CN113332309A - Application of high sulfated fucogalactan from brown algae in medicine and health product for preventing and treating pulmonary fibrosis - Google Patents
Application of high sulfated fucogalactan from brown algae in medicine and health product for preventing and treating pulmonary fibrosis Download PDFInfo
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- CN113332309A CN113332309A CN202110567851.4A CN202110567851A CN113332309A CN 113332309 A CN113332309 A CN 113332309A CN 202110567851 A CN202110567851 A CN 202110567851A CN 113332309 A CN113332309 A CN 113332309A
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- pulmonary fibrosis
- brown algae
- preventing
- high sulfated
- sulfated fucan
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Abstract
The invention discloses application of high sulfated fucan from brown algae in drugs and health care products for preventing and treating pulmonary fibrosis. The high sulfated fucan can remarkably improve pulmonary fibrosis degree, inhibit fibroblast hyperproliferation and activation, and reduce lung extracellular matrix (ECM) deposition. The high sulfated fucan is proved to be capable of down-regulating a PI3K-AKT pathway and reducing the expression of TGF-beta, beta-catenin and COL2A1 at cellular and animal levels. The high sulfated fucan from brown algae can relieve wet cold and damp heat, inflammatory reaction, viral environment and pulmonary fibrosis caused by PM2.5 inhalation, protect the structural integrity of lung, reduce the pulmonary epithelial-mesenchymal transition (EMT) phenomenon and collagen deposition and reduce the inflammatory reaction of organisms. The high sulfated fucan derived from brown algae has wide application in medicines, health products and foods for preventing and treating pulmonary fibrosis related symptoms/diseases.
Description
Technical Field
The invention belongs to the field of medicines, relates to a new application of brown algae polysaccharide, and particularly relates to an application of high sulfated fucogalactan in medicines and health products for preventing and treating pulmonary fibrosis.
Background
The novel coronavirus pneumonia (COVID-19) is caused by a novel beta coronavirus, namely a novel coronavirus, and the invasion of cells by using angiotensin converting enzyme 2(ACE2) as a receptor causes lung injury, so that severe patients with severe disease exacerbation and secondary systemic inflammatory response can have acute respiratory distress syndrome and septic shock and finally have multi-organ failure. The CODVID-19 has strong infectivity, can cause various serious complications and poses great threat to global public safety. At present, most of medicines such as balitinib, Reidesvir, chloroquine and the like are in a clinical test stage, and no specific medicine treatment exists.
Early manifestations of coronavirus pneumonitis disease may be associated with uncontrolled viral replication, while later lung injury may be immunopathological lesions due to the body's immune response. 2019-nCoV and SARS-CoV outbreak in 2003 act on ACE2 receptor, enter cells through receptor-mediated endocytosis, mainly infect ciliated bronchial epithelial cells and alveolar cells, increase pulmonary vascular permeability, and induce acute lung injury, which is manifested by diffuse alveolar injury and acute fibrinolytic pneumonia in later stage. In addition to the damage of lung tissue caused by virus directly, the inflammatory reaction is further aggravated by the induced cytokine storm, abnormally increased cytokines and over-activated immune cells are activated and recruited in the lung to cause diffuse damage of lung capillary endothelial cells and alveolar epithelial cells, a large amount of exudates are gathered to block the airway, the lung function is rapidly aggravated, the ARDS and respiratory-circulatory failure are caused, more serious cases can develop into uncontrolled systemic inflammatory reaction accompanied by shock, vascular leakage, disseminated intravascular coagulation and multi-organ failure, and the virus is an important factor for death of novel severe patients with coronary pneumonia. Therefore, in addition to active antiviral treatment, the treatment and improvement of alveolar injury and pulmonary fibrosis are important ways for improving the cure rate of patients with new coronary pneumonia and improving the survival quality of patients with pneumonia after recovery.
Highly sulfated fucogalactans (fucoidans) are a unique class of sulfated polysaccharides with fucose as the main constituent monosaccharide present in marine brown algae and in parts of marine echinoderms. The previous researches find that the high sulfated fucan has obvious activity of treating chronic renal failure and can obviously relieve and slow down the process of renal fibrosis. The action mechanism of the compound is to reduce the expression of fibronectin (Fn) or Connective Tissue Growth Factor (CTGF) in the renal tubular epithelial cells, maintain the normal cell morphology of the renal tubular epithelial cells and inhibit the TGF-beta 1 induced EMT transformation. The research shows that the high sulfated fucan can competitively inhibit the binding of SARS-CoV-2 virus or the receptor binding domain (S protein) of SARS virus and a target cell surface receptor (ACE2), effectively block the virus from entering cells, and has obvious SARS-CoV-2 virus or SARS virus resistance. Further research shows that on one hand, the high sulfated fucan inhibits TGF-beta dependent EMT induced by Akt pathway, and further down-regulates beta-catenin and COL2A1, and reduces collagen accumulation. On the other hand, the highly sulfated fucan can inhibit inflammatory pathways initiated by TGF-beta.
