Application of crude polysaccharide from blue calyx chrysanthemum

Abstract

The present invention relates to the use of crude polysaccharide of blue-calyx chrysanthemum, and specifically, the present invention relates to the use of crude polysaccharide of blue-calyx chrysanthemum in preparing a medicine for treating lung injury. The crude polysaccharide of blue-calyx chrysanthemum shows obvious inflammation-reducing effect in an animal model of acute lung injury in mice, and thus can be used as a potential medicine for treating acute lung injury.

Description

Technical Field

[0001] The invention belongs to the field of medicinal chemistry, and in particular relates to the use of crude polysaccharide of Isodon calyx. Background Art

[0002] Acute lung injury (ALI) is caused by various factors that damage alveolar epithelial cells and capillary endothelial cells, resulting in diffuse interstitial and alveolar edema. Clinically, it manifests as progressive hypoxemia and respiratory distress. Lung imaging shows heterogeneous exudative lesions. The more serious form is acute respiratory distress syndrome (ARDS), which is a clinical symptom associated with shock, sepsis, SARS, COVID-19, and pulmonary ischemia-reperfusion. Although the treatment strategy of ALI / ARDS is constantly improving, such as antimicrobial therapy and major progress in supportive treatment methods such as lung protective ventilation, improved ventilator synchrony, and extracorporeal membrane oxygenation, the mortality rate is as high as 30% to 40%, and there is still a lack of effective treatment measures and drugs. Inflammatory response plays a key role in the development of acute lung injury. The main pathological changes are the infiltration of inflammatory proteins into the alveoli, accumulation of inflammatory cells, interstitial edema, and damage to epithelial integrity. Therefore, it is of great significance to find drugs for the treatment of acute lung injury.

[0003] Rabdosia japonica (Burm.f.) Hara is a variant of Rabdosia japonica (Burm.f.) Hara, a plant of the genus Isodon in the family Labiatae. At present, the research on the chemical composition of Rabdosia japonica is also mainly focused on small molecules, such as diterpenes, triterpenes, flavonoids, organic acids, sterols and other components isolated from Rabdosia japonica, but there are few reports on the macromolecular components in Rabdosia japonica. Polysaccharides exist in almost all plants to play different functions, and have been determined to have a variety of biological functions, such as antioxidants, immunomodulatory, anti-tumor, radiation protection, anti-diabetic, anti-microbial and anti-tumor functions. More and more evidence shows that polysaccharides have anti-inflammatory activity. In the research of the inventor, the polysaccharide content in Rabdosia japonica is 6.3% of the dry weight, but there is no report on the protective effect of Rabdosia japonica polysaccharides on acute lung injury. Summary of the invention

[0004] The technical purpose of the present invention is to provide a medicine that can be used to treat lung injury.

[0005] The invention provides use of crude polysaccharide of Isodon calyx in preparing a medicine for treating lung injury.

[0006] In a specific embodiment, the crude polysaccharide of Isodon calyx is a water-extraction and alcohol-precipitation product of Isodon calyx.

[0007] In a specific embodiment, the crude polysaccharide of I. cerifera is prepared by the following method: boiling I. cerifera with water, filtering, concentrating the obtained filtrate, and adding ethanol to precipitate the obtained concentrate to obtain the crude polysaccharide of I. cerifera.

[0008] In a specific embodiment, the decoction with water is repeated twice, and the two filtrates are combined for subsequent ethanol precipitation.

[0009] In a specific embodiment, the concentrated solution is mixed with ethanol in a volume ratio of 1:2-5, preferably 1:4, and as the ethanol, 95 vol% ethanol can be used.

[0010] In a specific embodiment, the crude polysaccharide of Isodon calyx can alleviate the inflammatory symptoms of lung injury.

[0011] In a specific embodiment, based on the total weight of the crude polysaccharide of blue-calyx ophiopogon, the total sugar content of the crude polysaccharide of blue-calyx ophiopogon detected by the phenol-sulfuric acid method with glucose as the standard is 20.61±0.38wt%, and the uronic acid content detected by the m-hydroxybiphenyl method with galacturonic acid as the standard is 10.96±1.68wt%.

[0012] In a specific embodiment, the lung injury may be acute lung injury.

[0013] In a specific embodiment, the molecular weight of the crude polysaccharide of I. calyx may range from 2.0 kDa to 25.0 kDa.

