An active Inonotus obliquus polysaccharide IOP-1 against non-small cell lung cancer and its application

Abstract

The present invention discloses an anti-non-small cell lung cancer active birch inonotus polysaccharide IOP-1 and its application, belonging to the field of biomedicine. The present invention adopts a method of boiling water extraction and ethanol precipitation of birch inonotus polysaccharide, and combines DEAE cellulose column purification technology, and utilizes the principle that macromolecular polysaccharides are insoluble in organic solvents to successfully achieve the separation of birch inonotus polysaccharide and obtain a birch inonotus polysaccharide IOP-1 with anti-non-small cell lung cancer activity, wherein the monosaccharide composition of the birch inonotus polysaccharide IOP-1 includes fucose, rhamnose, arabinose, galactose, glucose, xylose and mannose with a molar mass ratio of 2.41:0.58:1.03:2.17:82.20:4.33:7.29. Its significant effect against non-small cell lung cancer was verified by in vitro and in vivo experiments, providing a new treatment method for the clinical treatment of non-small cell lung cancer.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to an Inonotus obliquus polysaccharide IOP-1 with anti-non-small cell lung cancer activity and an application thereof. Background Art

[0002] Inonotus obliquus, also known as Chaga, is a medicinal fungus that grows on birch bark and has been shown to have pharmacological activities such as lowering blood sugar, anti-oxidation and immune regulation.

[0003] Lung cancer is the malignant tumor with the highest morbidity and mortality in the world. Among them, non-small cell lung cancer (NSCLC) accounts for about 85% of the total number of lung cancer patients diagnosed, which is extremely harmful to human health. Many studies have found that fungal polysaccharides have proliferation inhibition activity on tumor cells, indicating that fungal polysaccharides have good anti-tumor activity and have certain value in the clinical treatment of diseases.

[0004] In recent years, research on birch polysaccharides has focused on antioxidant and hypoglycemic aspects, and there are few studies on the anti-tumor effects of birch polysaccharides. Studies have shown that birch polysaccharides have a certain therapeutic effect on liver cancer, and birch triterpenes also have a certain inhibitory effect on cervical cancer HeLa cells. However, due to the different tumor microenvironments of different types, treatment strategies are also different. Therefore, it is not known whether birch polysaccharides have a therapeutic effect on non-small cell lung cancer. In addition, the structural differences of fungal polysaccharides will lead to differences in their pharmacological activities and functions. Even if the same fungus is used as a raw material for homozygous extraction, a variety of polysaccharide components with different structures can be obtained, and the pharmacological activities and functions of these polysaccharide components are also different. Therefore, it is of great significance to explore whether birch polysaccharides have polysaccharide components with anti-lung cancer, especially non-small cell lung cancer activity, to enrich the treatment strategy of lung cancer. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-non-small cell lung cancer active Inonotus obliquus polysaccharide IOP-1 and its application to solve the problems existing in the above-mentioned prior art. The present invention separates and obtains an anti-non-small cell lung cancer active Inonotus obliquus polysaccharide IOP-1, verifies its significant anti-non-small cell lung cancer effect through in vivo and in vitro experiments, and provides a new treatment method for the clinical treatment of non-small cell lung cancer.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides an Inonotus obliquus polysaccharide IOP-1 with anti-non-small cell lung cancer activity. The monosaccharide composition of the Inonotus obliquus polysaccharide IOP-1 comprises fucose, rhamnose, arabinose, galactose, glucose, xylose and mannose, and the molar mass ratio of the fucose, the rhamnose, the arabinose, the galactose, the glucose, the xylose and the mannose is 2.41:0.58:1.03:2.17:82.20:4.33:7.29.

[0008] Furthermore, the preparation method comprises the following steps:

[0009] The fruiting bodies of Inonotus obliquus were ultrafinely crushed, soaked in ethanol, boiled and extracted with distilled water, the filtrate was filtered and concentrated, and the supernatant was taken after ultra-high-speed centrifugation. The supernatant was precipitated with ethanol solution and centrifuged again, and the precipitate was collected and chromatographed on a DEAE cellulose column, eluted with water, and then eluted with a 0.1 M NaCl solution to obtain the Inonotus obliquus polysaccharide IOP-1.