The patent reports that the high sulfated fucan from brown algae can effectively inhibit the transfer of cell epithelium to mesenchymal leaves induced by TGF-beta, has obvious treatment effect on pulmonary fibrosis induced by bleomycin and pulmonary fibrosis caused by inflammatory factors, and can be applied to prevention and treatment of pulmonary fibrosis.
Disclosure of Invention
The invention aims to provide application of high sulfated fucan from brown algae in medicaments and health care products for preventing and treating pulmonary fibrosis. The high sulfated fucan is proved to be capable of reducing the structural damage and the function loss of lung tissues through cell and animal experiments.
The high sulfated fucan mainly comprises monosaccharide including fucose and galactose, and also comprises a small amount of one or more of glucuronic acid, mannose, glucose, rhamnose, xylose, guluronic acid, mannuronic acid, etc.; the content of hydrolyzed sulfate group is 25-45%, the content of fucose is 25-45%, the content of uronic acid is 0.5-5%, and the molecular weight is 20-150 kDa.
The high sulfated fucogalactan is derived from marine brown algae, wherein the marine brown algae comprises fucoidan polysaccharide and is selected from one or more of marine brown algae including herba Zosterae Marinae, Undaria Pinnatifida, Sargassum, Fucus vesiculosus, Pelvetia siliquosa, Ascophyllum Nodosum, thallus laminariae, Cladosiphon okamuranus, Sargassum thunbergii, Sargassum fusiforme, Sargassum muticum, and Sargassum pallidum.
The high sulfated fucan brown algae polysaccharide can be used for preparing medicines, health products and foods for treating and preventing pulmonary fibrosis related symptoms/diseases.
The pulmonary fibrosis comprises pulmonary fibrosis caused by damp cold, damp heat, inflammatory reaction, virus environment, PM2.5 inhalation and the like.
The medicine, health product, food and biological material for preventing and treating fibrosis related symptoms/diseases can be added with pharmaceutically and biologically acceptable carriers or auxiliary materials.
Drawings
Figure 1. fucoidan reduces Bleomycin (BLM) -induced pulmonary fibrosis in mice. Lung photographs (a), fibrosis score (B), inflammation score (C), survival (D), representative images of IL-6, HE staining of mouse lung tissue (F) and Masson trichrome staining (G) lung sections in serum (E) were determined.
Figure 2 fucoidan attenuates the pulmonary EMT phenotype in a BLM-induced mouse model. Western blot analysis of collagen 1, β -catenin, P-PI3K, PI3K, P-AKT, AKT, TGF- β, TNF- α and IL-6 was performed for each experimental group.
Figure 3. fucoidan improves the TGF- β 1 induced fibrosis status of a549 cells. A549 cells were incubated with 10ng/mL TGF-. beta.1 and different concentrations of fucoidan (25, 50, 100, 200. mu.g/mL) for 36 hours. Cytotoxicity assay (a), cell morphology of a549 cells under ordinary light microscopy (B) and scanning electron microscopy (C).
FIG. 4 fucoidan attenuates the EMT phenotype of cells in a model of TGF- β 1-induced A549 cells. Western blot analysis was performed on Collagen 1, beta-catenin, P-PI3K, PI3K, P-AKT, AKT, TGF-beta, smad 4 and smad 2/3 for each experimental group.
Detailed Description
The invention is further illustrated by the following examples, but the scope of the invention as claimed is not limited to the examples.
Example 1 preparation and chemical composition analysis of highly sulfated fucan
(1) Removing silt from 5kg of kelp, extracting with 20 times of water in a pressure cooker for 3 hours at the temperature of 100 ℃ and 105 ℃ for 2 times. Removing algae, mixing extractive solutions for 2 times, vacuum filtering with diatomite, concentrating the filtrate, adding anhydrous ethanol 4 times the volume of the concentrated solution for precipitation, and filtering to obtain crude polysaccharide with yield: 4.84 percent.
