the control group was given an equal volume of physiological saline in the trachea.

[0058] 2. Administration method: The drug was administered on the first day after modeling. The blank group and the model group were given the same volume of physiological saline by gavage, and each drug administration group was given the corresponding dose of drug by gavage, once a day, for 21 consecutive days; the weight of the mice was recorded every 3 days.

[0059] 3. Sample collection: 3 hours after the last administration, mice were euthanized by cervical dislocation, and lung tissue was collected. Part of the tissue was fixed in 4% paraformaldehyde for pathological staining, and the other part was stored in an ultra-low temperature freezer at -80℃ for protein detection.

[0060] 4. Detection Indicators and Methods

[0061] (1) Mouse weight change: The weight change of mice from before modeling to 21 days after drug administration was recorded, the weight change curve was plotted, and the final weight of mice in each group was calculated.

[0062] (2) Pathological examination of lung tissue: The middle lobe of the right lung of mice was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and dewaxed. HE staining and Masson staining were performed respectively. The pathological structure, inflammatory cell infiltration and collagen deposition of lung tissue were observed under an optical microscope.

[0063] (3) Micro-CT imaging of small animals: 21 days after administration, mice were anesthetized and placed in a micro-CT instrument for small animals. The parameters were set as follows: voltage 90kV, current 88μA. The imaging changes of the mouse chest were observed to assess the degree of pulmonary fibrosis.

[0064] (4) Western Blot detection of fibrosis-related protein expression: Total protein was extracted from mouse lung tissue, quantified by BCA method, and then subjected to SDS-PAGE electrophoresis, membrane transfer, and blocking. Vimentin and α-SMA primary antibody were added and incubated overnight at 4℃. After washing, secondary antibody was added and incubated at room temperature for 1 h. The membrane was exposed by ECL chemiluminescence method, and the gray value of the band was quantified by ImageJ software. GAPDH was used as an internal reference to calculate the relative expression level of the protein.

[0065] 5. Statistical methods: GraphPad Prism 9.0 was used to analyze the data. Experimental data are expressed as x±s. One-way ANOVA was used for comparisons among multiple groups. P<0.05 was considered statistically significant.

[0066] 2.3 Experimental Results

[0067] 1. The composition of the present invention can significantly improve the weight loss in mice with pulmonary fibrosis.

[0068] Depend on Figure 1 It was found that after modeling, except for the blank group, the body weight of mice in all other groups decreased significantly. Six days after administration, the body weight of mice in each CASC-treated group showed an upward trend, significantly higher than that of the model group, and exhibited a clear dose-dependent effect. Twenty-one days after administration, the final body weight of mice in the model group was significantly lower than that of the blank group (P<0.01); the final body weight of mice in the CASC-M and CASC-H groups was significantly higher than that of the model group (P<0.05, P<0.01), and the CASC-H group was comparable to the PFD positive control group.

[0069] 2. The composition of the present invention can significantly reduce pathological damage and collagen deposition in the lung tissue of model mice.

[0070] Depend on Figure 2 HE staining results showed that the lung tissue of mice in the blank group was intact, the alveolar walls were smooth, and there was no inflammatory cell infiltration. The lung tissue of mice in the model group was severely disordered, with thickened alveolar walls, enlarged alveolar spaces, a large number of inflammatory cells infiltrating, and abnormal proliferation of alveolar epithelium. Each CASC administration group could improve the above pathological changes in a dose-dependent manner, reduce alveolar structural damage and inflammatory cell infiltration, and the CASC-H group had the most significant improvement effect, similar to that of the PFD group.

[0071] Masson staining results showed that in the blank group of mice, only a small amount of collagen fibers were seen around the bronchial walls in the lung tissue; in the model group of mice, extensive collagen fiber deposition was observed in the lung tissue, and a large number of blue-stained collagen fibers were found in the alveolar septa; the CASC administration groups significantly reduced collagen deposition in the lung tissue in a dose-dependent manner, confirming that the composition of the present invention can effectively inhibit abnormal collagen deposition in the process of pulmonary fibrosis.

[0072] 3. The composition of the present invention can significantly improve the pulmonary fibrosis changes observed in model mice.

[0073] Depend on Figure 3 The results of Micro-CT scans of the mouse chest showed that the lung fields of the blank group mice were clear and without abnormal density shadows; the lung fields of the model group mice showed obvious ground-glass opacities, reticular shadows and honeycomb lesions, which were consistent with the characteristic imaging changes of pulmonary fibrosis; the CASC administration groups could reduce the above imaging changes in a dose-dependent manner, and the ground-glass opacities and honeycomb lesions in the lung fields of the CASC-H group mice basically disappeared, which was consistent with the effect of the PFD positive control group.