[0010] Furthermore, the distilled water is added according to the material-liquid ratio of fruiting body powder: distilled water = 1 g: 50 mL.

[0011] Furthermore, the ultra-high speed centrifugation condition is 9000 rpm, 10 min.

[0012] Furthermore, when the supernatant is precipitated with an ethanol solution, the volume concentration of ethanol in the system is 80%.

[0013] Furthermore, the conditions for the second centrifugation are 8000 rpm, 15 min.

[0014] The present invention also provides the use of the Inonotus obliquus polysaccharide IOP-1 in preparing a medicine for treating non-small cell lung cancer.

[0015] The present invention also provides a medicine for treating non-small cell lung cancer, wherein the active ingredient comprises the Inonotus obliquus polysaccharide IOP-1.

[0016] The present invention discloses the following technical effects:

[0017] The present invention adopts the method of boiling water extraction and ethanol precipitation of Inonotus obliquus polysaccharide, combined with DEAE cellulose column purification technology, and utilizes the principle that macromolecular polysaccharides are insoluble in organic solvents to successfully separate Inonotus obliquus polysaccharide and obtain an Inonotus obliquus polysaccharide IOP-1 with anti-non-small cell lung cancer activity.

[0018] The present invention has confirmed through cell experiments that the birch polysaccharide IOP-1 has an inhibitory effect on non-small cell lung cancer cells, and the effect is better than that of glioma cells and cervical cancer cells; further analysis found that IOP-1 inhibits the growth and migration of non-small cell lung cancer by regulating cell apoptosis and migration-related pathways. At the same time, the present invention has verified through animal experiments that IOP-1 also plays a very significant anti-cancer effect in vivo, providing a new treatment method for the clinical treatment of non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 paying creative work.

[0020] Figure 1 This is the molecular weight detection diagram of IOP-1;

[0021] Figure 2 This is the HPLC detection diagram of the monosaccharide composition of IOP-1;

[0022] Figure 3 This is the FT-IR infrared detection diagram of the functional group structure of IOP-1;

[0023] Figure 4 This is a statistical diagram of the ability of IOP-1 to inhibit the proliferation of different tumor cells;

[0024] Figure 5 The figure shows the effect of IOP-1 on the proliferation of NCI-H460 cells, wherein A is the microscopic observation of the cell morphology of NCI-H460 cells after 24 and 48 hours of IOP-1 treatment; B is the inhibition rate of IOP-1 on the proliferation of NCI-H460 cells, *P<0.05 and ***P<0.001 compared with the control group;

[0025] Figure 6 The figure is the effect of IOP-1 on the colony-forming ability of NCI-H460 cells, wherein A is the colony-forming graph of NCI-H460 cells under the action of different concentrations of IOP-1; B is the change graph of the number of colonies formed by NCI-H460 cells under the action of different concentrations of IOP-1, **P<0.01 compared with the control group;

[0026] Figure 7 The diagrams are the effects of IOP-1 on cell nuclear morphology and the detection of cell apoptosis, wherein A is the microscopic observation of DAPI fluorescence staining; B is the observation of AO / EB staining;

[0027] Figure 8 Figure 2 shows the cell apoptosis analysis of NCI-H460 cells after drug treatment using flow cytometry (A) and the proportion of cells at different apoptotic stages after IOP-1 treatment of NCI-H460 cells for 48 hours (mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group);

[0028] Fig. 9 The figure shows the effect of IOP-1 on the migration and invasion of NCI-H460 cells, where A is the observation diagram of NCI-H460 cell migration after treatment with different concentrations of IOP-1; B is the diagram of NCI-H460 cell invasion after IOP-1 treatment; C is the statistical diagram of the migration changes of NCI-H460 cells before and after IOP-1 treatment; D is the statistical diagram of the invasion changes of NCI-H460 cells before and after IOP-1 treatment, mean ± SEM, n ≥ 3; *P < 0.05; **P < 0.01; ***P < 0.001 compared with the control group;

[0029] Fig.10 The effect of IOP-1 on the relative expression of Bax (A), Bcl-2 (B), P53 (C), Cyto-c (D), MMP-9 (E) and Parp-1 (F) proteins in NCI-H460 cells, *P<0.05; **P<0.01; ***P<0.001 compared with the control group;

[0030] Fig.11 This is a graph showing the effect of IOP-1 on ATP content in NCI-H460 cells;

[0031] Fig.12 This is a magnetic resonance imaging tracking image of IOP-1 inhibiting tumor growth in NCI-H460 model mice;

[0032] Fig.13 The analysis chart of IOP-1 on the food intake (A), water intake (B), tumor weight (C) and animal spleen index (D) of non-small cell lung cancer model mice, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Reagents and materials involved in the embodiments of the present invention:

[0039] Inonotus obliquus was collected from Changbai Mountain area of ​​Jilin Province and was identified as Inonotus obliquus by Professor Liu Yang of Jilin Agricultural University.

