Application of fagopyrum dibotrys freeze-dried powder in preparation of medicine for preventing or treating antimony pneumoconiosis

Abstract

The invention relates to the technical field of biological medicines, in particular to application of wild buckwheat rhizome freeze-dried powder in preparation of a medicine for preventing or treating antimony pneumoconiosis. A mouse antimony pneumoconiosis model is constructed by injecting an antimony (Sb) suspension into a trachea, and pulmonary fibrosis lesion caused by antimony exposure can be remarkably relieved by intragastric administration of the wild buckwheat rhizome freeze-dried powder prepared by the invention. Experimental results show that Hamp; e dyeing indicates that the freeze-dried powder can obviously relieve the fibrosis severity of the lung tissue; western Blot and PCR (Polymerase Chain Reaction) detection results show that the wild buckwheat rhizome can partially inhibit the expression up-regulation of Col-1 and alpha-SMA induced by antimony in protein and gene levels; in addition, the wild buckwheat rhizome can also obviously reduce the content of hydroxyproline in lung tissues. The results show that the wild buckwheat rhizome freeze-dried powder can effectively improve multiple pathological indexes and reduce the pulmonary fibrosis degree, has good potential in the aspect of preventing antimony pneumoconiosis, and has potential application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of a freeze-dried buckwheat powder in the preparation of a drug for the prevention or treatment of antimony pneumoconiosis. Background Technology

[0002] Dust from metallic antimony (Sb) and its compounds generated during mining, smelting, and processing operations can be inhaled and deposited in the lungs. Long-term exposure can lead to an occupational lung disease called antimony pneumoconiosis. This disease is characterized by chronic lung inflammation and progressive interstitial fibrosis. Early pathological changes include extensive infiltration of alveolar macrophages, oxidative stress damage caused by reactive oxygen species (ROS) bursts, and mitochondrial dysfunction. As the disease progresses, continuous stimulation from antimony dust leads to abnormal activation of pro-fibrotic factors such as transforming growth factor-β (TGF-β), which in turn promotes myofibroblast differentiation and excessive deposition of extracellular matrix, ultimately resulting in irreversible pathological changes, represented by diffuse interstitial pulmonary fibrosis. Notably, antimony pneumoconiosis often presents as diffuse small nodular shadows with a reticular pattern on imaging. Pathologically, collagen fibers are seen surrounding the metal dust deposition foci, forming a unique fibrotic structure different from silicotic nodules.

[0003] In terms of differential diagnosis and treatment strategies, antimony pneumoconiosis differs fundamentally from infectious pneumonia and other types of pulmonary fibrosis. Bacterial or viral pneumonia is usually caused by direct infection by pathogens, characterized by neutrophil infiltration and exudative inflammation in the alveolar spaces. The lesions are mostly acute and reversible, and antibiotic or antiviral treatment can effectively control the condition. Idiopathic pulmonary fibrosis (IPF), on the other hand, has an unknown etiology. High-resolution CT typically shows reticular shadows and honeycomb lung, predominantly in the subpleural region. Pathologically, it is characterized by fibroblastic foci. Although nintedanib or pirfenidone can be used for antifibrotic treatment, their drug response mechanism differs from that of fibrosis caused by metal dust. In contrast, antimony pneumoconiosis is driven by clearly defined metal toxic substances. Its fibrotic process is closely related to persistent oxidative damage and mitochondrial dysfunction caused by antimony. Currently, there are no specific drugs targeting this etiology. Clinical management still mainly focuses on removal from the exposure environment, glucocorticoid intervention for early inflammation, and symptomatic treatment for pulmonary rehabilitation. The efficacy in the middle and late stages is very limited.

