Cantharellus cibarius polysaccharide as well as preparation method and application thereof

Abstract

The invention provides cantharellus cibarius polysaccharide, the cantharellus cibarius polysaccharide is formed by polymerizing five monosaccharides including galactose, glucose, xylose, fucose and glucosamine hydrochloride, the chemical formula of the cantharellus cibarius polysaccharide is as shown in formula (I): # imgabs0, and the cantharellus cibarius polysaccharide provided by the invention is a single polysaccharide active substance and has the effect of effectively inhibiting lung cell inflammation. The invention further provides a preparation method of the chanterelle polysaccharide and application of the chanterelle polysaccharide in preparation of drugs for inhibiting lung cell inflammation.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a chanterelle polysaccharide and a preparation method and application thereof. Background Art

[0002] Acute lung injury and acute respiratory distress syndrome (ALI / ARDS) are acute inflammatory respiratory diseases caused by various forms of lung damage, resulting in high morbidity and mortality. The primary pathological feature of ALI / ARDS is pulmonary inflammatory infiltration. Human pulmonary epithelial cells protect against the invasion of heavy metals and microorganisms. Abnormalities in the epithelial barrier structure and function can be found in patients with certain lung diseases, such as acute lung injury and chronic obstructive pulmonary disease, and therefore serve as potential biomarkers for early signs.

[0003] ALI / ARDS treatment mainly focuses on mechanical ventilation and drug intervention. The former can cause varying degrees of lung tissue damage and only has a relieving effect on the disease. Many related drugs fail to achieve the expected results in clinical trials and have significant side effects. Therefore, it is necessary to find effective and low-toxic ALI / ARDS therapeutic drugs from natural products to achieve the treatment of lung inflammation.

[0004] Research has shown that large fungi and certain active ingredients have significant effects in relieving and treating lung inflammation. Fungal polysaccharides possess diverse biological activities, including hypoglycemic, immune-enhancing, antioxidant, anti-tumor, and anti-inflammatory properties, and hold enormous potential for application in diverse fields, including molecular biology, immunology, biotechnology, and medicinal chemistry. Cantharellus tabernensis is a rare medicinal fungus belonging to the genus Cantharellus. These fungi are nutritious, crisp, and delicious, with a texture similar to chicken fat. Research has shown that polysaccharides extracted from these fungi exhibit diverse biological activities, including antioxidant, immune-enhancing, anti-tumor, and hypoglycemic and lipid-lowering properties.

[0005] In the extraction of fungal polysaccharides, hot water extraction and complex enzyme methods are commonly used in the prior art to extract macrofungal polysaccharides. The hot water extraction process for extracting macrofungal polysaccharides is as follows: macrofungal powder → 80°C hot water extraction for 8 hours → filtration to remove residue → centrifugation (8,000 rpm, 10 minutes) → supernatant → 8% trichloroacetic acid added to the supernatant until the extract is turbid (to remove protein) → centrifugation (8,000 rpm, 20 minutes) → supernatant evaporated at 60°C to approximately 1 / 4 of its volume → 4 volumes of 95% ethanol added and allowed to stand for 24-48 hours → filtration → centrifugation of the filter residue (8,000 rpm, 20 minutes) to obtain the precipitate → washing with anhydrous ethanol and collecting the precipitate → dissolving the precipitate in distilled water and dialysis for 48 hours → freeze-drying → obtaining crude macrofungal polysaccharides. This method is simple to operate and easy to obtain the target polysaccharide, but the extraction temperature is high and the time is long, which causes more impurities to dissolve in the extract, increasing the difficulty of subsequent separation and purification. In addition, high temperature can cause changes in the polysaccharide structure, ultimately affecting its biological activity.

