Inonotus obliquus acidic polysaccharide IOP-2 with anti-thyroid cancer activity and application thereof

Abstract

The application discloses a chaga acidic polysaccharide IOP-2 with anti-thyroid cancer activity and application thereof, and belongs to the field of biological medicines. The application adopts a method for extracting chaga polysaccharide by boiling water and precipitating chaga polysaccharide by ethanol, and combines DEAE cellulose column purification technology, and successfully realizes separation of chaga polysaccharide by using the principle that macromolecular polysaccharide is insoluble in organic solvents, and obtains chaga acidic polysaccharide IOP-2 with anti-thyroid cancer activity. The monosaccharide composition of the chaga acidic polysaccharide IOP-2 includes fucose 0.71%, rhamnose 3.33%, arabinose 2.18%, galactose 2.92%, glucose 80.59%, xylan 3.46% and mannose 6.81% in terms of molar percentage. The significant effect of the chaga acidic polysaccharide IOP-2 on anti-thyroid cancer is verified through in-vivo and in-vitro experiments, and a new treatment method is provided for clinical treatment of thyroid cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an inonotus obliquus acidic polysaccharide IOP-2 with anti-thyroid cancer activity and application thereof. BACKGROUND

[0002] Inonotus obliquus, also known as white birch fungus, is a unique fungus with a long history of folk medicine. It has attracted much attention in modern scientific research due to its high anti-tumor and antioxidant activities and other bioactive components (polysaccharides, triterpenes, polyphenols, etc.). Existing research has shown its potential in immune regulation, anti-inflammatory, anti-tumor (cell and animal level), and blood glucose lowering. Many studies have used fungal monomer compounds or extracts to study tumor cells and found that the active ingredients of fungi can inhibit the proliferation and differentiation of tumor cells and promote cancer cell apoptosis, which has shown certain value in the clinical treatment of diseases. However, different active ingredients of the same fungus have significant differences in treatment effects on different types of tumors due to chemical structure, mechanism of action, and heterogeneity of tumor cells. For example, triterpenoids in Ganoderma lucidum (such as ganoderic acid) can resist liver cancer and breast cancer by inducing apoptosis and inhibiting angiogenesis, while polysaccharide components (such as β-glucan) can enhance the immune surveillance function of lung cancer or colon cancer by activating immune cells (such as macrophages and NK cells). Similarly, cordycepin in Cordyceps sinensis selectively inhibits the proliferation of leukemia cells by interfering with RNA synthesis, but its adenosine components may have a more significant effect on solid tumors (such as glioma) as it can penetrate the blood-brain barrier and regulate the tumor microenvironment. In addition, protein-bound polysaccharides (PSK) in Coriolus versicolor have been shown to enhance the sensitivity of chemotherapy drugs to gastric cancer, while another component, polysaccharopeptide (PSP), inhibits the progression of cervical cancer by regulating Th1 / Th2 immune balance. This difference is also reflected in the pharmacokinetic properties: lipid-soluble components (such as sterols in fungi) are more easily enriched in fatty tissues such as the breast or prostate, while water-soluble components (such as polysaccharides) have higher concentrations in organs with abundant blood flow such as the liver and kidneys. Therefore, the specificity of fungal anti-tumor effects depends not only on the molecular targets of the components (such as topoisomerase inhibition and epigenetic regulation), but also on the molecular subtypes of tumors (such as HER2-positive breast cancer sensitivity to fungal terpenes). This complexity suggests that more high-quality, large-scale experimental data are needed to confirm the anti-tumor effects of fungal active ingredients to achieve precision therapy.

