CN114099538A - Application of rabdosia glauca crude polysaccharide - Google Patents

Abstract

The invention relates to application of rabdosia glaucocalyx crude polysaccharide, in particular to application of rabdosia glaucocalyx crude polysaccharide in preparation of a medicine for treating lung injury. The rabdosia glaucocalyx crude polysaccharide shows an obvious inflammation relieving effect in an animal model of acute lung injury of mice, so that the rabdosia glaucocalyx crude polysaccharide can be used as a potential medicament for treating acute lung injury.

Description

Application of rabdosia glauca crude polysaccharide

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to application of rabdosia japonica crude polysaccharide.

Background

Acute Lung Injury (ALI) is caused by various factors, such as alveolar epithelial cell and capillary endothelial cell injury, causing diffuse interstitial pulmonary and alveolar edema, which is clinically manifested as progressive hypoxemia and respiratory distress, pulmonary imaging as non-uniform exudative disease, and more serious Acute Respiratory Distress Syndrome (ARDS), which is a clinical symptom accompanied by diseases such as shock, sepsis, SARS, covi-19 and pulmonary ischemia-reperfusion. Despite the major advances in ALI/ARDS treatment strategies, such as antimicrobial therapy and supportive therapies such as pulmonary protective ventilation, ventilator synchronization improvement, and extracorporeal membrane oxygenation, mortality rates of up to 30% to 40% remain lacking as effective therapeutic measures and drugs. The inflammatory response plays a key role in the development of acute lung injury, and the main pathological changes are infiltration of inflammatory proteins into alveoli, accumulation of inflammatory cells, interstitial edema and epithelial integrity damage, so that the discovery of a drug for treating acute lung injury is of great significance.

Isodon japonicus is an original variety of Isodon japonicus (Latin name Rabdosia japonica (burm. f.) Hara) belonging to family Labiatae (Labiatae) genus Isodon. At present, the research on the chemical components of rabdosia glaucocalyx is mainly focused on small molecules, for example, diterpenes, triterpenes, flavones, organic acids, sterols and other components are separated from the rabdosia glaucocalyx, but the large molecular components in the rabdosia glaucocalyx are only reported. Polysaccharides are present in almost all plants to perform different functions, and have been identified to have various biological functions, such as antioxidant, immunomodulating, antitumor, radioprotective, antidiabetic, antimicrobial and antitumor functions. There is increasing evidence that polysaccharides have anti-inflammatory activity. In the research of the inventor, the content of polysaccharide in rabdosia japonica is 6.3% of dry weight, but no report on the acute lung injury protection effect of rabdosia japonica polysaccharide exists so far.

Disclosure of Invention

The technical object of the present invention is to provide a drug which can be used for treating lung injury.

The invention provides application of rabdosia japonica crude polysaccharide in preparation of a medicine for treating lung injury.

In a specific embodiment, the rabdosia japonica crude polysaccharide is a water extraction and alcohol precipitation product of rabdosia japonica.

In a specific embodiment, the rabdosia japonica crude polysaccharide is prepared by the following method: decocting rabdosia glaucocalyx with water, filtering, concentrating the obtained filtrate, adding ethanol into the obtained concentrated solution for precipitation, and obtaining the rabdosia glaucocalyx crude polysaccharide.

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

In a specific embodiment, the resulting concentrate is mixed with ethanol in a volume ratio of 1:2 to 5, preferably 1:4, and as the ethanol, 95 vol% ethanol may be used.

In particular embodiments, the rabdosia japonica crude polysaccharide can alleviate inflammatory symptoms of lung injury.

In a specific embodiment, the rabdosia japonica crude polysaccharide has a total sugar content of 20.61 ± 0.38 wt% measured by a phenol-sulfuric acid method with glucose as a standard, and has an uronic acid content of 10.96 ± 1.68 wt% measured by a m-hydroxybiphenyl method with galacturonic acid as a standard, based on the total weight of the rabdosia japonica crude polysaccharide.

In a specific embodiment, the lung injury can be an acute lung injury.

In a specific embodiment, the rabdosia japonica crude polysaccharide may have a molecular weight ranging from 2.0kDa to 25.0 kDa.

Advantageous effects

The rabdosia japonica crude polysaccharide shows a determined acute lung injury treatment effect in an animal model, and can be used for relieving inflammation of acute lung injury, so that the rabdosia japonica crude polysaccharide can be used as a potential medicine for treating acute lung injury.