[0006] The process of extracting macrofungal polysaccharides by the composite enzyme method is: macrofungal powder → solid-liquid ratio 1:90, add papain, pectinase, and cellulase in a ratio of 1:1:1, heat at 62°C for 3h → filter and remove residue → centrifuge (8,000r / min, 10min) → take the supernatant → add 8% trichloroacetic acid to the supernatant until the extract is not turbid (to remove protein) → centrifuge (8,000r / min, 20min) → take the supernatant and rotary evaporate to about 1 / 4 of its volume at 60°C → add 4 times the volume of 95% ethanol and let stand for 24 to 48h → filter → centrifuge the residue (8,000r / min, 20min) and take the precipitate → wash with anhydrous ethanol and collect the precipitate → dissolve the precipitate in distilled water and dialyze for 48h → freeze-dry → obtain crude macrofungal polysaccharides. Enzyme-assisted extraction (EAE) has mild, environmentally friendly, and efficient operating conditions and usually does not destroy the three-dimensional molecular structure of polysaccharides, thus helping to maintain their biological activity. However, this method is costly and operating conditions such as extraction temperature, time, material-liquid ratio, pH value, etc. must be strictly controlled to ensure enzyme activity. Therefore, it has not been widely used in industrial production.

[0007] The polysaccharides obtained by the above methods are all crude polysaccharides. According to research, crude polysaccharides are not effective in reducing lung epithelial cell death and inhibiting the expression of inflammatory factors.

[0008] In view of this, it is necessary to provide a chanterelle polysaccharide for effectively inhibiting lung cell inflammation. Summary of the Invention

[0009] The purpose of the present invention is to provide a chanterelle polysaccharide and application thereof. The chanterelle polysaccharide is a single polysaccharide active substance and has the effect of effectively inhibiting lung cell inflammation.

[0010] The first aspect of the present invention is to provide a chanterelle polysaccharide.

[0011] The chanterelle polysaccharide is polymerized from five monosaccharides: galactose, glucose, xylose, fucose and glucosamine hydrochloride. The chemical formula of the chanterelle polysaccharide is shown in formula (I):

[0012]

[0013] Furthermore, the mass percentages of the monosaccharide components are as follows: galactose 45.40%, glucose 25.80%, xylose 18.30%, fucose 9.30%, and glucosamine hydrochloride 1.20%.

[0014] Furthermore, the xylose exists in three forms: Xylp-(1→, →3)-Xylp-(1→ and →4)-Xylp-(1→), and their molar percentages are 8.50:3.90:5.80 respectively;

[0015] The galactose exists in seven forms: Galp-(1→, →4)-Galp-(1→, →3)-Galp-(1→, →6)-Galp-(1→, →3,4)-Galp-(1→, →3,6)-Galp-(1→ and →3,4,6)-Galp-(1→), and their molar percentages are 2.60:11.00:2.50:7.90:2.80:16.20:7.40 respectively;

[0016] The glucose exists in five forms, namely Glcp-(1→, →2)-Glcp-(1→, →3)-Glcp-(1→, →6-Glcp-(1→ and →3,6)-Glcp-(1→), with molar percentages of 8.30:2.60:7.70:1.30:11.60 respectively.

[0017] Furthermore, the average molecular weight of the chanterelle polysaccharide is 13,722 g / mol.

[0018] The second aspect of the present invention is to provide a method for preparing the chanterelle polysaccharide described in the first aspect, comprising the following steps:

[0019] Step S1, preparing crude chanterelle polysaccharide:

[0020] Step S11, after the chanterelle fruiting bodies are crushed, the chanterelle powder is first defatted with 95% ethanol (v / v), and then 10 times the amount of deionized water is added;

[0021] Step S12, adding papain, pectinase, and cellulase (3%) in a ratio of 1:1:1, heating at 62°C for 3 hours, stirring evenly, sonicating at a low power of 475W for 20 minutes, filtering the filtrate, repeating this process twice, and collecting the resultant solution;

[0022] Step S13: After cooling, trichloroacetic acid is added to a concentration of 8% to remove protein from the supernatant, and the supernatant is collected after centrifugation and decolorized with hydrogen peroxide to a pH of 8;

[0023] Step S14, the extract is dialyzed with deionized water for 2 days, and then freeze-dried to obtain crude polysaccharide, wherein the dialysis molecular weight cut-off Mw is 3500 Da;

[0024] Step S2, re-dissolving the crude chanterelle polysaccharide in distilled water, and then purifying it on a DEAE-52 cellulose column, eluting with distilled water, 0.1 mol / L, 0.3 mol / L, and 0.5 mol / L NaCl at 1 mL / min, to obtain polysaccharide fractions F-1, F-2, F-3, F-4, and F-5, wherein F-1 refers to the fraction with the highest yield;

[0025] Step S3, eluting on a gel purification system column using 0.2 M / L NaCl as the mobile phase to obtain the polysaccharide component F-1 with the highest content;

[0026] Step S4: further purifying the polysaccharide component F-1 in a gel purification system to obtain the chanterelle polysaccharide as claimed in claim 1.