[0003] Thyroid cancer is a malignant tumor originating from thyroid tissue (mainly follicular epithelial cells or parafollicular C cells). The thyroid is located in the front of the neck, on both sides of the trachea, shaped like a butterfly, and is an important endocrine organ of the human body, responsible for secreting thyroid hormones, regulating key physiological processes such as metabolism and growth and development. In recent years, the incidence of thyroid cancer has shown a significant upward trend, and the age of onset is relatively young, and has become one of the more common malignant tumors. Current research has found that a variety of single components of traditional Chinese medicine show potential against thyroid cancer in laboratory studies, such as flavonoids (baicalin, wogonin and quercetin), saponins (ginsenosides and notoginsenosides), polyphenols (curcumin and resveratrol) and alkaloids (berberine and triptolide) and the like. The above-mentioned small molecule active ingredients mainly inhibit proliferation, induce apoptosis, inhibit metastasis and angiogenesis and the like. At present, these components are mainly explored as potential adjuvant therapy drugs or candidate molecules for new drug research and development. However, the focus of basic research is to find more effective and safer drugs, and to overcome the existing treatment difficulties (such as radioiodine resistance). The antitumor effect of Inonotus obliquus (especially its active component Inonotus obliquus polysaccharide component) on thyroid cancer has not been studied and no relevant reports have been found, which is worthy of further study. SUMMARY

[0004] The purpose of the present application is to provide an Inonotus obliquus acidic polysaccharide IOP-2 with anti-thyroid cancer activity and its application, so as to solve the problems existing in the prior art. The present application separates and obtains an Inonotus obliquus acidic polysaccharide IOP-2 with anti-non-small cell lung cancer activity, and verifies its significant effect on anti-thyroid cancer through in-vivo and in-vitro experiments, thereby providing a new treatment method for the clinical treatment of thyroid cancer

[0005] To achieve the above-mentioned purpose, the present application provides the following scheme:

[0006] The present application provides an Inonotus obliquus acidic polysaccharide IOP-2 with anti-thyroid cancer activity, wherein the monosaccharide composition of the Inonotus obliquus acidic polysaccharide IOP-2 comprises fucose 0.71%, rhamnose 3.33%, arabinose 2.18%, galactose 2.92%, glucose 80.59%, xylan 3.46% and mannose 6.81% in terms of molar percentage;

[0007] The preparation method comprises the following steps:

[0008] The fruiting body of Inonotus obliquus is ultra-micropulverized, soaked in ethanol, and boiled and extracted by adding distilled water; the extracted filtrate is filtered and concentrated, and the supernatant is obtained by high-speed centrifugation; the supernatant is precipitated by an ethanol solution and centrifuged again, and the precipitate is collected and subjected to DEAE cellulose column chromatography; the eluate of 0.3M NaCl solution is collected after elution with distilled water, 0.1M NaCl solution and 0.3M NaCl solution in sequence, to obtain the Inonotus obliquus acidic polysaccharide IOP-2.

[0009] When the supernatant is precipitated by an ethanol solution, the volume concentration of ethanol in the system is 80%.

[0010] Further, the distilled water is added in a solid-liquid ratio of 1g:50mL of fruiting body powder to distilled water.

[0011] Further, the high-speed centrifugation is performed at 9000rpm for 10min.

[0012] Further, the re-centrifugation is performed at 8000rpm for 15min.

[0013] The application further provides use of the Inonotus obliquus acidic polysaccharide IOP-2 in preparation of a medicament for treating thyroid cancer.

[0014] The application further provides a medicament for treating thyroid cancer, wherein the active ingredient comprises the Inonotus obliquus acidic polysaccharide IOP-2.

[0015] The application discloses the following technical effects:

[0016] The application adopts a method for extracting Inonotus obliquus polysaccharide by boiling water and precipitating by ethanol, and combines DEAE cellulose column purification technology, and successfully realizes separation of Inonotus obliquus polysaccharide and obtains an Inonotus obliquus acidic polysaccharide IOP-2 having an anti-thyroid cancer activity by using the principle that macromolecular polysaccharide is insoluble in an organic solvent.

[0017] The application proves that the Inonotus obliquus acidic polysaccharide IOP-2 has an inhibitory effect on thyroid cancer cells through a cell experiment; further analysis shows that IOP-2 inhibits occurrence and development of thyroid cancer by regulating the Bax / Bcl-2 / Caspase-3 mitochondrial apoptosis pathway of thyroid cancer. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative effort.