[0040] Other biological materials included NSCLC cell line NCI-H460 (CL-0299, Prosai Biotechnology Co., Ltd.), glioma cell line SHG-44 (ATCC cell bank), cervical cancer cell line HeLa (CL-0101, Prosai Biotechnology Co., Ltd.), fetal bovine serum (164210, Prosai Biotechnology Co., Ltd.), AO / EB fluorescent staining solution (CA1142, Beijing Solebeau Technology Co., Ltd., China), DAPI fluorescent staining solution (KGA1808-50, Jiangsu KeyGen Biotechnology Co., Ltd., China), and AnnexinV-FITC / PI double staining apoptosis detection kit (KGA1102-100, Jiangsu KeyGen Biotechnology Co., Ltd., China). β-actin, Bax, Bcl-2, P53, Cyto-c, Parp-1, and MMP-9 antibodies (AC026, A0207, A0208, A3185, A13430, A0942, and A11147, Wuhan Abotec Biotechnology Co., Ltd.).

[0041] Example 1 Extraction, separation and molecular weight detection of Inonotus obliquus polysaccharide IOP-1

[0042] 1. Extraction and separation of Inonotus obliquus polysaccharide IOP-1

[0043] The fruiting bodies of birch mushrooms were ultrafinely crushed, and the powder was soaked in ethanol for 3 days. After that, distilled water was added according to the material-liquid ratio of fruiting body powder: distilled water = 1g: 50mL, heated to boiling point for boiling, and the extraction was repeated 5 times, each time for 2 hours. The extraction filtrates were combined, filtered twice with filter cloth, concentrated to 1 / 5 of the original volume, and centrifuged at ultra-high speed 9000rpm for 10min, and the supernatant was reserved for use. 4 times the volume of 95% ethanol was added to the supernatant to precipitate the ethanol to a final concentration of 80%, stirred evenly with a glass rod, and allowed to stand at 4℃ overnight for alcohol precipitation. The next day, it was centrifuged at 8000r for 15min, and the precipitate was collected, dissolved with a small amount of water, and freeze-dried for use. 500mg of crude polysaccharide was fully dissolved, chromatographed on a DEAE cellulose DE-52 ion exchange column, and after the neutral polysaccharide was eluted with the water component, it was eluted with a 0.1M NaCl solution, and the final liquid was freeze-dried for use, which was recorded as birch mushroom polysaccharide IOP-1.

[0044] 2. Sugar content, monosaccharide composition and structural identification of IOP-1

[0045] (1) Sugar content of IOP-1

[0046] The sugar content in the prepared sample was determined by the classic phenol-sulfuric acid method. Before the experiment, the solution was prepared first. The detailed information is as follows:

[0047] Concentrated sulfuric acid: analytical grade, 99.8%.

[0048] 5% phenol solution: Use an analytical balance to accurately weigh 5g of phenol, place it in a 100mL volumetric flask, add distilled water and shake thoroughly to dissolve the phenol and adjust the volume to 100mL. After adjusting the volume, place it in a brown reagent bottle and refrigerate for use.

[0049] Glucose standard solution: Accurately weigh 20 mg of analytical grade glucose, dry it at 105°C to constant weight, place it in a 500 mL volumetric flask, dissolve it in double distilled water, shake well, and after constant volume, place it in a reagent bottle and refrigerate it for use.

[0050] After preparing the solution, draw the glucose standard curve. Take 0mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL, 0.8mL and 0.9mL of standard glucose solution respectively, and make up the volume to 1mL with double distilled water. Then, add 1mL of 5% phenol solution to each test tube, mix thoroughly, and then add 5mL of concentrated sulfuric acid, shake well and cool at room temperature. After leaving for 20 minutes, take 300μL of sample from each tube and transfer it to a 96-well plate. Use a full-wavelength microplate reader to detect the absorbance of the sample at a wavelength of 490nm. After completion, draw the standard curve.