[0004] In the application of traditional Chinese medicine, *Fagopyrum dibotrys* (D. Don) Hara is included in the Chinese Pharmacopoeia (2025 edition) and is traditionally used for pulmonary infectious diseases such as lung abscess with purulent sputum and cough due to lung heat. Its main functions are clearing heat and detoxifying, draining pus and removing blood stasis. No existing publicly available research or patents suggest its potential intervention in pulmonary fibrosis caused by metal dust. Although *Fagopyrum dibotrys* is known to contain antioxidant active substances such as flavonoids, there is still no experimental evidence to support whether it can be used to regulate antimony-induced mitochondrial dysfunction and specific fibrosis signaling pathways.

[0005] From the perspective of traditional Chinese medicine theory, antimony pneumoconiosis can be categorized as "lung collaterals damaged by mineral toxins." The pathogenesis involves the obstruction of lung collaterals by the dryness and toxicity of minerals, depleting qi and damaging yin, leading to phlegm and blood stasis forming masses. Although buckwheat can clear heat and toxins, its cooling nature makes it traditionally considered unsuitable for prolonged use in cases of deficiency with lingering pathogenic factors and established masses. Therefore, based on current knowledge, those skilled in the art lack the theoretical basis and experimental motivation to use buckwheat for the prevention and treatment of antimony pneumoconiosis.

[0006] In summary, using buckwheat extract for the prevention or treatment of antimony pneumoconiosis represents a novel application development not suggested by any existing technology. This not only fills a drug gap in the field of etiological prevention and treatment of metal pneumoconiosis, but also provides a new natural drug option for early intervention in antimony-exposed populations, demonstrating significant innovative value and promising clinical application prospects. Summary of the Invention

[0007] The purpose of this invention is to address the lack of effective prevention and treatment methods for antimony pneumoconiosis in the existing technology. This application provides the use of freeze-dried buckwheat powder in the preparation of drugs for the prevention or treatment of antimony pneumoconiosis. This use is proposed for the first time and provides a new solution for drug intervention for occupational pulmonary fibrosis caused by metallic antimony dust.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The use of a freeze-dried buckwheat powder in the preparation of a medicine for the prevention or treatment of antimony pneumoconiosis.

[0010] Preferably, the drug is used to alleviate pulmonary fibrosis caused by antimony exposure.

[0011] Preferably, the drug is used to reduce collagen deposition in lung tissue in the antimony exposure model group, and / or to downregulate the expression levels of Col-1 and α-SMA proteins and genes in lung tissue.

[0012] Preferably, the drug is used to reduce the content of hydroxyproline in the lung tissue of the antimony exposure model group.

[0013] The freeze-dried powder of golden buckwheat is made from the dried rhizomes of golden buckwheat (Fagopyrum dibotrys (D. Don) Hara) through water extraction, concentration and freeze-drying. Each gram of freeze-dried powder is equivalent to 15 g of the original medicinal material. The preparation is stable and easy to preserve for a long time and for subsequent drug development.

[0014] To verify the preventive and therapeutic effects of lyophilized buckwheat powder on antimony pneumoconiosis, this invention established a mouse model of antimony pneumoconiosis by intratracheal instillation of a suspension of metallic antimony dust. SPF-grade male C57BL / 6 mice were randomly divided into a control group (Group C), an antimony dust exposure group (Sb group), a lyophilized buckwheat intervention group (Sb group), and a combined antimony dust and lyophilized buckwheat treatment group (Sb & lyophilized buckwheat group). The combined treatment group received daily oral gavage administration of 300 mg / kg body weight of lyophilized buckwheat powder suspension for 28 days while being exposed to antimony dust, simulating the early intervention process under occupational exposure conditions.