[0019] Figure 1 Molecular configuration of IOP-2;

[0020] Figure 2 Molecular weight and homogeneity detection results of IOP-2;

[0021] Figure 3 Monosaccharide composition analysis results of IOP-2;

[0022] Figure 4 Functional group results of FT-IR detection of IOP-2;

[0023] Figure 5 Effect of crude polysaccharide IOP from Inonotus obliquus on the proliferation inhibition of four tumor cells;

[0024] Figure 6 Effect of IOP-W, IOP-1 and IOP-2 (1000 μg / mL, 48 h) on the morphology of thyroid cancer C643 cells;

[0025] Figure 7 Effect of IOP-W, IOP-1 and IOP-2 on the proliferation inhibition of thyroid cancer C643 cells;

[0026] Figure 8 Effect of IOP-2 (1000 μg / mL, 48 h) on the nuclear morphology of thyroid cancer C643 cells;

[0027] Figure 9 Effect of IOP-2 (1000 μg / mL, 48 h) on the apoptosis of thyroid cancer C643 cells;

[0028] Figure 10 Effect of IOP-2 on the tumor growth of thyroid cancer C643 tumor-bearing mice;

[0029] Figure 11 Effect of IOP-2 on the survival state of thyroid cancer C643 tumor-bearing mice; wherein, A is the average body weight; B is the food intake; C is the water intake;

[0030] Figure 12 Laser speckle system detection of blood supply around the tumor of tumor-bearing mice after administration of IOP-2;

[0031] Figure 13 Effect of IOP-2 on the expression of mitochondrial apoptosis-related proteins in the tumor tissues of thyroid cancer tumor-bearing mice. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] The Inonotus obliquus was collected from the Changbai Mountain area of ​​Jilin Province and identified as Inonotus obliquus by Professor Liu Yang of Jilin Agricultural University.

[0038] Other biological materials include thyroid cancer cell line C643 (CL-0648, Promocell Life Science Technology Co., Ltd.), liver cancer cell HepG2 (CL-0627, Promocell Life Science Technology Co., Ltd.), lung cancer cell NCI-H460 (CL-0299, Promocell Life Science Technology Co., Ltd.), pancreatic cancer cell MIAPaCa-2 (CL-0627, Promocell Life Science Technology Co., Ltd.), fetal bovine serum (164210, Promocell Life Science Technology Co., Ltd.), complete culture medium 1640 (PM150110, Promocell Life Science Technology Co., Ltd., China). DAPI fluorescent staining solution (KGA1808-50, Jiangsu KeyGene Biotech Co., Ltd., China) and Annexin V-FITC / PI double staining cell apoptosis detection kit (KGA1102-100, Jiangsu KeyGene Biotech Co., Ltd., China). Immunofluorescence and protein antibodies (ABclonal Biotechnology Co., Ltd., Wuhan, China).

[0039] Example 1 Extraction, isolation and molecular weight detection of Inonotus obliquus polysaccharide IOP-2

[0040] 1. Extraction and isolation of Inonotus obliquus polysaccharide IOP-2

[0041] The fruiting body of Inonotus obliquus was ultra-finely ground, and the powder was soaked in ethanol for 3 days. Then, distilled water was added according to the ratio of fruiting body powder:distilled water = 1 g:50 mL, heated to boiling point for extraction, and repeated 5 times for 2 hours each time. The filtrate was combined and filtered with filter cloth twice, concentrated to 1 / 5 of the original volume, and centrifuged at 9000 rpm for 10 min. The supernatant was retained for use. Four times the volume of 95% ethanol was added to the supernatant to precipitate the polysaccharides, and the final concentration of ethanol was adjusted to 80%. The mixture was stirred uniformly with a glass rod and allowed to stand overnight at 4°C. The next day, the mixture was centrifuged at 9000 r for 15 min, and the precipitate was collected. The precipitate was dissolved in a small amount of water and lyophilized for use. The crude polysaccharide IOP was obtained.