[0051] The same method (n≥3) was used to prepare the Inonotus obliquus polysaccharide IOP-1 sample in Example 1. The sugar content in the sample was calculated by comparing with the standard curve to be 82.8%.

[0052] (2) Protein content determination

[0053] The protein content in the birch polysaccharide IOP-1 was detected by the Coomassie Brilliant Blue Bradford method. Coomassie Brilliant Blue G-250 is a blue dye with a maximum absorbance at a wavelength of 465nm. The dye can form a blue complex with protein, and the absorbance of this complex is the maximum at 595nm. Under the line of sight, the darker the color of the complex, the higher the protein content. This method is easy to operate, has stable color and high sensitivity, and is very suitable for the detection of trace proteins. The Coomassie Brilliant Blue Bradford method is one of the conventional methods for detecting protein content in polysaccharide extracts.

[0054] 0μL, 2μL, 4μL, 6μL, 8μL, 10μL, 12μL, 14μL, 16μL, 18μL and 20μL of standard protein solution were respectively added to a 96-well plate, and the volume was supplemented to 20μL with double distilled water. Then, 200μL of Coomassie Brilliant Blue G-250 dye solution was added to each well, and the pipette was fully mixed. After standing at room temperature for 5 minutes, the absorbance of the sample was detected at 595nm using a full-wavelength microplate reader. A standard curve was drawn to calculate the concentration of the target protein (n≥3).

[0055] After detection, no blue complex was formed, indicating that the Inonotus obliquus polysaccharide IOP-1 sample prepared in Example 1 did not contain protein.

[0056] (3) Polysaccharide molecular weight detection

[0057] The molecular weight of polysaccharides was detected by gel permeation chromatography. The samples were detected by Wyatt Sec-Mals Dawn Heleos-II laser detector and differential refractometer. The chromatographic column was Shodex OHpak sb-806, with 0.1M sodium sulfate and 0.2% sodium azide as the mobile phase, and the flow rate was set to 0.5mL / min. The concentration of the polysaccharide sample to be tested was 2mg / mL, and the total injection volume of the sample solution was 500μL. Dextran 40000 was used as a reference substance for normalization, and the running time was set to 35min. Finally, the absolute molecular weight of the four polysaccharides in this study was calculated using the principle of light scattering.

[0058] The results are as follows Figure 1 The molecular weight of Inonotus obliquus polysaccharide IOP-1 is 1513.341 kDa.

[0059] (4) Determination of monosaccharide composition

[0060] The monosaccharide composition of the polysaccharide samples was determined by HPLC, and the monosaccharide standards included mannose, rhamnose, glucuronic acid, glucose, arabinose, galacturonic acid, galactose, xylose, and fucose.

[0061] Before the experiment, the solution was prepared first. The detailed steps are as follows:

[0062] Preparation of standard: Take a clean 15mL centrifuge tube, add 8mL sterile water, add 100mg each of fucose, arabinose, glucose, fructose, galactose, ribose, xylose, mannose, galacturonic acid, glucuronic acid and mannuronic acid in turn, dissolve and make up to 10mL, and prepare a 10mg / mL mixed standard stock solution. Dilute the above mixed standard stock solution 100 times to prepare a working solution with a concentration of 100μg / mL, dilute the above solution according to the following gradient, and put it into a 1.5mL EP tube.

[0063] Sample pretreatment: weigh 5 mg of polysaccharide sample, add the prepared 2M TFA solution, and heat at 121℃ for 2h. Pass nitrogen and blow dry. Add methanol to wash, blow dry again, and repeat the methanol washing 2 to 3 times. Add sterile water to dissolve, transfer to a clean chromatographic bottle and wait for detection on the machine.

[0064] The chromatographic detection system used was Thermo ICS5000+ ion chromatography system (Thermo Fisher Scientific, USA), using DionexTM CarboPac TM PA20 liquid chromatography column, injection volume 20μL. Mobile phase A (H 2 O), mobile phase B (100 mM NaOH), column temperature was set at 30°C, and monosaccharide components were analyzed and detected using an electrochemical detector.