[0074] 4. The composition of this invention can significantly downregulate the expression of fibrosis-related proteins in the lung tissue of model mice.

[0075] Depend on Figure 4 Western blot results showed that, compared with the blank group, the expression of Vimentin and α-SMA proteins in the lung tissue of mice in the model group was significantly upregulated (P<0.01); compared with the model group, the expression of Vimentin and α-SMA proteins in the CASC-M and CASC-H groups was significantly downregulated (P<0.05, P<0.01), and the downregulation effect of the CASC-H group on the two proteins was comparable to that in the PFD positive control group, confirming that the composition of the present invention can effectively inhibit the expression of pulmonary fibrosis marker proteins and block the fibrosis process.

[0076] Example 3

[0077] Verification of the anti-pulmonary fibrosis mechanism of the composition prepared in Example 1

[0078] 3.1 Experimental Materials

[0079] Experimental animals and CASC composition were the same as in Example 2; miRNA library construction kit (NEB, USA); total RNA extraction kit, reverse transcription kit, and qPCR kit (Nanjing Novizan Biotechnology Co., Ltd.); primers were synthesized by Wuhan Saive Biotechnology Co., Ltd.; QuantStudio 7Flex real-time PCR instrument (ABI, USA).

[0080] 3.2 Experimental Methods

[0081] 1. Sample preparation: Take lung tissue samples from mice in the blank group, model group, and CASC-H group in Example 2, with 3 mice in each group, for miRNA transcriptome sequencing and Real-time PCR verification.

[0082] 2. miRNA Transcriptome Sequencing and Analysis: Total RNA was extracted from lung tissue using the Trizol method. After detecting RNA concentration, purity, and integrity, a miRNA library was constructed and sequenced using the Illumina platform. Differential expression analysis was performed using the DEG algorithm in the R package, with screening criteria of P < 0.05 and |log2FC| > 1. The starBase, miRDB, and Targetscan databases were integrated to predict target genes for differentially expressed miRNAs. KEGG pathway enrichment analysis was performed after taking the intersection. The mouse mitochondrial autophagy pathway (mmu04137) related target in the KEGG database was obtained, and the intersection with the differentially expressed miRNA target genes was taken to construct a PPI protein interaction network. Core targets were screened through topological analysis.

[0083] 3. Real-time PCR verification of core target mRNA expression: Total RNA was extracted from lung tissue, reverse transcribed to synthesize cDNA, and detected by real-time quantitative PCR. The reaction conditions were: 95℃ pre-denaturation for 1 min, 94℃ denaturation for 10 s, 55℃ annealing for 30 s, for 40 cycles. The relative gene expression level was calculated using the 2-ΔΔCt method. GAPDH was used as an internal reference gene to detect the mRNA expression levels of core targets Bnip3l, Kras, Becn1, Rela, Jun, Nbr1, and Foxo3.

[0084] 4. Statistical methods: Same as in Example 2.

[0085] 3.3 Experimental Results

[0086] 1. Screening differentially expressed miRNAs by miRNA transcriptome sequencing

[0087] Compared with the model group, a total of 121 differentially expressed miRNAs were screened in the blank group, of which 34 were upregulated and 87 were downregulated; compared with the model group, a total of 144 differentially expressed miRNAs were screened in the CASC-H group, of which 54 were upregulated and 90 were downregulated; taking the intersection of the two groups of differentially expressed miRNAs, a total of 89 differentially expressed miRNAs with common regulation were obtained, and the core differentially expressed miRNAs included miR-30b-5p, miR-30e-5p, miR-30c-5p, miR-29a-3p, miR-29c-3p, miR-155-5p, miR-381-3p, miR-132-3p, etc.

[0088] 2. Target gene prediction and pathway enrichment analysis

[0089] Target gene prediction was performed on 89 core differentially expressed miRNAs, resulting in 5207 miRNA-mRNA pairs, including 2331 target genes. KEGG pathway enrichment analysis showed that the target genes of differentially expressed miRNAs were significantly enriched in the mitophagy pathway, confirming that the composition of the present invention mainly exerts its anti-pulmonary fibrosis effect by regulating the mitophagy pathway.