[0042] 500 mg of crude polysaccharide was dissolved and subjected to DEAE cellulose DE-52 ion exchange column chromatography. Distilled water, 0.1 mol / L and 0.3 mol / L NaCl solutions were used as eluents in sequence, and the flow rate was kept stable. An automatic sample injector was used to collect the eluents, and three components, IOP-W, IOP-1 and IOP-2, were obtained. The sugar content of each tube in the automatic sample injector was determined by the phenol-sulfuric acid method, and the same components were combined. After lyophilization, G200 dextran gel column was used for molecular sieve chromatography, and finally three components of lyophilized powder were obtained. IOP-W and IOP-1 have been disclosed in Chinese patents with publication numbers CN118615316B and CN119350519B, respectively.

[0043] 2. Molecular weight, monosaccharide composition and structural identification of IOP-2

[0044] (1) Molecular configuration of IOP-2

[0045] After IOP-2 was purified by G200 Sephadex gel column, the molecular configuration analysis showed that the slope was 0.03±0.00, indicating that the molecular configuration of Inonotus obliquus acidic polysaccharide IOP-2 tended to be spherical, as shown in Figure 1 .

[0046] (2) Detection of polysaccharide molecular weight

[0047] The gel permeation chromatography was used to detect the molecular weight of polysaccharide, Wyatt Sec-Mals Dawn Heleos-II laser detector and differential refractive detector were used to detect the sample together, the chromatographic column was Shodex OHpak sb-806, 0.1M sodium sulfate and 0.2% sodium azide were used as the mobile phase, the flow rate was set to 0.5mL / min. The concentration of polysaccharide sample to be tested was 2mg / mL, the total amount of sample solution was 500μL. Dextran 40000 was used as the control, the running time was set to 35min. Finally, the absolute molecular weight of the four polysaccharides in this study was calculated by light scattering principle.

[0048] The results are shown in Figure 2 , the molecular weight of Inonotus obliquus polysaccharide IOP-2 was 55.1822kDa.

[0049] (3) Determination of monosaccharide composition

[0050] The monosaccharide composition of polysaccharide sample was determined by high performance liquid chromatography, and the monosaccharide standard included mannose, rhamnose, glucuronic acid, glucose, arabinose, galacturonic acid, galactose, xylose and fucose.

[0051] Before the experiment, the solution was prepared first, the detailed steps are as follows:

[0052] Preparation of standard: take a clean 15mL centrifuge tube, add 8mL sterile water, add fucose, arabinose, glucose, fructose, galactose, ribose, xylose, mannose, galacturonic acid, glucuronic acid and mannuronic acid 100mg respectively, dissolve and constant volume to 10mL, prepare 10mg / mL mixed standard solution mother liquor. Dilute the above mixed standard mother solution by 100 times to prepare a working solution with a concentration of 100μg / mL, take the above solution and dilute according to the following gradient, and load into 1.5mL EP tube.

[0053] Sample pretreatment: 5 mg of polysaccharide sample was weighed, and 2M TFA solution was added. It was heated at 121℃ for 2h. Nitrogen was blown to dry. Methanol was added for washing, and then dried. Methanol washing was repeated for 2-3 times. Sterile water was added for dissolution, and then transferred into a clean chromatographic bottle for machine detection.

[0054] The chromatographic detection system used Thermo ICS5000+ ion chromatography system (Thermo Fisher Scientific, USA), Dionex TM CarboPac TM PA20 liquid chromatography column, sample size 20μL. Mobile phase A (H2O), mobile phase B (100mM NaOH), column temperature set to 30℃, and single sugar components were analyzed and detected by electrochemical detector.