[0065] According to the monosaccharide composition test, IOP-1 includes fucose, rhamnose, arabinose, galactose, glucose, xylose and mannose, which account for the molar mass ratio of IOP-1 of 2.41:0.58:1.03:2.17:82.20:4.33:7.28 ( Figure 2 ). After FT-IR analysis, it was found that the -1 There is an obvious strong absorption peak in the molecule, which is composed of intermolecular and internal hydrogen bonds, making it at 3288cm -1 There is a broad peak at 3288cm -1 and 2929cm -1 They represent the hydroxyl group (-OH) and CH on the sugar chain of IOP-1. 1452cm -1 and 1379cm -1 The absorption peak at 1076cm is the CH angle vibration absorption of the methyl group connected to the carboxyl group. -1 and 1018cm -1 The absorption peak of is the angular vibration absorption peak of the hydroxyl group of pyranose ring, indicating that the monosaccharide exists in the form of pyranose. These are the characteristic peaks of polysaccharides. In addition, at 827cm -1 The absorption peak at indicates that the glycosidic bond in the sugar chain of IOP-1 is of α-configuration ( Figure 3 ).

[0066] Example 2 Antitumor activity of Inonotus obliquus polysaccharide IOP-1

[0067] 1. Inhibitory activity of Inonotus obliquus polysaccharide IOP-1 on various tumor cells

[0068] Cell line and cell culture: NCI-H460 was cultured in RPMI-1640 (containing 100 μg / mL streptomycin and 100 units / mL penicillin G) and 10% fetal bovine serum at 37°C constant temperature and humidity, with a carbon dioxide concentration of 5%. When the cell density reached 90%, the cells were subcultured for 1 minute and 3 passages.

[0069] Cell proliferation assay: NCI-H460 cells (2×10 3 cells / well), SHG-44 cells (2×10 3 cells / well) and HeLa cells (2×10 3 / well) were cultured in 96-well plates. After 24 hours of cell culture, the original culture medium was replaced with a drug culture medium containing 0, 250, 500 and 1000 μg / mL IOP-1. The experiment set up negative control, positive control and blank control: the negative control cells were cultured normally without any drugs; doxorubicin hydrochloride (DOX) was added to the positive control instead of IOP-1 drugs, and the DOX dose was set to 5μM; the blank control was not inoculated with cells and no drugs were added. After 24 hours of inoculation of NCI-H460 cells, 5μM DOX and different concentrations of IOP-1 (0, 250, 500, 1000μg / mL) were co-incubated with the cells. After 24h and 48h of co-incubation, the CCK-8 kit was used to detect cell viability. The CCK-8 reagent was added to the culture medium at a ratio of 1:10, incubated at 37°C for 30 minutes, and the absorbance A at 450nm of the microplate reader was calculated as follows:

[0070] Proliferation inhibition rate (%) = ([A 阴性对照 -A 实验组 ] / [A 阴性对照 -A 空白对照 ])×100%.

[0071] Each of the above groups was set up with 5 replicate wells, and the parallel experiments were repeated more than 3 times.

[0072] The results are as follows Figure 4 As shown in the figure, with the increase of IOP-1 concentration, the proliferation inhibition rate of glioma cells SHG-44 and cervical cancer cells HeLa increased less than that of non-small cell lung cancer NCI-H460 cells. The inhibition rate of NCI-H460 cells was 75.12±6.8%. Therefore, IOP-1 has the strongest inhibitory effect on non-small cell lung cancer NCI-H460 cells, and further studies on the anti-tumor activity and mechanism of birch polysaccharide IOP-1 on non-small cell lung cancer were carried out.

[0073] 2. Inhibitory activity and anti-tumor mechanism of Inonotus obliquus polysaccharide IOP-1 on non-small cell lung cancer NCI-H460 cells

[0074] (1) Inonotus obliquus polysaccharide IOP-1 significantly inhibits the proliferation of non-small cell lung cancer NCI-H460 cells

[0075] According to the cell proliferation experimental protocol in "1. Inhibitory activity of Inonotus obliquus polysaccharide IOP-1 on various tumor cells", non-small cell lung cancer NCI-H460 cells were co-incubated with IOP-1 for 24h and 48h respectively to further explore the inhibitory effect of IOP-1 on the proliferation of non-small cell lung cancer NCI-H460 cells. The results are as follows Figure 5 shown.