(2) Dissolving the crude polysaccharide in water to prepare a solution with the mass concentration of 1.5%, and adding 2mol/L MgCl2Making MgCl2Adding absolute ethanol to the final mass concentration of 0.05mol/L to enable the final weight concentration of the ethanol to be 20%, stirring to generate precipitate, centrifuging to remove the precipitate, taking supernate, adding 95% ethanol to enable the final weight concentration of the ethanol to be 70-75%, stirring to generate precipitate, centrifuging to collect the precipitate, dissolving the precipitate in water to prepare an aqueous solution with the mass concentration of 2%, filling the aqueous solution into a dialysis bag, dialyzing for 2 days by using a 3500Da dialysis bag, concentrating the solution in the dialysis bag, and freeze-drying to obtain the brown algae polysaccharide, wherein the yield is 2.71%.
(3) Dissolving the brown algae polysaccharide obtained by freeze-drying in water to prepare a water solution with the concentration of 2.5%, loading the solution to a carrier column chromatography by using DEAE-Sepharose-CL-6B, eluting with 0.1mol/L NaCl, 0.5mol/L NaCl, 1.0mol/L NaCl, 1.5mol/L NaCl and 2.0mol/L NaCl solution in sequence, collecting each eluent, and filling the eluent into a 3500Da dialysis bag for dialysis and freeze-drying the solution in a concentrated dialysis bag respectively. Wherein the white flocculent precipitate eluted by 1.0mol/L NaCl is the high sulfated fucan. The chemical composition results are shown in table 1.
TABLE 1 chemical composition analysis (weight%) of highly sulfated fucogalactan (SGF)
The results show that the molecular weight of the high sulfated fucan is 82100Da, the contents of fucose and sulfuric acid groups are higher and are respectively 43.43 percent and 41.80 percent, and the content of glucuronic acid is lower and is only 0.86 percent. The neutral monosaccharide composition results show that in addition to fucose and galactose, mannose, glucose and rhamnose are contained in small amounts.
Application example 1: brown algae polysaccharide for preventing and treating pulmonary fibrosis and relieving Bleomycin (BLM) -induced pulmonary inflammation and fibrotic lesions in mice
The experiment uses a fucoidan extracted from Laminaria japonica for preventing and treating pulmonary fibrosis, wherein the fucose content is 34.8%, the uronic acid content is 2.5%, and the average molecular weight of the high sulfated fucogalactan is 80.1kDa.
Eight-week-old C57BL/6J male mice were selected, weighed 25. + -.2 g, and divided into 5 groups (blank group-Neg, BLM model group-BLM, fucoidan high dose group-BLM + DFPSH, fucoidan low dose group-BLM + DFPSH, Nintedanib group-BLM + Nintedanid), and 8 mice were each group. The model group and the administration group were administered with bleomycin (5mg/kg) through trachea, and the blank group was subjected to a sham operation and administered with an equal amount of physiological saline. The blank group is perfused with normal saline, the low-dose group is perfused with 40 mg/kg/day, the high-dose group is perfused with 80 mg/kg/day, and the positive drug is selected from the group consisting of 200 mg/kg/day of nebrodensis. Mice were monitored for weight and survival during gavage. After 14 days of administration, the mice were dissected and treated with serum and lung fluid nitrogen, and then placed in a refrigerator at-80 ℃ for later use, for biochemical determination and tissue morphology observation.
FIG. 1 shows that fucoidan reduces the decrease in survival rate and lung swelling of mice induced by BLM perfusion in the trachea. The brown algae polysaccharide can improve lung pathological condition, reduce cell proliferation, reduce collagen deposition, and reduce alveolar space and lung structure. The brown algae polysaccharide can reduce inflammation of mice caused by BLM and reduce IL-6 content in serum of mice.
Example 2: brown algae polysaccharide for preventing and treating pulmonary fibrosis reduces lung EMT and TGF-beta phenotype in BLM-induced mouse model
Figure 2 shows that a brown algae polysaccharide for prevention and treatment of pulmonary fibrosis reduced lung EMT and TGF- β phenotypes in a BLM-induced mouse model. The fucoidan can down-regulate PI3K-AKT pathway. On one hand, the inhibition of Akt pathway down-regulates beta-catenin and COL2A1, which reduces the occurrence of EMT and reduces collagen accumulation. In another aspect, the fucoidan reduces inflammatory pathways that may be initiated by TGF- β.