[0055] Through monosaccharide composition detection, IOP-2 includes fucose, rhamnose, arabinose, galactose, glucose, xylan and mannose, accounting for 0.71%, 3.33%, 2.18%, 2.92%, 80.59%, 3.46% and 6.81% of IOP-2 respectively Figure 3 )。

[0056] (4) Structure identification of IOP-2

[0057] The main functional groups in IOP-2 were detected by infrared spectroscopy to understand its structural characteristics. After FT-IR analysis, it was found that there was a strong absorption peak at 3416.76cm -1 , indicating the existence of intermolecular and internal hydrogen bonds, which is a typical characteristic of hydroxyl stretching vibration. The absorption peak at 3134.2cm -1 further confirmed the hydrogen bonding of hydroxyl on the sugar chain. The absorption peak at 2918.25cm -1 corresponds to the stretching vibration of C-H bond. The absorption peak at 1617.02cm -1 indicates the existence of C=O stretching vibration, so it is indicated that IOP-2 may have acid connected glucan. The absorption peak at 1263.62cm -1 is the methyl C-H angle vibration absorption of carboxyl group. The absorption peak at 1067.41cm -1 is the hydroxyl stretching vibration in pyranose ring, indicating that monosaccharide exists in the form of pyranose. The absorption peak at 619.52cm -1 indicates that the glycosidic bond in the sugar chain may be β-configuration, which is one of the typical characteristics of polysaccharide structure Figure 4 )。

[0058] Example 2 Antitumor activity of Inonotus obliquus polysaccharide IOP-2

[0059] 1. Inhibitory activity of crude polysaccharide from Inonotus obliquus against different tumor cell types

[0060] Four tumor cell lines were selected: HepG2 liver cancer cells, NCI-H460 lung cancer cells, MIAPaCa-2 pancreatic cancer cells, and C643 thyroid cancer cells. The effect of crude polysaccharide IOP from Inonotus obliquus on the proliferation ability of tumor cells was detected by the CCK-8 assay.

[0061] Cell lines and cell culture: HepG2 liver cancer cells, NCI-H460 lung cancer cells, MIAPaCa-2 pancreatic cancer cells, and C643 thyroid cancer cells were cultured at 37°C under constant temperature and humidity and 5% carbon dioxide in RPMI-1640 (containing 100 μg / mL streptomycin and 100 units / mL penicillin G) and 10% fetal bovine serum, respectively. Cells were passaged 1:3 and cultured continuously after reaching a confluence of 90%.

[0062] Cell proliferation assay: HepG2 liver cancer cells, NCI-H460 lung cancer cells, MIAPaCa-2 pancreatic cancer cells, and C643 thyroid cancer cells (all 2×10⁻⁶ cells) were detected. 3 Cells were cultured in 96-well plates (cells / well) for 24 hours. The original culture medium was then replaced with a drug-treated medium containing different concentrations of crude polysaccharide IOP (0, 500, and 1000 μg / mL). Negative, positive, and blank controls were included: negative control cells were cultured normally without any drugs; positive control cells were treated with doxorubicin hydrochloride (DOX) instead of Inonotus obliquus polysaccharide (DOX) at a dose of 5 μM; blank control cells were not seeded and no drugs were added. After 24 hours of seeding, C643 cells were co-incubated with 5 μM DOX and different concentrations of IOP (0, 500, and 1000 μg / mL). Cell viability was assessed using a CCK-8 assay kit after 48 hours of co-incubation. The CCK-8 reagent was added to the culture medium at a 1:10 ratio, and the cells were incubated at 37°C for 30 minutes. The absorbance (A) at 450 nm was calculated using the following formula:

[0063] Proliferation inhibition rate (%) = ([A) 阴性对照 -A 实验组 ] / [A 阴性对照 -A 空白对照 ])×100%.

[0064] Each of the above groups has 5 replicate wells, and the experiment is repeated at least 3 times in parallel.

[0065] The results are as follows Figure 5, IOP had inhibitory activity on the proliferation of four tumor cells, but the inhibitory ability on thyroid cancer cell C643 was the strongest (48h), reaching 85.42±4.91%, the inhibition rates of liver cancer cell HepG2, lung cancer cell NCI-H460, and pancreatic cancer cell MIAPaCa-2 were 39.82±3.46%, 42.31±4.61%, and 59.24±6.87%, respectively. This shows that the inhibitory ability of IOP on thyroid cancer cell C643 is significantly better than that on other tumor cells. Further selection of thyroid cancer cell C643 for in-depth study.