[0015] Experimental results showed that *Fagopyrum esculentum* intervention significantly alleviated pathological damage to lung tissue caused by antimony dust. Histological analysis revealed that the combined treatment group showed milder alveolar structural damage, significantly reduced inflammatory cell infiltration, and reduced collagen fiber deposition. Molecular biological analysis further revealed that *Fagopyrum esculentum* effectively inhibited the overexpression of antimony dust-induced collagen I (Col-1) and α-smooth muscle actin (α-SMA) at both the protein and gene levels, while significantly reducing the hydroxyproline content in lung tissue. Furthermore, *Fagopyrum esculentum* intervention also alleviated the weight loss and increased lung coefficient in mice exposed to antimony dust. These results collectively indicate that freeze-dried *Fagopyrum esculentum* powder has a clear inhibitory effect on the occurrence and development of antimony pneumoconiosis.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1) This invention is the first to reveal the application value of freeze-dried buckwheat powder in the prevention and treatment of antimony pneumoconiosis. Currently, there are no specific drugs for the treatment of antimony pneumoconiosis, and clinical treatment is mainly symptomatic. This invention provides a new drug intervention approach for this disease and fills a technological gap in related fields.

[0018] 2) Buckwheat is a traditional medicinal plant with a good foundation for food safety. Developing it into a preventive drug for people exposed to antimony dust has advantages such as readily available raw materials, low toxicity, and good compliance among the applicable population, providing a new approach for the primary prevention of occupational antimony pneumoconiosis.

[0019] 3) The freeze-dried powder preparation process used in this invention is mature and the quality is controllable. It has good potential for industrialization and transformation, which is conducive to promoting the practical application of related preventive drugs. Attached Figure Description

[0020] Figure 1HE staining images of lung tissue sections from mice in different treatment groups [control group (C group), antimony exposure group (Sb group), buckwheat intervention group (buckwheat group), combined exposure intervention group (Sb & buckwheat group)].

[0021] Figure 2 Western blot results of Col-1 and α-SMA protein expression in lung tissue of mice in different treatment groups [control group (C group), antimony exposure group (Sb group), buckwheat intervention group (buckwheat group), combined exposure intervention group (Sb & buckwheat group)]. * P<0.05, **** (P<0.001, n=8)

[0022] Figure 3 The image shows the real-time quantitative PCR results of Col-1 and α-SMA gene expression in the lung tissue of mice in different treatment groups [control group (C group), antimony exposure group (Sb group), buckwheat intervention group (buckwheat group), combined exposure intervention group (Sb & buckwheat group)]. * (P<0.05, n=8)

[0023] Figure 4 The graph shows the results of hydroxyproline content measurement in lung tissue of mice in different treatment groups [control group (C group), antimony exposure group (Sb group), buckwheat intervention group (buckwheat group), combined exposure intervention group (Sb & buckwheat group)]. ** P<0.01, n=8). Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the scope of protection of the present invention. The embodiments described in this invention are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0025] Example 1: Preparation of freeze-dried buckwheat powder

[0026] 12.5 kg of dried rhizomes of Fagopyrum dibotrys (D. Don) Hara (purchased from Nantong Sanyue Chinese Medicine Pieces Co., Ltd., batch number: 240313) were sliced ​​and soaked in 50 L of water for 1 hour. After sealing, the mixture was steamed for 4 hours and the resulting liquid was cooled.

[0027] After the above-mentioned cooled medicinal solution was filtered to remove insoluble matter, the filtrate was filtered through a 0.45 μm filter membrane and then freeze-dried to obtain lyophilized buckwheat powder (approximately 840 g).

[0028] Example 2: Construction of an Antimony Exposure Model

[0029] Laboratory animals:

[0030] Thirty-two healthy male SPF-grade C57BL / 6 mice, 6 weeks old and weighing 20–22 g, were purchased from the Experimental Animal Center of Nantong University. All mice were acclimatized for one week in an SPF-grade animal experimental barrier environment before the experiment. The rearing conditions were: ambient temperature 22 ± 1℃, relative humidity 50 ± 10%, 12 h / 12 ​​h light / dark cycle, and free access to standard laboratory feed and sterile drinking water.

