Citrus flavonoid compound and application thereof in treating acute lung injury

Abstract

The invention discloses a citrus flavonoid compound and application thereof in treating acute lung injury. The compound consists of naringenin and apigenin in a mass ratio of (1-10): 1. An in-vitro cell experiment shows that the compound can be used for remarkably inhibiting the expression of RAW264.7 and J774A.1 cell inflammatory factors induced by LPS (Lipopolysaccharide); an in-vivo mouse acute lung injury model further proves that the compound can effectively relieve pathological injury of lung tissues and reduce the level of inflammatory factors in the lung tissues and alveolar perfusion fluid, the effect of the compound is superior to that of a single compound, and a remarkable synergistic effect is shown. The compound provided by the invention is derived from natural citrus flavonoids, has the advantages of high safety, small toxic and side effects, definite curative effect, low administration dosage and the like, can be used for preparing medicines for treating acute lung injury, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a flavonoid complex derived from citrus and its application in the preparation of drugs for treating acute lung injury. Background Technology

[0002] Acute lung injury (ALI) is a common and critical clinical condition, often triggered by factors such as infection, trauma, and shock. In severe cases, it can develop into acute respiratory distress syndrome (ARDS), with a high mortality rate. Currently, clinical treatment mainly involves symptomatic relief using mechanical ventilation, antibiotics, and corticosteroids, lacking highly specific and effective targeted therapies. Inflammatory responses play a crucial role in the development and progression of ALI, especially the excessive release of inflammatory factors such as TNF-α, IL-6, and IL-β, which exacerbates lung tissue damage. Therefore, inhibiting the inflammatory response is one of the important strategies for treating ALI.

[0003] Citrus flavonoids are widely found in citrus fruits and have various biological activities such as antioxidation, anti-inflammation, and antitumor. Studies have shown that quercetin can reduce the expression of inflammatory factors such as TNF-α and IL-6 induced by TLR7 [Yasui, M., Matsushima, M., Omura, A. et al. The suppressive effect of quercetin on Toll-like receptor 7-mediated activation in alveolar macrophages. Pharmacology 96,201–209 (2015).]; Naringin can effectively improve LPS-induced acute lung injury in mice through intestinal flora metabolism [Li Wenwen et al. Naringin improves acute lung injury in mice by regulating the AMPK / SIRT1 / NF-κB signaling pathway through intestinal flora metabolites. Chinese Traditional and Herbal Drugs, 2025, 56(20):7384-7394.]. However, the development of flavonoid anti-inflammatory drugs is still in its early stages, with significant drawbacks such as unclear efficacy and large dosage. In particular, systematic evaluation in acute lung injury models is still lacking.

[0004] As a natural medicinal citrus, the anti-inflammatory and antitussive effects of Huazhou pomelo have always attracted much attention. This study selected the aglycones of naringin and apigenin, which are abundant in Huazhou pomelo, as research subjects. Through in vitro cell experiments and mouse experiments, the therapeutic effect of the combination of the two on acute lung injury was systematically evaluated, thus providing feasibility for the development of natural plant-derived anti-inflammatory drugs. Summary of the Invention

[0005] The present invention aims to provide a citrus flavonoid complex composed of naringenin and apigenin for use in the preparation of a drug for treating acute lung injury.

[0006] This invention is achieved through the following technical solution: A citrus flavonoid complex, the complex being composed of naringenin and apigenin, wherein the mass ratio of naringenin to apigenin is (1-10):1.

[0007] Preferably, the mass ratio of naringin to apigenin is (3-8):1.

[0008] More preferably, the mass ratio of naringin to apigenin is 5:1.

[0009] The effects of the complex in in vitro cell models and in vivo acute lung injury models were systematically evaluated. Results showed that the two flavonoids significantly inhibited the expression of inflammatory factors in LPS-induced mouse monocytes / macrophages AW264.7 and J774A.1; effectively alleviated acute lung injury in Balb / c mice, and reduced the levels of inflammatory factors in lung tissue and blood. Furthermore, the complex was more effective than the single compound, and naringenin and apigenin exhibited a significant synergistic effect in anti-inflammation and treatment of acute lung injury. Therefore, the citrus flavonoid complex provided by this invention can be used to prepare drugs for treating acute lung injury.