[0014] Beneficial Effects

[0015] The crude polysaccharide of the blue-calyx spatholobiaceae of the present invention shows a definite therapeutic effect on acute lung injury in an animal model, and can be used to reduce the inflammation of acute lung injury. Therefore, it can be used as a potential drug for treating acute lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the HPGPC diagram of the crude polysaccharide of Ipomoea aquatica (hereinafter referred to as XPS) prepared in the present invention.

[0017] Figure 2 The results show that the crude polysaccharide of Ilex calyx prepared by the present invention has a therapeutic effect on acute lung injury in mice. Figure 2 A is the H&E staining photos of the lung pathological sections of mice in the normal group, LPS model group, XPS administration group (80 mg / kg) and DEX positive drug group; Figure 2B shows the effect of XPS at different concentrations (20 mg / kg, 40 mg / kg and 80 mg / kg) on ​​the number of mononuclear cells in the alveolar lavage fluid of mice, where: * P<0.05, ** P<0.01, *** P < 0.001, compared with the LPS model group; Figure 2 C shows the effect of XPS at different concentrations (20 mg / kg, 40 mg / kg and 80 mg / kg) on ​​proteins in mouse bronchoalveolar lavage fluid, where: * P<0.05, ** P<0.01, *** P<0.001, compared with the LPS model group. DETAILED DESCRIPTION

[0018] The following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0019] Materials and instruments

[0020] Blue-calyx chrysanthemum (5.0 kg) was from Fengyang, Anhui (collected on November 20, 2015). Pullulan P-82 standard set: P-5, P-10, P-20, P-50, P-100, P-200, P-400, P-800, Shodex; water was ultrapure water (self-made in the laboratory); reagents were all analytically pure. Agilent 1260 series high performance liquid chromatograph (including autosampler, infusion pump, degasser, DAD detector, IR detector and Agilent Cirrus GPC software).

[0021] Preparation Example: Preparation of Crude Polysaccharide from Ipomoea aquatica

[0022] 5.0 kg of blue calyx fragrant tea leaves, add 50 L of water, boil for three hours, filter, add 50 L of water to the filter residue, repeat the boiling for three hours, filter, combine the two filtrates, concentrate the filtrate to an appropriate volume, add 4 times the volume of 95 vol% ethanol to precipitate, and leave overnight. The supernatant is concentrated under reduced pressure, vacuum dried at 65℃-70℃, powdered and packaged in aluminum foil, and packaged in aluminum foil; the ethanol precipitate is freeze-dried to obtain crude polysaccharide recorded as XPS (315 g, yield 6.3%). The total sugar content of XPS is 20.61±0.38wt% detected by phenol-sulfuric acid method using glucose as the standard, and the uronic acid content in XPS is 10.96±1.68wt% detected by m-hydroxybiphenyl method using galacturonic acid as the standard.

[0023] Test Example: XPS Molecular Weight Distribution Determination

[0024] High performance gel permeation chromatography (HPGPC) was used to detect the polysaccharide. A standard curve was prepared using different known molecular weight dextran standards. The molecular weight standard curve was then prepared using Agilent GPC analysis software based on the elution time of the sample and the standard curve. The XPS test results can be used to obtain the molecular weight of the polysaccharide 2.0k-25.0k Da ( Figure 1 ).

[0025] Experimental Example: Protective effect of crude polysaccharide of Xanthoceras cyanobacteria (XPS) on acute lung injury (ALI) model in mice

[0026] The experimental model animals, male BALB / c mice (16-18 g), were purchased from Shanghai Slake Laboratory Animal Co., Ltd. with license number SCXK2017-0005.

[0027] Establishment of acute lung injury model in mice

[0028] After intraperitoneal anesthesia, the mouse was fixed in a supine position on a mouse board. The fur layer of the mouse neck was cut longitudinally by 1-2 cm to expose the trachea. The fascia was separated, and a small hole was drilled in the soft tissue between the cartilage rings close to the thyroid cartilage of the trachea with a 1mL disposable injection needle. A 0.1mL microinjector with a smoothed blade was used to insert the needle for tracheal instillation. Each mouse was injected with 3mg / kg lipopolysaccharide (LPS) (0.05mL / 20g). The infusion time was controlled at 5-6min. The mouse was placed on the table and carefully turned over so that the LPS was evenly distributed in the lungs. After the end, the skin incision was sutured with silk thread and the light was turned on to keep warm. After waking up, it was placed in a mouse cage.