Drawings

FIG. 1 is a HPGPC chart of rabdosia japonica crude polysaccharide (hereinafter referred to as XPS) prepared by the present invention.

FIG. 2 shows the therapeutic effect of rabdosia glaucocalyx crude polysaccharide prepared by the invention on acute lung injury of mice. Wherein: FIG. 2A is a lung pathology section of mice in the normal group, LPS model group, XPS administration group (80mg/kg) and DEX positive drug groupH&E, dyeing a photo; FIG. 2B shows the effect of XPS at different concentrations (20mg/kg,40mg/kg and 80mg/kg) on the number of mononuclear cells in mouse alveolar lavage fluid, wherein,^*P<0.05，^**P<0.01，^***P<0.001, compared to LPS model group; FIG. 2C shows the effect of XPS at different concentrations (20mg/kg,40mg/kg and 80mg/kg) on protein in mouse alveolar lavage fluid, wherein,^*P<0.05，^**P<0.01，^***P<0.001, compared to LPS model group.

Detailed Description

The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention.

Materials and instruments

Isodon japonicus (5.0kg) was derived from the Aphanthus annuus (collected in 2015 at 11 months and 20 days) pullulan P-82 standard set: p-5, P-10, P-20, P-50, P-100, P-200, P-400, P-800, Shodex; water is ultrapure water (self-made in laboratories); all reagents are analytically pure. Agilent 1260 series high performance liquid chromatography (including autosampler, infusion pump, degasser, DAD detector, IR detector, and Agilent Cirrus GPC software).

Preparation examples: preparation of rabdosia glauca crude polysaccharide

5.0kg of rabdosia japonica, 50L of water is added, the decoction is carried out for three hours, the filtration is carried out, 50L of water is added to the filter residue, the decoction is carried out for three hours repeatedly, the filtration is carried out, the two filtrates are combined, the filtrate is concentrated to the proper volume, and the 4 times volume of 95 vol% ethanol is added for precipitation and stays overnight. Concentrating the supernatant under reduced pressure, vacuum drying at 65-70 deg.C, pulverizing into powder, packaging with aluminum foil, and packaging with aluminum foil; the ethanol precipitate was freeze dried to give crude polysaccharide as XPS (315g, 6.3% yield). The total sugar content of XPS is 20.61 + -0.38 wt% by phenol-sulfuric acid method with glucose as standard, and uronic acid content of XPS is 10.96 + -1.68 wt% by m-hydroxybiphenyl method with galacturonic acid as standard.

Test examples: XPS molecular weight distribution determination

A standard curve is prepared by using dextran standards with different known molecular weights through High Performance Gel Permeation Chromatography (HPGPC), and then a molecular weight standard curve is prepared by using Agilent GPC analysis software according to the comparison of the elution time of a sample and the standard curve, and the molecular weight of the polysaccharide is 2.0k-25.0k Da through XPS (figure 1).

Experimental examples: protection effect of rabdosia japonica crude polysaccharide (XPS) on mouse Acute Lung Injury (ALI) model

Male BALB/c mice (16-18g) of the experimental model animal were purchased from Shanghai Slek laboratory animals Co., Ltd, license number SCXK 2017-0005.

Establishment of mouse acute lung injury model

After the abdominal cavity of the mouse is anesthetized, the mouse is fixed on the mouse plate in an upward position. The mouse neck cortex was cut longitudinally 1-2cm, exposing the trachea. The fascia was separated, a small hole was drilled in the soft tissue between the cartilage rings immediately adjacent to the trachea thyroid cartilage using a 1mL disposable needle, the trachea instilled using a 0.1mL microsyringe needle with a flat edge, and each mouse was infused with 3mg/kg Lipopolysaccharide (LPS) (0.05mL/20 g). The dripping time is controlled within 5-6 min. Mice were placed on a table and carefully inverted so that LPS was evenly distributed in the lungs. After finishing, the skin incision is sutured with silk thread, and the lamp is used for keeping warm. And putting the mouse cage after waking up.