[0010] In the preparation of a drug for treating acute lung injury, the drug is preferably an injectable, oral, or inhaled formulation.

[0011] Furthermore, the drug also contains a pharmaceutically acceptable carrier.

[0012] The beneficial effects of this invention are: This invention confirms that naringin and apigenin possess excellent anti-inflammatory potential, providing a basis for the treatment of acute lung injury with natural plant-derived flavonoids. The complex provided by this invention is derived from natural citrus flavonoids, exhibiting advantages such as high safety, low toxicity, definite efficacy, and low dosage, demonstrating promising prospects for clinical application. Attached Figure Description

[0013] Figure 1 . Pathological section staining of acute lung injury in mice. Detailed Implementation

[0014] The present invention will now be described in detail through specific embodiments. It should be noted that the specific embodiments described are merely illustrative of the invention and are not intended to limit the invention. Furthermore, the various experimental operations involved in the embodiments are all conventional techniques in the art. For parts not specifically described herein, those skilled in the art can refer to various commonly used reference books, scientific and technological documents, or related instructions and manuals prior to the filing date of this invention to perform the operations.

[0015] Example 1: Anti-inflammatory cell assay of flavonoid complex 1. Test Methods (1) Cell resuscitation, medium change and passage Frozen RAW264.7 and J774A.1 cells were thawed at 37°C, centrifuged, and the supernatant was discarded. The cells were resuspended in complete culture medium. The cell suspension was transferred to a T25 culture flask, and complete culture medium was added to a final volume of 5 mL. The flask was then incubated at 37°C in a 5% CO2 incubator. After the cells adhered to the flask, the old culture medium was discarded, and 4 mL of fresh complete culture medium was added. The cells were then cultured until the cell density reached 80%. The flasks were then passaged.

[0016] (2) Cellular drug treatment Cells were seeded in 12-well plates and allowed to grow until they adhered to the plate and reached a density of about 80%. DMEM culture medium containing LPS (100 μg / mg) and different drug concentrations was added to each well. After incubation for 24 hours, the culture medium was discarded, and 800 μL of Trizol was added to each well. The plates were then placed on ice for RNA extraction, reverse transcription, and indicator detection.

[0017] (3) qRT-PCR method to detect the expression level of anti-inflammatory factors The supernatant culture medium was discarded, and Trizol reagent was added to extract RNA. RNA was then reverse transcribed into cDNA using the HiScript III All-in-one RTSuperMix Perfect for qPCR reverse transcription kit. The mRNA expression levels of inflammatory factors TNF-α, IL-6, IL-β, and iNOS were detected using qRT-PCR. Primer sequences are shown in Table 1.

[0018] Table 1. Primer sequences for real-time quantitative PCR

[0019] 2. Experimental Results Based on previous drug concentration screening results, we selected 100 μM naringenin and 1 μM apigenin, which have strong anti-inflammatory effects, to form a complex at a ratio of 100:1. The anti-inflammatory effect of the combination was evaluated by detecting the expression levels of inflammatory factors. The results showed that, compared to the LPS group, treatment with naringenin and apigenin, respectively, reduced the expression of LPS-induced inflammation in RAW264.7 and J774A.1 cells. TNF-α The expression levels of inflammatory factors decreased significantly, indicating that naringenin and apigenin have anti-inflammatory effects. When naringenin and apigenin were used in combination, the expression of each inflammatory factor decreased further, indicating that the anti-inflammatory effect of the complex was stronger than that of the single component at the same concentration (Table 2, Table 3).

[0020] Table 2. Quantitative analysis of naringenin, apigenin and their complexes on inflammatory factors in RAW264.7 cells.