[0066] 2. Inhibitory activity of different inonotus obliquus polysaccharides on tumor cells

[0067] Cell line and cell culture: thyroid cancer cell C643 was cultured in RPMI-1640 (containing 100 μg / mL streptomycin and 100 units / mL penicillin G) and 10% fetal bovine serum at 37°C in a constant temperature and humidity environment with a carbon dioxide concentration of 5%. When the cell density reached 90%, the cells were subcultured at a ratio of 1:3 and continuously cultured.

[0068] Cell proliferation detection: C643 cells (2×10 3 were cultured in 96-well plates. After 24 hours of cell culture, the original culture medium was replaced with drug culture medium containing different concentrations of IOP-W, IOP-1, and IOP-2 (0, 500, and 1000 μg / mL). Negative control, positive control, and blank control were set up: negative control cells were normally cultured without any drugs; in the positive control, doxorubicin hydrochloride (DOX) was added instead of inonotus obliquus polysaccharides, and the DOX dose was set to 5 μM; the blank control did not inoculate cells and did not add drugs. After 24 hours of C643 cell inoculation, 5 μM DOX and different concentrations of IOP-W, IOP-1, and IOP-2 (0, 500, 1000 μg / mL) were incubated with the cells. After 24h and 48h of co-incubation, the cell viability was detected by CCK-8 kit. 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 was detected by a microplate reader. The calculation formula is as follows:

[0069] Inhibition rate (%) = ([A 阴性对照 -A 实验组 ] / [A 阴性对照 -A 空白对照 ])×100%.

[0070] Each group of the above was set up with 5 replicate wells, and the parallel experiment was repeated more than 3 times.

[0071] After 48h of co-incubation, microscopic observation showed that the C643 cells co-cultured with IOP-2 had abnormal morphology, appeared shrunk and broken, and the cell density was significantly reduced, as shown in Figure 6The CCK-8 method was used to detect the effects of the separated and purified IOP components on the proliferation inhibition of thyroid cancer C643 (48 h), and the results are shown in Table 1 Figure 7 The results show that the activity of IOP-2 is the best, and when the acting concentration is 1000 μg / mL, the proliferation inhibition rate reaches 87.89 ± 2.76%, which is significantly higher than that of IOP-W and IOP-1 two polysaccharide components. At the same time, the anti-tumor activities of the three components all have the characteristics of concentration dependence.

[0072] 3. IOP-2 significantly induces C643 cell apoptosis

[0073] To verify the effect of IOP-2 on the apoptosis of thyroid cancer C643 cells, DAPI fluorescent staining was performed using 500 and 1000 μg / mL of IOP-2.

[0074] DAPI fluorescent staining experiment: C643 cells were seeded in culture dishes containing culture medium at a density of 1 × 10 4 cells / well and cultured for 24 hours. Then, the original culture medium was replaced with a drug culture medium, and the cells were co-incubated with IOP-2 for 24 and 48 hours. Subsequently, the cells were washed with PBS, fixed with methanol, and washed again. 80 μL of DAPI was added to each well, and the cells were stained in the dark for 5 minutes, followed by washing with PBS once. Finally, photographs were taken using a Nikon inverted fluorescence microscope (magnification of 100x and 200x), and 5 photographs were retained for each drug concentration.

[0075] The results show that after IOP-2 administration, the nuclear morphology of C643 cells began to change, the chromatin aggregated, the nucleus pyknosis, and the blue light apoptotic bodies appeared. The number of apoptotic bodies in the experimental group was significantly more than that in the control group, and increased with the increase of drug concentration (see Figure 8 ).

[0076] Inducing apoptosis is an important way to inhibit tumor cell proliferation. Flow cytometry was used to analyze the proportion of apoptosis:

[0077] C643 (2 × 10 5 cells / well) was cultured in RPMI-1640 medium for 24 h, and then the drug culture medium was used to replace it. After IOP-1 (1000 μg / mL) was co-incubated with the cells for 48 h, PBS was used to wash the cells twice, the reagents were added according to the instructions, mixed thoroughly, and incubated in the dark at room temperature for 5-15 min. The experiment was repeated more than 3 times.