[0076] The morphology of cells co-incubated with IOP-1 was observed under a microscope. Figure 5 The results of A) showed that the cells in the IOP-1 group had abnormal morphology, shrunken and broken, and the cell density was significantly reduced. The CCK-8 results showed that the inhibitory effect of IOP-1 on the proliferation of NCI-H460 cells was time-dependent and concentration-dependent ( Figure 5 The maximum inhibition rate reached 87.99±2.01%. 50 ) and the IC values ​​of IOP-1 on NCI-H460 cells at 24h and 48h 50 They were 1068.10±167.15μg / mL and 341.35±31.49μg / mL respectively.

[0077] (2) Inonotus obliquus polysaccharide IOP-1 significantly inhibited the colony formation ability of non-small cell lung cancer NCI-H460 cells

[0078] Cell colony formation ability test: NCI-H460 (2×10 2 The cells were cultured in RPMI-1640 medium (0, 250, 500 and 1000 μg / mL) for 24 h. The medium was replaced with drug medium containing IOP-1 (0, 250, 500 and 1000 μg / mL), and at least 3 parallel experiments were performed for each dosing concentration. The cells were co-cultured with IOP-1 for 28 days, and the medium was replaced every 5 days. Crystal violet staining was used, and the cells were photographed under a microscope to count the number of cell colonies. Photos were taken with a Nikon inverted microscope (100×, 200×), and 3 photos were retained for each concentration.

[0079] like Figure 6 As shown in A, as the concentration of IOP-1 gradually increased, the density of NCI-H460 cell colonies in the culture plate gradually decreased. Statistical analysis found that IOP-1 significantly inhibited the colony-forming ability of NCI-H460 cells at concentrations of 250, 500, and 1000 μg / mL ( Figure 6 B).

[0080] (3) Inonotus obliquus polysaccharide IOP-1 induces apoptosis in non-small cell lung cancer NCI-H460 cells

[0081] Cell apoptosis assay: NCI-H460 (2×10 5 The cells were incubated with RPMI-1640 medium for 24 h, and then replaced with drug medium. After IOP-1 and cells were incubated for 48 h, PBS was washed twice, and reagents were added according to the instructions, mixed thoroughly, and kept at room temperature in the dark for 5-15 min. The experiment was repeated more than 3 times.

[0082] DAPI fluorescence staining: NCI-H460 (1×10 4Each well was incubated with RPMI-1640 medium for 24 hours, and then replaced with drug medium. After IOP-1 was co-incubated with cells for 24 hours and 48 hours, the cells were washed with PBS. After methanol fixation, the cells were washed again, and 80 μL of DAPI was added to each well, stained for 5 minutes in the dark, and then washed once with PBS. Photos were taken with a Nikon inverted fluorescence microscope (100×, 200×), and 5 photos were retained for each concentration.

[0083] Acridine Orange (AO) / Ethylpyridine Bromide (EB) fluorescence staining: NCI-H460 (1×10 4 After 24h and 48h of incubation of IOP-1 and cells, AO / EB dye solution (1:1) was prepared, 20 μL of dye solution was added to each well, mixed evenly, and reacted in the dark for 5 min. Photos were taken with a Nikon inverted fluorescence microscope (100×, 200×), and 5 photos were retained for each concentration.

[0084] DAPI fluorescence staining experiments showed that as the concentration of IOP-1 increased, the morphology of the cell nucleus began to change, chromatin aggregation, cell nucleus condensation, and blue apoptotic bodies began to appear. The number of apoptotic bodies in the experimental group was significantly higher than that in the control group, and increased with the increase of drug concentration ( Figure 7 A).

[0085] AO / EB fluorescent staining reflects the apoptosis of cells at different stages of apoptosis. Four different cell morphologies can be observed under a fluorescent microscope: nuclear chromatin is green, which is the typical shape of living cells; in early apoptotic cells, nuclear chromatin is green, solidified or beaded; nuclear chromatin of non-apoptotic dead cells is orange and has a normal shape; chromatin of apoptotic cells is orange, solid or beaded. In the IOP-1 group, AO / EB staining showed that the number of apoptotic cells increased in a concentration-dependent manner, while the number of living cells decreased ( Figure 7 B).