[0021] Note: Letters a, b, c, d, etc., represent the results of multiple comparisons (Duncan's test) after one-way ANOVA. If two groups of data have no identical letters, it means that the difference between the two groups is statistically significant (P<0.05), and the same applies below.

[0022] Table 3. Quantitative analysis of naringenin, apigenin and their complexes on inflammatory factors in J774A.1 cells.

[0023] Example 2: Intervention effect of flavonoid complex on a mouse model of acute lung injury 1. Test Methods (1) Animal grouping and model establishment Forty female BALB / c mice (SPF grade, weighing 24-25g) were acclimatized for one week and then randomly divided into five groups of eight mice each: CK group, LPS group, naringenin group (50mg / kg), apigenin group (10mg / kg), and a complex group (naringenin 25mg / kg + apigenin 5mg / kg). The experimental groups received intraperitoneal injections of different doses of the drugs daily for 7 consecutive days. The CK and LPS groups received equal volumes of solvent (70% DMSO + physiological saline) for 7 consecutive days as controls. On day 7, except for the CK group, all other groups were anesthetized with tribromoethanol and then infused with LPS (5mg / kg) via tracheal infusion to establish an acute lung injury model in mice. Lung tissue was collected 12 hours later for pathological analysis to assess the level of lung injury in each group.

[0024] (2) Preparation of pathological sections of mouse lungs Left lungs were harvested from mice in each group, fixed in 4% FAA solution (for more than 24 hours), dehydrated with ethanol, cleared with xylene, embedded in paraffin, cut into 4μm thin sections, dewaxed and hydrated, stained with hematoxylin and eosin (HE), mounted, and the lesions in the mouse lung tissue were observed and recorded.

[0025] (3) Detection of expression levels of inflammatory factors in mouse lungs and blood To assess pulmonary and systemic inflammatory responses, inflammatory factors were detected at both gene transcription and protein expression levels. Transcriptional level detection: Right lung tissue samples from mice were collected and detected using qRT-PCR. TNF-α, IL-6, IL-β, iNOS The mRNA expression levels of inflammatory factors were measured. Protein level detection: Alveolar perfusion fluid (BALF) samples from mice were collected and subjected to enzyme-linked immunosorbent assay (ELISA) by Wuhan HYCEZMBIO Biotechnology Co., Ltd. to detect the concentrations of inflammatory proteins TNF-α, IL-6, IL-β, and iNOS.

[0026] 2. Test Results HE staining results showed that ( Figure 1 ): CK group: intact lung tissue structure, fine and uniform alveolar septa, no edema, hemorrhage or inflammatory cell infiltration; LPS group: lung tissue showed typical pathological changes of acute lung injury, including alveolar collapse, significant thickening of alveolar septa, severe congestion of lung parenchyma and a large number of inflammatory cell infiltrations; Naringenin group (50mg / kg) and apigenin group (10mg / kg): the degree of lung tissue damage was significantly lower than that of the LPS group, the alveolar structure was more intact, and the inflammatory cell infiltration was significantly reduced; Complex group (naringenin 25mg / kg + apigenin 5mg / kg): intact lung tissue structure, fine and uniform alveolar septa, almost no hemorrhage or inflammatory cell infiltration, its protective effect was better than that of the single naringenin or apigenin group, and there was no significant difference from the CK group, indicating that the two produced a significant synergistic effect when used in combination at low doses.

[0027] Further investigation into the effects of flavonoid drugs on the expression levels of relevant inflammatory factors in mice revealed the following results (Tables 4 and 5): In mice with an acute lung injury model, both naringenin and apigenin significantly reduced the expression of key inflammatory factors in mouse lung tissue. TNF-α , IL-6 , IL-β ,and iNOS The mRNA and protein levels of naringenin were reduced, and the combination of low concentrations of naringenin and apigenin showed comparable or even better effects, further suggesting that the two have a synergistic effect of low dosage and high efficiency in this model.

[0028] Table 4. Quantification of naringin, apigenin and their complexes on inflammatory factors in lung tissue

[0029] Table 5. ELISA quantification of inflammatory protein levels in bronchoalveolar perfusion fluid (BALF) by naringenin, apigenin and their complexes.