[0027] The third aspect of the present invention is to provide the use of the chanterelle polysaccharide described in the first aspect in the preparation of a drug for inhibiting lung cell inflammation.

[0028] The fourth aspect of the present invention is to provide the use of the chanterelle polysaccharide described in the first aspect in the preparation of a drug for treating lung inflammation.

[0029] Compared with the prior art, the chanterelle polysaccharide provided by the present invention and its preparation method and application have the following beneficial effects:

[0030] The chanterelle polysaccharide provided by the present invention is a single polysaccharide active substance, which has an inhibitory effect on lung inflammation caused by lipopolysaccharide-induced lung epithelial cells, and can therefore be used as a potential drug for treating lung inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 This is the elution separation diagram of crude polysaccharide on DEAE-52 cellulose column;

[0033] Figure 2 It is the HPGPC map of CTPS;

[0034] Figure 3 is the FT-IR spectrum of CTPS;

[0035] Figure 4 is the IC chromatogram of standard monosaccharides and CTPS in the present invention;

[0036] Figure 5 This is a graph showing the effect of the chanterelle polysaccharide CTPS of the present invention on the viability of lung epithelial cells;

[0037] Figure 6 This is a bar graph showing the effect of the chanterelle polysaccharide CTPS of the present invention on the ROS content in polysaccharide-induced lung epithelial cells;

[0038] Figure 7 This is a flow cytometric graph showing the effect of the chanterelle polysaccharide CTPS of the present invention on polysaccharide-induced apoptosis of lung epithelial cells;

[0039] Figure 8 It is a bar graph showing the effect of the chanterelle polysaccharide CTPS of the present invention on the mRNA and protein expression of apoptosis-related genes in lung epithelial cells induced by the polysaccharide;

[0040] Figure 9 is the mRNA and protein expression levels of IL-6, IL-1β, and TNF-α in LPS-induced Beas-2B cells;

[0041] Figure 10 It is a bar graph showing the effects of the chanterelle polysaccharide CTPS of the present invention on NF-κB and AP-1 in lung epithelial cells induced by the polysaccharide. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention and to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation methods of the present invention are further described below.

[0043] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0044] Example 1

[0045] A method for preparing chanterelle polysaccharide comprises the following steps:

[0046] Step S1, preparing crude chanterelle polysaccharide:

[0047] Step S11, after the chanterelle fruiting bodies are crushed, the chanterelle powder is first defatted with 95% ethanol (v / v), and then 10 times the amount of deionized water is added;

[0048] Step S12, adding papain, pectinase, and cellulase (3%) in a ratio of 1:1:1, heating at 62°C for 3 hours, stirring evenly, sonicating at a low power of 475W for 20 minutes, filtering the filtrate, repeating this process twice, and collecting the resultant solution;

[0049] Step S13: After cooling, trichloroacetic acid is added to a concentration of 8% to remove protein from the supernatant, and the supernatant is collected after centrifugation and decolorized with hydrogen peroxide to a pH of 8;

[0050] Step S14, the extract is dialyzed with deionized water for 2 days, and then freeze-dried to obtain crude polysaccharide, wherein the dialysis molecular weight cut-off Mw is 3500 Da;

[0051] Step S2, re-dissolving the crude chanterelle polysaccharide in distilled water, and then purifying it on a DEAE-52 cellulose column to obtain polysaccharide fractions F-1, F-2, F-3, F-4, and F-5;

[0052] Specifically, the crude polysaccharide was redissolved in distilled water and purified on a DEAE-52 cellulose column (3.5 cm × 60 cm), eluting with distilled water, 0.1 mol / L, 0.3 mol / L, and 0.5 mol / L NaCl, sequentially at 1 mL / min. The components were determined using the phenol-sulfuric acid method, and the major polysaccharide fractions were collected, dialyzed, concentrated, and lyophilized.

[0053] See also Figure 1 Figure 2 shows the elution separation of crude polysaccharides on a DEAE-52 cellulose column. The five major polysaccharide fractions obtained from the DEAE-52 cellulose column are designated F-1, F-2, F-3, F-4, and F-5. The major polysaccharide fractions are F-1, F-2, and F-3, with F-1 being the fraction with the highest yield.