[0029] The experiment divided the mice into a normal group, a model group, a polysaccharide group (XPS) high, medium, and low dose groups, and a positive drug dexamethasone group (DEX). The mouse model was prepared as described above. The normal group mice were instilled with normal saline through the trachea, and the other groups of mice were instilled with LPS through the trachea. After the mice woke up from anesthesia (2h after tracheal instillation), the normal group and the model group were gavaged with normal saline. The three dose groups of the polysaccharide group (XPS) were gavaged, and the doses were 20mg / kg, 40mg / kg, and 80mg / kg, respectively. The positive drug dexamethasone group (DEX) was injected into the tail vein, and the dose of DEX injection was 4mg / kg.

[0030] 24 hours after tracheal instillation, the mice were killed and the samples were collected. The left lung of the mice was lavaged and the lavage fluid was recovered and centrifuged. The supernatant was collected as the alveolar lavage fluid (BALF) for determination of extravasated protein. The lower cell pellet was resuspended with 100 μL 0.01 mol / L phosphate buffer (PBS) and counted. The right upper lobe of the lung was immersed in formalin solution.

[0031] The effect of crude polysaccharide XPS from Scutellaria baicalensis on lung pathological damage in mice with acute lung injury was observed by H&E staining of mouse lung pathological sections. The alveolar structure of the normal control group was clear and complete. Due to the injection of a small amount of normal saline into this group, some alveoli showed mild exudation and alveolar wall thickening. In the LPS-induced model group, the alveolar wall was significantly thickened, with bleeding and inflammatory cell infiltration accompanied by obvious alveolar collapse. The lung pathological damage in the drug-treated group (80 mg / kg) was significantly reduced. Although the alveolar wall still showed thickening, most of the alveolar structures remained basically normal ( Figure 2 A).

[0032] The results of mononuclear cell counts in mouse bronchoalveolar lavage fluid (BALF) showed that the number of mononuclear cells in the model group was significantly higher than that in the normal group ( Figure 2 B, P<0.001); compared with the LPS-induced model group, the number of mononuclear cells in the bronchoalveolar lavage fluid of the XPS-treated group was significantly decreased ( Figure 2 B, P<0.05). The results were consistent with the pathological observation, indicating that the model group mice had obvious lung inflammation and increased leukocyte infiltration. Administration of the drug significantly improved the inflammatory state of increased leukocyte infiltration in the lungs of mice.

[0033] The BCA method was further used to determine the content of extravasated protein in BALF of each group. The results showed that compared with the normal control group, the model group had obvious lung inflammation and the total protein concentration in the alveolar lavage fluid was significantly increased (P<0.001); compared with the LPS group, the total protein concentration in the alveolar lavage fluid of the XPS-treated group was significantly reduced (both P<0.05), indicating that XPS significantly reduced the exudation of lung tissue protein ( Figure 2 C).

[0034] From the above results, it can be seen that the crude polysaccharide XPS from the blue-calyx spatholobi can significantly reduce the inflammatory state of acute lung injury. Therefore, it can be effectively used in the treatment of lung injury, especially acute lung injury.

Claims

1. Use of crude polysaccharide of Isodon calyx in the preparation of a medicament for treating acute lung injury, in, The crude polysaccharide of Isodon calyx is a water-extraction and alcohol-precipitation product of Isodon calyx, and has a molecular weight range of 2.0 kDa-25.0 kDa.

2. The use according to claim 1, wherein The crude polysaccharide of Ipomoea aquatica is prepared by the following method: boiling Ipomoea aquatica with water, filtering, concentrating the obtained filtrate, and adding ethanol to precipitate the obtained concentrated solution.

3. The use according to claim 2, wherein The decoction with water was repeated twice, and the two filtrates were combined for subsequent ethanol precipitation.

4. The use according to claim 2, wherein The obtained concentrated solution is mixed with ethanol in a volume ratio of 1:2-5.

5. The use according to claim 1, wherein The uses include alleviating inflammatory symptoms of lung injury.

6. The use according to claim 1, wherein Based on the total weight of the crude polysaccharide of blue-calyx ophiopogon, the total sugar content of the crude polysaccharide of blue-calyx ophiopogon was 20.61±0.38wt% as detected by the phenol-sulfuric acid method using glucose as the standard, and the uronic acid content was 10.96±1.68wt% as detected by the m-hydroxybiphenyl method using galacturonic acid as the standard.

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