[0030] Terminology Explanation: 1. Naringenin This is a natural flavonoid compound, mainly found in citrus fruits, and is the aglycone form of naringin. It possesses various biological activities, including antioxidant, anti-inflammatory, and anti-apoptotic effects. It exerts its anti-inflammatory effect by inhibiting signaling pathways such as NF-κB and MAPK, thereby reducing the expression of inflammatory factors.

[0031] 2. Apogenin A natural flavonoid compound widely found in plants such as celery, chamomile, and citrus, it is the aglycone form of rhubarb glycoside. It possesses anti-inflammatory, antioxidant, and antitumor pharmacological activities, and can inhibit the release of inflammatory factors such as TNF-α and IL-6, thus regulating immune responses.

[0032] 3. Acute Lung Injury (ALI) Acute diffuse lung parenchymal injury induced by factors such as infection, trauma, and shock is clinically manifested as hypoxemia, pulmonary edema, and inflammatory cell infiltration. In severe cases, it can develop into acute respiratory distress syndrome (ARDS) with a high mortality rate.

[0033] 4. Lipopolysaccharide (LPS) The main component of the cell wall of Gram-negative bacteria is a potent immune activator that can induce macrophages to release large amounts of inflammatory factors. It is often used to construct in vitro and in vivo inflammation models, such as acute lung injury models.

[0034] 5. RAW264.7 cells / J774A.1 cells Both are mouse-derived mononuclear macrophage cell lines, widely used in inflammation-related research. LPS stimulation can mimic the activation state of macrophages in inflammatory responses, allowing for the evaluation of drug anti-inflammatory activity.

[0035] 6. Inflammatory Cytokines Pro-inflammatory proteins are a class of small molecules secreted by immune cells that participate in regulating inflammatory responses. Common pro-inflammatory factors include TNF-α, IL-6, IL-β, and iNOS. Overexpression of these proteins can exacerbate tissue damage and is a key indicator for evaluating the efficacy of anti-inflammatory drugs.

[0036] 7. qRT-PCR (qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction) A molecular biology technique for detecting gene expression levels, which uses fluorescence signals to monitor the PCR amplification process in real time and quantitatively analyzes the mRNA expression level of the target gene. It is often used to assess the transcriptional level of inflammatory factors.

[0037] 8. ELISA (Enzyme-Linked Immunosorbent Assay) A protein quantification detection technique based on antigen-antibody specific binding is used to determine the protein concentration of inflammatory factors in cell supernatant, serum or bronchoalveolar perfusion fluid, reflecting the inhibitory effect of drugs on inflammatory responses.

[0038] 9. HE staining (hematoxylin-eosin staining) Histopathology commonly uses staining methods to observe changes in tissue structure and cell morphology. In this patent, these methods are used to assess the degree of damage to mouse lung tissue, such as alveolar structure, inflammatory cell infiltration, and alveolar septal thickness.

[0039] 10. Synergistic Effect This refers to the combined use of two or more drugs, where the therapeutic effect is significantly better than the sum of the effects of using each drug alone. In this patent, the combined use of naringenin and apigenin at low doses exhibits a superior anti-inflammatory effect compared to high doses of a single drug, demonstrating a synergistic effect.

Claims

1. A citrus flavonoid complex, characterized in that, The complex is composed of naringenin and apigenin, wherein the mass ratio of naringenin to apigenin is (1-10):

1.

2. The citrus flavonoid complex as described in claim 1, characterized in that, The mass ratio of naringin to apigenin is (3-8):

1.

3. The citrus flavonoid complex as described in claim 1, characterized in that, The mass ratio of naringin to apigenin is 5:

1.

4. The use of the citrus flavonoid complex according to any one of claims 1-3 in the preparation of a medicament for treating acute lung injury.

5. The application as described in claim 4, characterized in that, The drug is an injectable, oral, or inhaled formulation.

6. The application as described in claim 4, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

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