[0054] Step S3, eluting on a gel purification system column using 0.2 M / L NaCl as the mobile phase to purify the polysaccharide component F-1 with the highest content;

[0055] Specifically, the polysaccharide component was eluted on a gel purification system column (3 cm×100 cm) with 0.2 M / L NaCl at a flow rate of 0.1 mL / min, and the polysaccharide component F-1 with the highest content was purified.

[0056] In step S4, the polysaccharide component F-1 is further purified in a gel purification system to obtain purified chanterelle polysaccharide, which is named CTPS. The purification parameters in this step are the same as those in step S3.

[0057] See also Figure 2 , is the HPGPC spectrum of CTPS. The purity and molecular weight of CTPS were determined using an HPGPC system. A single peak was detected on the column, indicating that CTPS is a homogeneous polysaccharide. GPC-MALS-RI analysis revealed an average molecular weight of 13,722 g / mol for the purified CTPS. Furthermore, the number average molecular weight (Mn) of CTPS was determined to be 13,410 g / mol, and the Mw / Mn, or polydispersity index (PDI), was 1.02, indicating that CTPS is relatively uniform in molecular size.

[0058] Example 2

[0059] Structural Characterization of Chanterelle Polysaccharide CTPS

[0060] (1) FT-IR spectrum of CTPS

[0061] FT-IR spectroscopy is a qualitative analysis method that predicts the structure of polysaccharides based on their specific functional groups. Figure 3 As shown. In the functional group area (4000~500cm -1 ), 3600-3200cm -1 Within the range, this band is the characteristic peak of sugars, and 3436cm -1 The broad peak at 1637 cm-1 indicates the presence of -OH stretching vibration, indicating that CTPS has typical characteristics of carbohydrate compounds. -1 There is an absorption peak at 1432cm -1 There is an absorption peak at 1128cm, indicating the presence of CO stretching vibration. -1 1033cm -1 There is an absorption peak at 877cm -1 There is an absorption peak at , which is caused by the CH angle vibration of the equatorial bond other than the terminal diisomeric CH.

[0062] (2) Monosaccharide composition

[0063] The monosaccharide composition of polysaccharides was determined by ion chromatography. Ion chromatography determined the composition of 16 monosaccharides in CTPS. The results showed that CTPS was mainly composed of galactose, glucose, xylose, fucose and glucosamine hydrochloride, such as Figure 4 As shown, Figure 4 A represents the IC chromatogram of standard monosaccharides, Figure 4B shows the IC chromatogram of CTPS. The contents of each monosaccharide are shown in Table 1. The main chain of CTPS is formed by galactose (45.40) and glucose (25.80), because these two monosaccharides are the main monosaccharides.

[0064] Table 1: Monosaccharide composition of CTPS

[0065]

[0066] (3) Sugar chain composition

[0067] A total of 15 sugar alcohol acetates were identified after polysaccharide methylation. Among them, xylose existed in three forms: Xylp-(1→, →3)-Xylp-(1→ and →4)-Xylp-(1→), with molar percentages of 8.50:3.90:5.80, respectively. Galactose existed in three forms: Galp-(1→, →4)-Galp-(1→, →3)-Galp-(1→, →6)-Galp-(1→, →3,4)-Galp-(1→, →3,6)-Galp-(1→ and →3 ,4,6)-Galp-(1→) exists in seven forms, with molar percentages of 2.60:11.00:2.50:7.90:2.80:16.20:7.40 respectively; glucose exists in five forms, with molar percentages of 8.30:2.60:7.70:1.30:11.60 respectively.

[0068] (4) Nuclear magnetic resonance analysis

[0069] The structure of CTPS was further investigated by 1H- and 13C-NMR and 2D-NMR (HSQC, HMBC and COSY spectra). Based on the methylation combined with NMR analysis, the main glycosidic bond structure of the polysaccharide was inferred to be:

[0070] The main chain connection method is

[0071] →[6)-α-D-Galp-(1]2→6)-α-D-Galp-(1→6)-α-D-Galp-(1→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→[6)-β-D-Glcp-(1]2→; and the side chains are connected to the main chain through the O-2 bond of →2,6)-α-D-Galp-(1→), the O-3 bond and O-4 bond of →3,4,6)-α-D-Galp-(1→), and the O-3 bond of →3,6)-β-D-Glcp-(1→.