The experiment divided the mice into normal, model, polysaccharide (XPS) high, medium and low dose groups and a positive drug Dexamethasone (DEX). Mouse models were prepared as described above. Normal mice were instilled intratracheally with saline, and the remaining mice were instilled intratracheally with LPS. After the mice were anesthetized and recovered (2 h after tracheal instillation), the normal group and the model group were administered by gastric gavage with physiological saline. The polysaccharide group (XPS) three dose groups were administered by gavage, at doses of 20mg/kg,40mg/kg and 80mg/kg respectively. The Dexamethasone (DEX) group as the positive drug is administrated by tail vein injection, and the dosage of DEX injection is 4 mg/kg.

24 hours after tracheal instillation, the mice were sacrificed and the left lungs were lavaged and the lavages recovered and centrifuged, the supernatant was collected as the alveolar lavage fluid (BALF) for determination of extravasated protein, the lower cell pellet was resuspended in 100. mu.L of 0.01mol/L Phosphate Buffered Saline (PBS) and counted, and the upper right lung lobe was soaked in formalin solution.

The influence of XPS on the rabdosia japonica crude polysaccharide on the lung pathological injury of the mice with acute lung injury is observed through H & E staining of the lung pathological sections of the mice. The normal control group had a clear and intact alveolar structure, and some of the alveoli exhibited mild exudation and alveolar wall thickening due to the small saline injection in this group. The model group induced by administration of LPS showed significant thickening of alveolar walls, bleeding and inflammatory cell exudation with significant alveolar collapse. The pathological injury of the lung in the administration group (80mg/kg) is obviously reduced, although the alveolar wall is still thickened, most alveolar structures are basically maintained to be normal (figure 2A).

The counting of the number of mononuclear cells in mouse alveolar lavage fluid (BALF) shows that the number of mononuclear cells in the model group is obviously increased compared with that in the normal group (FIG. 2B, P < 0.001); whereas the number of mononuclear cells in alveolar lavage fluid was significantly reduced in the XPS-dosed group compared to the LPS-induced model group (FIG. 2B, P < 0.05). The results are consistent with pathological observation, which shows that the lung inflammation of the model group mice is obvious and the infiltration of white blood cells is increased. The administration obviously improves the inflammatory state of the increase of the white blood cell exudation of the lung of the mouse.

The BCA method was further used to determine the amount of extravasated protein in each group of BALF. The results show that compared with the normal control group, the lung inflammation of the model group is obvious, and the total protein concentration in the alveolar lavage fluid is obviously increased (P < 0.001); the total protein concentration in alveolar lavage fluid of rabdosia glaucocalyx crude polysaccharide XPS-administered group was significantly reduced (both P <0.05) compared to LPS group, indicating that XPS significantly reduced the exudation of lung tissue proteins (fig. 2C).

From the above results, it can be seen that XPS, a rabdosia japonica crude polysaccharide, can significantly reduce the inflammatory state of acute lung injury, and thus, can be effectively used for the treatment of lung injury, particularly acute lung injury.

Claims (9)

1. Application of rabdosia japonica crude polysaccharide in preparation of medicines for treating lung injury.

2. The use of claim 1, wherein the rabdosia japonica crude polysaccharide is a water extraction and alcohol precipitation product of rabdosia japonica.

3. The use of claim 1, wherein the rabdosia glaucocalyx crude polysaccharide is prepared by the following method: decocting Rabdosia glaucocalyx with water, filtering, concentrating the filtrate, and precipitating with ethanol.

4. The use according to claim 3, wherein the water decoction is repeated twice, and the two filtrates are combined for subsequent ethanol precipitation.

5. Use according to claim 3, wherein the concentrate obtained is mixed with ethanol in a volume ratio of 1: 2-5.

6. The use of claim 1, wherein the use comprises alleviating an inflammatory symptom of lung injury.

7. The use according to claim 1, wherein the rabdosia japonica crude polysaccharide has a total sugar content of 20.61 ± 0.38 wt% measured by phenol-sulfuric acid method using glucose as a standard, and an uronic acid content of 10.96 ± 1.68 wt% measured by m-hydroxybiphenyl method using galacturonic acid as a standard, based on the total weight of the rabdosia japonica crude polysaccharide.

8. The use of claim 1, wherein the lung injury is acute lung injury.

9. The use of claim 1, wherein the rabdosia japonica crude polysaccharide has a molecular weight in the range of 2.0kDa to 25.0 kDa.

Images