[0078] The results are shown in Table 2 Figure 9When C643 cells were treated with IOP-2 at a concentration of 1000 μg / mL, the number of living cells was significantly reduced, the rate of apoptosis changed significantly (P < 0.05), and the total rate of apoptosis increased significantly, with a maximum total rate of apoptosis of 18.92 ± 2.19%. Fluorescent staining of the nucleus and flow cytometry results showed that IOP-2 could significantly induce apoptosis in C643 cells.

[0079] Example 3 Effect of Inonotus obliquus polysaccharide IOP-2 on thyroid cancer-bearing mice

[0080] 1. Modeling of tumor-bearing mice and administration of drugs

[0081] Male C57BL / 6 mice aged 4-6 weeks were randomly divided into groups of 5 mice each, and C643 cells were inoculated into the right axillary of the mice once every 7 days at a quantity of 10 7 cells. This was repeated for 3 weeks, and IOP-2 (400 mg / Kg of polysaccharide concentration) was administered orally once daily from the fourth week, for a total of 28 days of treatment. The control group (Control) was administered normal saline. The mice were measured for food intake, water consumption, and body weight once every week, and the size of the tumor was monitored once every 2 weeks using a small-animal nuclear magnetic resonance system. The growth of the tumor in the mice was tracked using a small-animal nuclear magnetic resonance system. The mice were measured for body weight 72 h after the last administration of the drug, and were then sacrificed. After confirming the death of the mice, the mice were dissected, and the tumor tissue was completely peeled off.

[0082] The survival status, body weight, food intake, and tumor size of the tumor-bearing mice were tracked during the modeling period. The results are shown in Figure 10 After 4 weeks of treatment, the mice in the control group were administered normal saline, and the tumor volume of the mice increased significantly, from 33.645 mm 2 to 67.834 mm 2 . In contrast, the tumor volume of the mice in the IOP-2 treatment group decreased significantly, from 46.011 mm 2 to 29.539 mm 2 . Based on the above results, it can be concluded that IOP-2 can inhibit the proliferation of C643 tumor cells in tumor-bearing mice. At the same time, the survival status of the mice was observed, and it was found that the average body weight, food intake, and water consumption of the mice in the control group decreased as the tumor developed, while the mice in the administration group showed an increase in all indicators. The results show that IOP-2 not only inhibits the growth of tumors in tumor-bearing mice, but also effectively improves the survival status of the mice, and has a significant therapeutic effect, as shown in Figure 11

[0083] 2. Effect of IOP-2 on blood flow around the tumor tissue of tumor-bearing mice

[0084] ​The blood supply around the tumor tissue of tumor-bearing mice was detected by laser speckle blood flow imaging system. The results showed that the blood supply around the tumor tissue of mice was rich before treatment, and the laser intensity value was large, which was 215.00±37.97 and 221.05±24.10 for the control group and the drug group, respectively. After treatment, with the decrease of tumor size, the blood flow around the tumor tissue decreased, and the values of the control group and the drug group changed to 231.45±21.75 and 194.72±12.98 (P<0.05), respectively. It can be seen that the blood flow around the tumor tissue of IOP-2 group decreased, which may be related to the decrease of tumor tissue volume. The results are shown in Figure 12 and Table 1.

[0085] Table 1 Effect of IOP-2 on the blood supply around the tumor tissue of thyroid cancer-bearing mice

[0086] Control IOP-2 0 weeks 215.00±37.97 221.05±24.10 4 weeks 231.45±21.75 194.72±12.98*

[0087] Note: mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0088] 3, IOP-2 affects the expression of apoptosis-related genes in the tumor tissue of thyroid cancer C643-bearing mice

[0089] Through the above interaction of IOP-2 and C643 cells, combined with animal experiments, it was found that IOP-2 can inhibit the proliferation and apoptosis of C643 cells, and inhibit tumor growth, which may be achieved by regulating multiple genes and proteins. Therefore, the removed tumor tissue of tumor-bearing mice was subjected to Q-PCR study of apoptosis genes to detect the expression levels of apoptosis-related genes Bcl-2, Bax and Caspase-3 in the tumor, and to clarify its anti-tumor mechanism. The results are shown in Table 2. Compared with the control, the expression levels of apoptosis-related genes Bcl-2, Bax and Caspase-3 in the tumor were significantly changed, specifically, the pro-apoptotic gene Bax was significantly increased, while Bcl-2 was significantly decreased, and the expression of Caspase-3 was also significantly increased.