Example 3: brown algae polysaccharide for preventing and treating pulmonary fibrosis relieves A549 cell fibrosis state induced by TGF-beta 1
A549 cells were supplemented with 10% fetal bovine serum and 5% CO21640 in (5). Cells were plated at 5X 10 per well4Cell density was seeded into 6-well plates at 1X 10 per well4The density of cells was seeded in 24-well plates and 2000 cells per well in 96-well plates. Starved for 24 hours in serum-free medium until 60% confluence was reached, and then they were treated with TGF- β 1(10ng/mL), or co-treated with fucoidan (50, 100, 200ug/mL) or TGF- β 1 inhibitor (4 μ M) at different concentrations for 36 hours for cell viability assay, scanning electron microscopy, morphological analysis and western blot analysis.
Fig. 3 shows that a brown algae polysaccharide for preventing and treating pulmonary fibrosis can reduce the proliferation of A549 cells, increase the contact and connection among the cells and reduce the appearance of spindle-shaped fibroblasts. Electron microscopy shows that the brown algae polysaccharide can protect the epithelial appearance of A549 cells.
Figure 4 shows that the fucoidan can reduce the EMT phenotype at the cellular level. The fucoidin can down-regulate PI3K-AKT pathway, on one hand, the inhibition of Akt pathway can down-regulate beta-catenin and COL2A1, thus reducing the occurrence of EMT and collagen accumulation. In another aspect, the fucoidan reduces the likelihood that TGF- β will initiate the smad pathway.
Supplemental content
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (6)
1. Application of high sulfated fucogalactan derived from brown algae in medicine and health product for preventing and treating pulmonary fibrosis is provided.
2. Use according to claim 1, characterized in that: the high sulfated fucan mainly comprises monosaccharide including fucose and galactose, and may also include one or more of glucuronic acid, mannose, glucose, rhamnose, xylose, guluronic acid, mannuronic acid, etc.; the content of hydrolyzed sulfate group is 25-45%, the content of fucose is 25-45%, the content of uronic acid is 0.5-5%, the molecular weight is 20-150 kDa, and the preferable molecular weight is 70-150 kDa.
3. Use according to claim 1, 2, characterized in that: the high sulfated fucogalactan is derived from marine brown algae, which comprises one or more of herba Zosterae Marinae, thallus laminariae, Sargassum, sea oak, cornu Cervi, Ascophyllum Nodosum, thallus laminariae, Cladosiphon okamuranus, Sargassum thunbergii, Sargassum integrifolium, Cyrtymenia Sparsa, Sargassum sal eminensis, Sargassum pallidum, and Sargassum pallidum.
4. The use according to claim 1, characterized in that the hyper-sulfated fucan can be used in drugs, health products and foods for treating and preventing symptoms/diseases associated with pulmonary fibrosis.
5. Use according to claim 4, characterized in that: the pulmonary fibrosis comprises pulmonary fibrosis caused by damp cold, damp heat, inflammatory reaction, virus environment, PM2.5 inhalation and the like.
6. Use according to claim 4, characterized in that: the medicine, health product, food and biological material for preventing and treating fibrosis related symptoms/diseases can be added with pharmaceutically and biologically acceptable carriers or auxiliary materials.
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| CN115844928A (en) * | 2022-12-16 | 2023-03-28 | 中国科学院上海药物研究所 | Seaweed sulfated polysaccharide and preparation method and application thereof |
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| CN115569145A (en) * | 2022-03-28 | 2023-01-06 | 上海交通大学医学院附属新华医院 | Use of poly (propyl guluronate) sulfate for the preparation of a product for the prevention and treatment of atrial fibrillation |
| CN115569145B (en) * | 2022-03-28 | 2023-09-12 | 上海交通大学医学院附属新华医院 | Application of polyguluronic acid propyl sulfate in preparation of products for preventing and treating atrial fibrillation |
| CN115844928A (en) * | 2022-12-16 | 2023-03-28 | 中国科学院上海药物研究所 | Seaweed sulfated polysaccharide and preparation method and application thereof |
| CN115844928B (en) * | 2022-12-16 | 2024-03-01 | 中国科学院上海药物研究所 | Seaweed sulfated polysaccharide and preparation method and application thereof |
| WO2024125540A1 (en) * | 2022-12-16 | 2024-06-20 | 中国科学院上海药物研究所 | Seaweed-derived sulphated polysaccharide, preparation method therefor, and use thereof |
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