[0072] (5) Chemical structure characterization

[0073] After analysis, the chemical structure of the chanterelle polysaccharide CTPS of the present invention is shown in formula (I):

[0074]

[0075] Example 3 Effect of CTPS on the Viability of Human Lung Epithelial Beas-2B Cells

[0076] 1. Experimental methods

[0077] BEAS-2B cells were seeded in 96-well plates and incubated for 24 hours. Different concentrations of CTPS solution were then added to the well-grown BEAS-2B cells, resulting in final CTPS concentrations of 6.25 μg / ml, 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml. After incubation for 24 hours at 37°C in a cell culture incubator with 5% CO2, the cells were washed twice with PBS. Cell morphology was observed and photographed using an inverted microscope to examine the effects of CTPS on BEAS-2B cell morphology. 10 μL of MTS solution was added to each well, and the plates were incubated in the incubator for 1 hour. The absorbance at 490 nm was measured using a microplate reader. The cell viability of the control group was defined as 100%. The cell viability of the other groups was calculated by comparing the viability of the control group with that of the control group to examine the effects of CTPS on cell viability. By examining the effects of CTPS on cell morphology and viability, it was determined whether CTPS was toxic to BEAS-2B cells.

[0078] 2. Experimental results

[0079] Beas-2B cells were treated with different concentrations of CTPS (0-400 μg / ml) and the cell activity was detected. Figure 5 Compared with the control group, Beas-2B cells adhered well under different CTPS concentrations, showing a long spindle shape with smooth edges, and the morphology was the same as that of the control group, as shown in Figure 2. Figure 5 As shown in A; MTS method was used to detect cell viability, and the results were as shown in Figure 5 As shown in Figure B, with the cell viability of the control group defined as 100%, the MTS assay results showed that the cell viabilities of the 6.25-400 μg / mL CTPS treatment groups were 100.32% ± 3.2%, 99.78% ± 1.8%, 99.09% ± 3.04%, 99.54% ± 1.74%, 99.37% ± 1.76%, 99.52% ± 2.97%, and 100.03% ± 1.27%, respectively, with no significant differences among the groups (p>0.05). The test results indicate that CTPS has no significant cytotoxicity against Beas-2B cells.

[0080] Example 4 Detection of Cellular Reactive Oxygen Species (ROS)

[0081] 1. Experimental methods

[0082] A reactive oxygen species detection kit was used to detect ROS levels in cells. BEAS-2B cells were seeded in 6-well plates and incubated for 24 hours. CTPS sample solutions of varying concentrations were added to the BEAS-2B cells, resulting in final CTPS concentrations of 0, 25 μg / ml, 50 μg / ml, and 100 μg / ml. LPS (60 μM) was added 4 hours later and incubated for 12 hours. DCFH-DA was diluted 1:1000 in serum-free medium and incubated in a cell culture incubator at 37°C for 30 minutes. BEAS-2B cells were seeded in 96-well plates and loaded with the DCFH-DA fluorescent probe according to the above procedures. Fluorescence intensity was measured using a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. ROS levels were expressed as mean fluorescence intensity. The ROS level in the control group was defined as 100%, and the ROS levels in the other groups were calculated by comparison with the control group.

[0083] 2. Experimental results

[0084] Effects of CTPS on LPS-induced Beas-2B cell activity Figure 6 As shown in Figure 3 . Compared with the control group, the fluorescence intensity and ROS levels in Beas-2B cells in the LPS group were significantly higher (con group: 0.135±0.003, LPS group: 0.161±0.003, p < 0.05), indicating significant oxidative stress damage. The fluorescence intensity of Beas-2B cells treated with CTPS (25μg / mL, 50μg / mL, 100μg / mL) was significantly lower than that in the LPS group. ROS concentrations were significantly increased in the LPS group. After treatment with CTPS (25μg / mL, 50μg / mL, 100μg / mL), ROS concentrations gradually decreased with increasing sample concentrations to levels similar to those in the control group (25μg / mL: 0.144±0.002, 50μg / mL: 0.138±0.003, 100μg / mL: 0.128±0.004, p < 0.05). These results indicate that LPS can induce oxidative damage and CTPS can reduce the release of ROS after LPS-induced oxidative damage in Beas-2B cells.