[0031] Experimental Groups:

[0032] After the acclimatization period, mice were randomly divided into four groups of eight mice each using a random number table based on their body weight: Control group (C group): sham surgery was performed (intratracheal instillation of an equal volume of PBS), and an equal volume of PBS was administered daily by gavage; Antimony exposure group (Sb group): antimony suspension was instilled via the trachea, and an equal volume of PBS was administered daily by gavage; Buckwheat intervention group (Buckwheat group): sham surgery was performed, and lyophilized buckwheat powder suspension (300 mg / kg) was administered daily by gavage; Combined exposure intervention group (Sb & Buckwheat group): antimony suspension was instilled via the trachea, and lyophilized buckwheat powder suspension (300 mg / kg) was administered daily by gavage.

[0033] Preparation and identification of antimony suspension:

[0034] Amorphous antimony (purity ≥ 99%, particle size ≤ 5 μm) was used to prepare a suspension with a concentration of 0.05 g / mL using sterile PBS. The particles were thoroughly dispersed by ultrasonic cell disruption (40 kHz) for 30 minutes. Dynamic light scattering particle size analysis showed that the average hydrated particle size of the suspension was 180 ± 25 nm, and the polydispersity index (PDI) was less than 0.2, indicating uniform particle dispersion and system stability.

[0035] Establishment of antimony pneumoconiosis model and drug administration:

[0036] Mice were anesthetized with 2% isoflurane inhalation, fixed in a supine position, and the neck skin was disinfected. A 1 cm incision was made along the midline, and the muscle tissue was bluntly dissected to expose the trachea. 50 μL of antimony suspension (containing 2.5 mg of antimony per mouse) was slowly injected via a 26G intravenous catheter. Immediately after injection, the mice were rotated upright to ensure even distribution of the particles in the lungs. The control group and the FD group received an equal volume of PBS using the same method. Postoperatively, the skin incision was sutured with sterile sutures, and 0.5% povidone-iodine ointment was applied to the incision to prevent infection. The mice's mental state and incision healing were observed for 3 consecutive days postoperatively. Frozen buckwheat powder was freshly prepared into a suspension of the appropriate concentration with PBS before each gavage. Starting from the day after model establishment, the frozen buckwheat powder intervention group and the combined exposure intervention group were administered the frozen buckwheat powder suspension by gavage at a fixed time daily, while the control group and the antimony exposure group were administered an equal volume of PBS by gavage, for a total of 4 weeks.

[0037] Compared with the control group (Group C), the lung tissue of mice in the antimony exposure group (Sb group) showed typical fibrotic pathological changes. Figure 1 H&E staining results showed that the alveolar structure of mice in the antimony-exposed group (Sb group) was disrupted, with a large number of inflammatory cell infiltrations and collagen fiber deposition in the interstitium, and the degree of fibrosis was significantly higher than that in the control group. Western Blot ( Figure 2 ) and real-time quantitative PCR ( Figure 3 The test results showed that the protein and mRNA expression levels of COL-1 and α-SMA in the lung tissue of the antimony exposure group (Sb group) were significantly upregulated (p < 0.05). Simultaneously, the hydroxyproline content in the lung tissue was also significantly increased. Figure 4 (p < 0.01), indicating increased collagen deposition. These results collectively demonstrate that this embodiment successfully established an antimony-induced mouse model of antimony pneumoconiosis.

[0038] Example 3: Evaluation of the protective / therapeutic effects of lyophilized buckwheat powder on a mouse model of antimony pneumoconiosis.

[0039] Drug intervention plan:

[0040] The lyophilized buckwheat powder prepared as described in Example 1 was freshly prepared into a 30 mg / mL suspension with phosphate-buffered saline (PBS) before each gavage administration. The buckwheat intervention group (b& buckwheat group) and the combined exposure intervention group (Sb&b group) were administered the powder daily by gavage at a dose of 300 mg / kg body weight. The control group (C group) and the antimony exposure group (Sb group) were given an equal volume of PBS as controls. All gavage solutions were freshly prepared and used immediately, and stored at 4°C protected from light to ensure the stability of the active ingredients. The intervention began on day 1 after model establishment and continued for 4 weeks.