[0090] 3. Screening of core targets for mitochondrial autophagy

[0091] Intersections between differentially expressed miRNA target genes and mitophagy pathway targets were obtained, resulting in 33 intersection targets. A PPI network was constructed and topological analysis was performed to screen out 10 core genes, which were ranked from highest to lowest MCC score as follows: Becn1, Bnip3l, Kras, Gabarap, Foxo3, Rela, Hif-1α, Jun, Nbr1, and Tfeb. These genes are the core targets for the regulation of mitophagy by the composition of this invention.

[0092] 4. Real-time PCR verification of core target mRNA expression

[0093] The results in Table 1 show that, compared with the control group, the mRNA expression levels of Bnip3l, Kras, Becn1, Rela, Jun, Nbr1, and Foxo3 in the lung tissue of mice in the model group were significantly downregulated (P<0.05, P<0.01); compared with the model group, the mRNA expression levels of the above genes in the CASC-H group were significantly upregulated (P<0.05, P<0.01). This confirms that the composition of the present invention can significantly upregulate the expression of core genes of mitophagy, activate the mitophagy pathway, and exert an anti-pulmonary fibrosis effect.

[0094] Table 1. Effects of genes related to the CASC mitophagy pathway ( (n=3)

[0095]

[0096] Note: Compared with the blank group 1） P < 0.05 2） P < 0.01; compared with the model group 3） P < 0.05 4） P < 0.01.

[0097] Example 4

[0098] A method for preparing a tablet of the composition includes the following steps:

[0099] (1) 300g of the active composition prepared in Example 1, 120g of lactose, 80g of microcrystalline cellulose, 20g of sodium carboxymethyl starch, an appropriate amount of 5% povidone K30 aqueous solution, and 3g of magnesium stearate were used to make 1000 tablets, each containing 300mg of the active composition.

[0100] (2) Pass the composition, lactose, microcrystalline cellulose and sodium carboxymethyl starch through a 100-mesh sieve, mix them evenly in equal increments, add 5% povidone K30 aqueous solution to make soft material, granulate through a 24-mesh sieve, dry at 60℃, granulate through a 22-mesh sieve, add magnesium stearate and mix for 3 minutes, compress into tablets to obtain the composition tablets.

[0101] Example 5

[0102] A method for preparing a capsule formulation of the composition includes the following steps:

[0103] (1) 150g of the active composition prepared in Example 1, 80g of microcrystalline cellulose, 60g of pregelatinized starch, 8g of cross-linked polyvinylpyrrolidone, and 2g of magnesium stearate were used to make 1000 capsules.

[0104] (2) Mix the composition with microcrystalline cellulose, pregelatinized starch and cross-linked polyvinyl ketone in equal increments, add 80% ethanol to make soft material, pass through a 20-mesh sieve to make wet granules, dry in a forced air at 60°C, and then pass the dry granules through an 18-mesh sieve to granulate.

[0105] (3) Add magnesium stearate to the granulated particles and mix for 5 minutes.

[0106] (4) Fill the granules into No. 0 gelatin empty capsules using a capsule filling machine to obtain the composition capsules, each containing 150mg of active composition.

[0107] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, It is composed of synephrine, hesperidin, naringenin and nosenoside.

2. The pharmaceutical composition according to claim 1, characterized in that: The mass ratio of synephrine, hesperidin, naringenin and nosanthin is 3:5:5:

2.

3. The pharmaceutical composition according to claim 1, characterized in that: Includes excipients, which are selected from one or more of fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrices.

4. The pharmaceutical composition according to claim 3, characterized in that: The pharmaceutical composition is an oral preparation, selected from any one of capsules, tablets, and granules.

5. A method for preparing the pharmaceutical composition according to any one of claims 1 to 4, characterized in that: Synephrine, hesperidin, naringenin, and novo terpene are each passed through an 80-100 mesh sieve and then mixed evenly according to the specified ratio to obtain the drug composition.

6. The method for preparing the pharmaceutical composition according to claim 5, characterized in that: The mixing speed is 15~25 r / min, and the mixing time is 20~40 min.

7. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for the prevention or treatment of pulmonary fibrosis.

8. The application according to claim 7, characterized in that: The pulmonary fibrosis includes any one of the following: idiopathic pulmonary fibrosis, bleomycin-induced pulmonary fibrosis, post-viral infection pulmonary fibrosis, radiotherapy-associated pulmonary fibrosis, drug-induced pulmonary fibrosis, and connective tissue disease-associated pulmonary fibrosis.

9. The application according to claim 7, characterized in that: The pharmaceutical composition exerts its anti-pulmonary fibrosis effect by regulating the miRNA-mediated mitophagy pathway.

10. The application according to claim 7, characterized in that: The drug is administered orally, and the daily dose for adults, based on the active composition, is 75-300 mg / kg.

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