[0085] Example 5 Cell apoptosis detection

[0086] 1. Experimental methods

[0087] Apoptosis was detected using an apoptosis detection kit. BEAS-2B cells were seeded in a 6-well plate and incubated for 24 hours. The plates were washed once with PBS, and 2 mL of serum-free medium was added. Different concentrations of CTPS sample solution were added to achieve final CTPS concentrations of 0, 25 μg / mL, 50 μg / mL, and 100 μg / mL. LPS (60 μM) was added 4 hours later and incubated for 12 hours. The medium was discarded, and the cells were washed three times with PBS. The PBS was aspirated, and the cells were digested with EDTA-free trypsin to prepare a cell suspension. The suspension was collected in a 15 mL centrifuge tube. The cell count per sample was approximately 3 × 106 cells / mL. The suspension was centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded. The cells were washed once with PBS and centrifuged at 800 rpm for 10 minutes. To 100 μL of binding buffer, 5 μL of Annexin V-F22 was added, and the cells were incubated at room temperature in the dark for 15 minutes. After completion, 5 μL of propidium iodide staining solution was added for staining, and the cells were incubated in the dark for 5 minutes. After completion, flow cytometry analysis was performed.

[0088] 2. Experimental results

[0089] Apoptosis usually has different morphological characteristics and energy-dependent biochemical mechanisms. Figure 7 As shown in the figure, flow cytometry results showed that LPS induced Beas-2B cell apoptosis, and CTPS treatment (25 μg / mL, 50 μg / mL and 100 μg / mL) significantly alleviated the LPS-induced cell apoptosis in a dose-dependent manner.

[0090] Example 6 Detection of cellular inflammatory factors—qRT-PCR detection and apoptosis protein expression

[0091] When cells are stimulated by LPS, an inflammatory response occurs, and the genes for related inflammatory factors, such as IL-1B, IL-6, and TNF-a, are activated and expressed in large quantities. To explore the anti-inflammatory effects of CTPS in vitro, RT-qPCR and western blot were used to analyze the expression of inflammatory factors in Beas-2B cells to evaluate the anti-inflammatory efficacy of CTPS.

[0092] Total RNA was extracted from BEAS-2B cells and tested for RNA integrity. RNA reverse transcription was then performed (according to the kit instructions). Primers for IL-6, IL-1β, TNF-α, bax, bcl-2, and β-Actin were designed, and cDNA was amplified by real-time fluorescence quantitative PCR. After the reaction, the relative expression level of the target gene mRNA was calculated based on the Ct value (β-actin was selected as the reference gene).

[0093] BEAS-2B cells were incubated with CTPS sample solutions at varying concentrations (25, 50, and 100 μg / ml) and induced with LPS (60 μM). Total cellular protein was extracted using RIPA lysis buffer, and anti-IL-6, anti-IL-1β, anti-TNF-α, anti-bax, anti-bcl-2, and anti-β-actin antibodies (Promega, USA) were used. Protein concentration was determined by immunoblotting using the BCA assay. Samples were loaded according to protein concentration and subjected to SDS-PAGE electrophoresis. After separation, the gel was cut according to the molecular weight of the target gene protein and transferred to a PVDF membrane. The PVDF membrane containing the target protein was blocked in 5% bovine serum albumin blocking buffer prepared in TBS / T for 1 hour. The membrane was then immersed in primary antibody dilution (1:5000) and incubated overnight at 4°C on a shaker. Wash the PVDF membrane three times with TBS / T solution, add the secondary antibody dilution (1:10,000), incubate on a shaker at room temperature for 1 hour, and then wash three times with TBS / T solution. Finally, add developer to visualize the protein bands. Use an imaging system to calculate the integrated optical density of each band to determine the relative expression of the target protein and the control protein.