[0041] Physiological indicator monitoring:

[0042] During the experiment, the weight changes of mice in each group were recorded daily at regular intervals. The respiratory rate (breaths / minute) of mice in a resting state was measured using a non-invasive respiratory rate monitor, and changes in vesicular sounds were assessed by auscultation. Mice exhibiting abnormal signs such as rapid breathing or moist rales, or whose weight loss exceeded 20% of their initial weight, were excluded.

[0043] Sample collection and processing:

[0044] Euthanasia was performed 24 hours after the last intervention. Lung tissue was rapidly dissected under aseptic conditions and the surface blood was washed away with pre-cooled PBS. The right middle lobe was fixed in 4% paraformaldehyde solution for subsequent paraffin embedding, sectioning, and histopathological analysis including H&E staining and Masson staining. The remaining lung tissue was flash-frozen in liquid nitrogen and then transferred to a -80°C cryopreservation freezer for subsequent protein, gene, and hydroxyproline content detection.

[0045] Test results:

[0046] The H&E staining results are attached. Figure 1 As shown in the figure, compared with the control group (Group C), the alveolar structure of mice in the antimony exposure group (Sb group) was significantly damaged, with a large number of inflammatory cell infiltrations and collagen fiber depositions visible in the interstitium, indicating that the antimony pneumoconiosis mouse model was successfully established. The lung tissue structure of the buckwheat intervention group (Buckwheat group) was intact, with no obvious pathological changes, indicating that buckwheat itself has no adverse effect on the alveolar structure of mice. Compared with the antimony exposure group (Sb group), the degree of alveolar structural damage in the combined exposure intervention group (Sb & Buckwheat group) was significantly reduced, and inflammatory cell infiltration and fibrosis were significantly improved, indicating that the Buckwheat freeze-dried powder intervention involved in this invention can effectively alleviate antimony-induced lung tissue damage.

[0047] Western Blot results (see attached) Figure 2 Compared with the antimony exposure group (Sb group), the combined exposure intervention group (Sb & buckwheat group) showed significantly lower levels of Col-1 and α-SMA protein expression in lung tissue (p < 0.05). Real-time quantitative PCR further confirmed this. Figure 3 In the combined exposure intervention group (Sb & buckwheat group), the mRNA expression of COL1 and α-SMA was also significantly inhibited (p < 0.05). Hydroxyproline content detection results ( Figure 4 The results showed that intervention with buckwheat can effectively inhibit antimony-induced collagen deposition in lung tissue (p < 0.01).

[0048] In summary, the freeze-dried buckwheat powder provided by this invention can effectively inhibit the expression of key indicators of pulmonary fibrosis induced by Sb exposure, reduce collagen deposition and lung tissue damage, and demonstrate a good preventive effect against antimony pneumoconiosis in animal models. This invention provides reliable experimental evidence for the clinical prevention of antimony pneumoconiosis using buckwheat and lays a theoretical foundation for the development of antimony pneumoconiosis prevention and treatment drugs with traditional Chinese medicine characteristics.

[0049] The descriptions and practices disclosed in this invention are readily apparent and understandable to those skilled in the art, and various modifications and refinements can be made without departing from the principles of this invention. Therefore, any modifications or improvements made without departing from the spirit of this invention should also be considered within the scope of protection of this invention.

Claims

1. The use of a freeze-dried buckwheat powder in the preparation of a medicine for the prevention or treatment of antimony pneumoconiosis.

2. The use as described in claim 1, characterized in that, The drug is used to alleviate pulmonary fibrosis caused by antimony exposure.

3. The use as described in claim 1 or 2, characterized in that, The drug is used to reduce collagen deposition in lung tissue in the antimony exposure model group, and / or to downregulate the expression levels of Col-1 and α-SMA proteins and genes in lung tissue.

4. The use as described in claim 1, characterized in that, The drug was used to reduce the content of hydroxyproline in the lung tissue of the antimony exposure model group.

Images