[0086] In order to quantitatively analyze apoptotic cells, flow cytometry was used to analyze cell apoptosis ( Figure 8 ). When IOP-1 at concentrations of 250 and 500 μg / mL was co-incubated with NCI-H460 cells, the number of live cells was significantly reduced, the proportion of apoptotic cells was significantly increased (P<0.05), and the maximum total apoptotic rate reached 19.68±1.34%. The above results show that IOP-1 can significantly induce apoptosis of NCI-H460 cells.

[0087] (4) Inonotus obliquus polysaccharide IOP-1 inhibited the migration and invasion of NCI-H460 cells

[0088] Tumor metastasis and invasion are one of the difficulties in current tumor treatment. Therefore, the effect of IOP-1 on tumor cell migration and invasion was evaluated using scratch assay and invasion assay.

[0089] Cell migration assay (scratch): NCI-H460 cells (6×10 5 / well) for 24h. When the cell density proliferated to 90%, the cell surface was evenly scratched with a pipette tip, and then the regular culture medium was replaced with a culture medium containing IOP-1 (0-1000μg / mL), and photos were taken at 0h and 72h. Fixed positions were marked on the cell well plate so that the captured areas at two time points were the same, the scratch healing rate was calculated, and more than 3 parallel experiments were performed before statistical analysis.

[0090] Cell invasion assay: NCI-H460 cells (1×10 5 The cells were inoculated into the Transwell insert with matrix gel. 10% serum was added to the lower chamber for induction, and the cells were incubated with medium containing IOP-1 (0-1000 μg / mL) in the upper chamber for 24 hours. The cells that passed through the chamber were stained with crystal violet, photographed and counted. Five groups of photos were taken for each well, and three parallel experiments were performed for each concentration.

[0091] The results showed that NCI-H460 cells in the control group almost covered the scratched area after 72 hours of culture, while the IOP-1 treated group did not heal ( Fig. 9 A) The results of the Transwell experiment are shown in Fig. 9 As shown in B, the matrix was placed in the chamber, allowing cancer cells to gradually migrate from the upper chamber to the lower chamber containing fetal bovine serum. The results showed that IOP-1 significantly reduced the number of NCI-H460 cells passing through the lower chamber of the matrix gel and the migration of cell membranes ( Fig. 9 C and D).

[0092] (5) Inonotus obliquus polysaccharide IOP-1 regulates protein expression in cells

[0093] Through the above studies, IOP-1 can inhibit the proliferation of NCI-H460 cells and induce apoptosis of tumor cells. Therefore, protein immunoblotting was used to detect the expression levels of related proteins in cells to clarify the anti-tumor mechanism of IOP-1 and its application in the development of anti-tumor drugs.

[0094] Western blotting: Cells from each group of culture conditions were collected, protein samples were extracted, and analyzed by western blotting. Protein samples were separated by electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes for 1.5 h. They were blocked with skim milk for 2 h, used with primary antibodies overnight, washed 4 times with PBST, incubated with secondary antibodies for 2 h, washed 4 times with PBST, developed with ECL kit, and visualized and quantitatively analyzed for protein expression using Tanon-5200 imaging system (Tanon, China).

[0095] The present invention detects Bax, Bcl-2, P53, Cyto-c and Parp-1 which are closely related to cell apoptosis. The study found that IOP-1 intervention significantly changes the expression of the above proteins in cells. The expression of MMP-9 which is related to migration and invasion is also abnormal ( Fig.10 ). These results indicate that IOP-1-induced apoptosis of NCI-H460 cells may be related to the regulation of P53, Cyto-c, Bax, Bcl-2 and Parp-1 proteins. At the same time, IOP-1 may participate in the regulation of cell migration and invasion by inhibiting the expression of MMP-9.

[0096] (6) Inonotus obliquus polysaccharide IOP-1 regulates ATP production in NCI-H460 cells to induce apoptosis

[0097] Intracellular adenosine triphosphate (ATP) assay: After cells were incubated with IOP-1 (500 and 1000 μg / mL) for 48 hours, the intracellular ATP level was determined using an ATP detection kit according to the manufacturer's instructions. The absorbance value was measured at 636 nm using an ELISA reader to determine the ATP content in the cells of the drug-treated groups under each condition.