[0094] 2. Experimental results

[0095] like Figure 8 As shown, Figure 8 A represents the expression level of Bcl-2 mRNA, a gene related to cell apoptosis. Figure 8 B represents the expression level of Bax mRNA, apoptosis-related gene. Figure 8 C represents the expression of apoptosis-related proteins detected by Western blot. Figure 8 D represents the quantitative data of the relative protein expression of Bcl-2 and Bax. Figure 8 LPS inhibited the expression of Bcl-2 mRNA and protein, with mRNA expression 0.71-fold and protein expression 0.72-fold compared to the Con group. CTPS treatment upregulated Bcl-2 RNA and protein expression (p < 0.05). At increasing CTPS concentrations (25, 50, and 100 μg / mL), mRNA expression increased by 0.82, 0.92, and 1.02 times, respectively, and protein expression increased by 0.7, 0.89, and 1.1 times, respectively. These results suggest that CTPS treatment can inhibit the downregulation of Bcl-2 expression induced by LPS injury.

[0096] like Figure 9 As shown in Figure 3, compared with the Con group, the mRNA and protein expression levels of IL-6, IL-1β, and TNF-α in LPS-induced Beas-2B cells were significantly increased, with the mRNA expression levels increasing by 2.18, 2.39, and 2.51 times, respectively. Figure 9A. Figure 9 B and Figure 9 As shown in C, the protein expression levels increased by 1.28, 1.17, and 1.77 times, respectively. Figure 9 D and Figure 9 As shown in E, this indicates a significant inflammatory response, with LPS-induced inflammation in Beas-2B cells. CTPS treatment significantly reduced the RNA and protein expression of IL-6, IL-1β, and TNF-α (p < 0.05), with a dose-response relationship. CTPS treatment concentrations increased from low to high (25, 50, and 100 μg / mL) with 1.87-, 1.62-, and 1.54-fold increases in IL-6 mRNA expression, and 0.77-, 0.7-, and 0.47-fold increases in protein expression. IL-1β mRNA expression increased by 1.99-, 1.71-, and 1.35-fold increases in IL-1β protein expression, and 1.52-, 0.96-, and 1.15-fold increases in TNF-α mRNA expression, and 1.93-, 1.58-, and 1.24-fold increases in TNF-α protein expression. This suggests that CTPS can alleviate inflammatory symptoms by downregulating LPS-induced IL-6, IL-1β, and TNF-α mRNA and protein expression in Beas-2B cells. By reducing the expression of inflammatory factors, it is confirmed that CTPS has an inhibitory effect on LPS-induced inflammation, and CTPS is beneficial for alleviating inflammation by regulating inflammatory factors.

[0097] Example 7 Detection of cellular inflammatory factors

[0098] 1. Experimental methods

[0099] RNA extraction: BEAS-2B cells were seeded in 24-well plates and incubated for 24 hours. Serum-free medium was then added and transfected with a transfection mixture (1.5 μL of nuclear factor-κB (NF-κB) or activator protein 1 (AP-1)-luc plasmid dissolved in 1.5 μL of Lipofectamine 2000 reagent) for 4 hours. The medium containing the transfection mixture was then removed, and the cells were incubated in complete medium for 4 hours. CTPS (25, 50, or 100 μg / mL) was added and incubated with the cells for 2 hours. LPS (60 μM) was then added and induced for 12 hours. Cells were harvested with 200 μL of 1× reporter lysis buffer for 5–10 minutes, centrifuged at 5000–10000 rpm for 5 minutes, and the supernatant was collected. 80 μL / well luciferase (Luciferase Assay substrate, Lot No. 0000291388) and 30 μL / well liquid supernatant were added to a 96-well plate. Luciferase activity was measured by Software (Promega Biosystem Sunnyvale, Inc., USA). Luciferase expression was expressed as relative light units (RLU) / mg protein.

[0100] 2. Experimental results

[0101] NF-κB and activator protein-1 (AP-1) are important transcription factors that regulate the expression of pro-inflammatory genes during the inflammatory response. Figure 10 As shown, Figure 10 A represents the expression level of AP-1, Figure 10 B represents NF-κB expression. Compared with the Con group, in LPS-induced Beas-2B cells, the transcriptional activities of NF-κB and AP-1 were significantly increased (1.93- and 1.5-fold, respectively) (P < 0.05). Treatment with CTPS (25, 50, and 100 μg / mL) decreased the transcriptional activities of NF-κB and AP-1 by 0.75-, 0.8-, and 0.97-fold, respectively, and by 0.87-, 0.73-, and 0.8-fold, respectively. This indicates that CTPS can inhibit the activation of NF-κB and AP-1 in LPS-stimulated Beas-2B cells, with a gradual decrease in the transcriptional activities of NF-κB and AP-1 genes (P < 0.05). Consistent with the results of proinflammatory cytokine expression, the inhibition of NF-κB and AP-1 activity was accompanied by a decrease in the expression of inflammatory factors. This suggests that CTPS may exert its anti-inflammatory effects by blocking the activity of NF-κB and AP-1.

[0102] In summary, the chanterelle polysaccharide CTPS obtained by separation and purification using DEAE-52 cellulose and gel purification system columns in the present invention is a single polysaccharide active substance that has an inhibitory effect on pulmonary inflammation caused by lipopolysaccharide-induced lung epithelial cells, and can therefore be used as a potential drug for treating lung inflammation.

[0103] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations made to these embodiments without departing from the principles and spirit of the present invention are still within the scope of protection of the present invention.

Claims

1. A chanterelle polysaccharide, characterized in that The chanterelle polysaccharide is polymerized from five monosaccharides: galactose, glucose, xylose, fucose and glucosamine hydrochloride. The chemical formula of the chanterelle polysaccharide is shown in formula (I):

2. The chanterelle polysaccharide according to claim 1, characterized in that The mass percentages of the monosaccharide components are as follows: galactose 45.40%, glucose 25.80%, xylose 18.30%, fucose 9.30%, and glucosamine hydrochloride 1.20%.

3. The chanterelle polysaccharide according to claim 1, wherein The xylose exists in three forms: Xylp-(1→, →3)-Xylp-(1→ and →4)-Xylp-(1→, with molar percentages of 8.50:3.90:5.80 respectively; The galactose exists in seven forms: Galp-(1→, →4)-Galp-(1→, →3)-Galp-(1→, →6)-Galp-(1→, →3,4)-Galp-(1→, →3,6)-Galp-(1→ and →3,4,6)-Galp-(1→), and their molar percentages are 2.60:11.00:2.50:7.90:2.80:16.20:7.40 respectively; The glucose exists in five forms, namely Glcp-(1→, →2)-Glcp-(1→, →3)-Glcp-(1→, →6-Glcp-(1→ and →3,6)-Glcp-(1→), with molar percentages of 8.30:2.60:7.70:1.30:11.60 respectively.

4. The chanterelle polysaccharide according to claim 1, wherein The average molecular weight of the chanterelle polysaccharide is 13,722 g / mol.

5. A method for preparing chanterelle polysaccharide according to claim 1, characterized in that: The steps include: Step S1, preparing crude chanterelle polysaccharide: Step S11, after the chanterelle fruiting bodies are crushed, the chanterelle powder is first defatted with 95% ethanol (v / v), and then 10 times the amount of deionized water is added; Step S12, adding papain, pectinase, and cellulase (3%) in a ratio of 1:1:1, heating at 62°C for 3 hours, stirring evenly, sonicating at a low power of 475W for 20 minutes, filtering the filtrate, repeating this process twice, and collecting the resultant solution; Step S13: After cooling, trichloroacetic acid is added to a concentration of 8% to remove protein from the supernatant, and the supernatant is collected after centrifugation and decolorized with hydrogen peroxide to a pH of 8; Step S14, the extract is dialyzed with deionized water for 2 days, and then freeze-dried to obtain crude polysaccharide, wherein the dialysis molecular weight cut-off Mw is 3500 Da; Step S2, re-dissolving the crude chanterelle polysaccharide in distilled water, and then purifying it on a DEAE-52 cellulose column, eluting with distilled water, 0.1 mol / L, 0.3 mol / L, and 0.5 mol / L NaCl at 1 mL / min, to obtain polysaccharide fractions F-1, F-2, F-3, F-4, and F-5, wherein F-1 refers to the fraction with the highest yield; Step S3, eluting on a gel purification system column using 0.2 M / L NaCl as the mobile phase to obtain the polysaccharide component F-1 with the highest content; Step S4: further purifying the polysaccharide component F-1 in a gel purification system to obtain the chanterelle polysaccharide as claimed in claim 1.

6. Use of the chanterelle polysaccharide according to claim 1 in preparing a drug for inhibiting lung cell inflammation.

7. Use of the chanterelle polysaccharide according to claim 1 in preparing a medicine for treating lung inflammation.

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