[0098] Effects of IOP-1 on ATP content in NCI-H460 cells Fig.11 When IOP-1 was co-incubated with NCI-H460 cells, the ATP content in the cells decreased from 0.00203±0.00024 to 0.0009±0.00004 (μmol / 10 4 The above results show that IOP-1 inhibits ATP production in NCI-H460 cells, affects the function of mitochondria, and thus induces apoptosis of tumor cells, achieving the purpose of tumor suppression.

[0099] Example 3 Effect of Inonotus obliquus polysaccharide IOP-1 on non-small cell lung cancer tumor-bearing mice

[0100] Tumor-bearing mouse modeling and drug administration: Male C57BL / 6 mice aged 4 to 6 weeks were randomly divided into groups, with 6 mice in each group. NCI-H460 cells were inoculated once in the right armpit of the mice every 7 days, with a number of 10 7 The mice were inoculated for 3 consecutive weeks. From the 4th week, IOP-1 was used for daily gavage treatment. The low dose (IOP-1-L) used a polysaccharide concentration of 200 mg / Kg, and the high dose (IOP-1-H) used a polysaccharide concentration of 400 mg / Kg. The treatment lasted for 30 days. The negative control group (Control) was gavaged with normal saline; the positive control group (DDP) was gavaged with 3 mg / kg cisplatin. The mice's food intake, drinking water, and body weight were measured weekly, and the tumor size was monitored once every 2 weeks using small animal nuclear magnetic resonance. A small animal nuclear magnetic resonance system was used to track the growth of mouse tumors.

[0101] By tracking the tumor growth process of mice, it was found that the tumors of mice in the IOP-1-L and IOP-1-H groups stopped growing and the tumors gradually shrank ( Fig.12 ). Analysis of the survival status of tumor-bearing mice revealed that the daily water intake, daily food intake, weekly body weight changes, and tumor weight of tumor-bearing mice treated with IOP-1 were significantly better than those of the negative control group. Survival curve analysis showed that IOP-1 can prolong the lifespan of tumor-bearing mice. At the same time, statistics of immune organs (spleen index) found that the spleen index of the IOP-1 group increased significantly, indicating that IOP-1 played a bidirectional regulatory role, not only inducing tumor cell apoptosis, but also improving the immune regulation ability of mice. The above results show that IOP-1 significantly inhibits tumor growth in tumor-bearing mice and plays a therapeutic role ( Fig.13 ).

[0102] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A polysaccharide from Inonotus obliquus with anti-non-small cell lung cancer activity, characterized in that: The monosaccharide composition of the birch inonotus polysaccharide IOP-1 includes fucose, rhamnose, arabinose, galactose, glucose, xylose and mannose, and the molar mass ratio of the fucose, the rhamnose, the arabinose, the galactose, the glucose, the xylose and the mannose is 2.41:0.58:1.03:2.17:82.20:4.33:7.29; The preparation method comprises the following steps: The fruiting bodies of Inonotus obliquus were ultrafinely crushed, soaked in ethanol, boiled and extracted with distilled water, the filtrate was filtered and concentrated, and the supernatant was taken after ultra-high-speed centrifugation. The supernatant was precipitated with ethanol solution and centrifuged again, and the precipitate was collected and chromatographed on a DEAE cellulose column, eluted with water, and then eluted with a 0.1 M NaCl solution to obtain the Inonotus obliquus polysaccharide IOP-1; When the supernatant is precipitated with ethanol solution, the volume concentration of ethanol in the system is 80%.

2. The Inonotus obliquus polysaccharide IOP-1 according to claim 1, characterized in that The distilled water was added according to the material-liquid ratio of fruiting body powder: distilled water = 1 g: 50 mL.

3. The Inonotus obliquus polysaccharide IOP-1 according to claim 1, characterized in that The ultra-high speed centrifugation conditions are 9000 rpm for 10 min.

4. The Inonotus obliquus polysaccharide IOP-1 according to claim 1, characterized in that The conditions for the second centrifugation are 8000 rpm, 15 min.

5. Use of the Inonotus obliquus polysaccharide IOP-1 according to any one of claims 1 to 4 in the preparation of a medicament for treating non-small cell lung cancer.

6. A drug for treating non-small cell lung cancer, characterized in that: The active ingredient comprises the Inonotus obliquus polysaccharide IOP-1 according to any one of claims 1 to 4.

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