[0090] Table 2 Effect of IOP-2 on the expression of apoptosis-related genes in the tumor tissue of thyroid cancer-bearing mice

[0091] Control IOP-2 Bcl-2 1 0.72±0.13* Bax 1 3.15±1.02** Caspase-3 1 3.98±2.14**

[0092] Note: mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0093] 4, IOP-2 affects the expression of mitochondrial apoptosis-related proteins in the tumor tissue of thyroid cancer C643-bearing mice

[0094] According to the Q-PCR results, the Bax, Bcl-2 and Caspase-3 proteins closely related to mitochondrial apoptosis were first detected.

[0095] Western Blot: Cell samples were collected from each group of culture conditions, protein samples were extracted and analyzed using Western Blot. After electrophoretic separation, the protein samples were transferred to a polyvinylidene fluoride (PVDF) membrane for 1.5 hours. Subsequently, the membrane was blocked with skim milk for 2 hours, incubated with primary antibody overnight, then washed 4 times with PBST, and co-incubated with secondary antibody for 2 hours. Finally, color development was performed with an ECL kit, and the protein expression was visualized and quantitatively analyzed using a Tanon-5200 imaging system (Tanon, China).

[0096] The results showed that the pro-apoptotic protein Bax was significantly increased, while Bcl-2 was significantly decreased, and Caspase-3 expression was also significantly increased, indicating an increased trend of mitochondrial apoptosis in cells. The Western Blot results are shown in Figure 6 and Table 3. The above results indicate that IOP-2 induces apoptosis of C643 cells, which may be related to the expression of mitochondrial apoptosis-related proteins such as Bax, Bcl-2 and Caspase-3. Figure 13 and Table 3. The above results indicate that IOP-2 induces apoptosis of C643 cells, which may be related to the expression of mitochondrial apoptosis-related proteins such as Bax, Bcl-2 and Caspase-3.

[0097] Table 3 Analysis of mitochondrial apoptosis-related protein expression in tumor tissues of IOP-2-treated thyroid cancer-bearing mice

[0098] Control IOP-2 Bcl-2 1 0.32±0.25* Bax 1 4.26±1.78** Caspase-3 1 5.81±1.92***

[0099] Note: Mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0100] The above examples show that the Inotodiasic Acid Polysaccharide IOP-2 component has significant inhibitory activity on thyroid cancer C643 cells, and the antitumor activity of IOP-2 is confirmed in vivo by tumor-bearing mouse experiments. Mechanism studies have preliminarily explored that IOP-2 inhibits the occurrence and development of thyroid cancer by regulating the Bax / Bcl-2 / Caspase-3 mitochondrial apoptosis pathway of thyroid cancer. In summary, IOP-2 provides a new treatment method for the clinical treatment of thyroid cancer.

[0101] The above-described examples are only descriptions of preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. An inonotus obliquus acidic polysaccharide IOP-2 having anti-thyroid cancer activity, characterized in that, The monosaccharide composition of the Inonotus obliquus acidic polysaccharide IOP-2 includes fucose 0.71%, rhamnose 3.33%, arabinose 2.18%, galactose 2.92%, glucose 80.59%, xylan 3.46% and mannose 6.81% in terms of molar percentage; The preparation method comprises the following steps: After the Inonotus obliquus fruiting body is ultra-micro pulverized, the fruiting body is soaked in ethanol, and distilled water is added for decocting and extracting; the extracted filtrate is filtered and concentrated, and the supernatant is obtained after high-speed centrifugation; the supernatant is precipitated by an ethanol solution and centrifuged again, and the precipitate is collected and subjected to DEAE cellulose column chromatography; the eluate of the 0.3M NaCl solution is collected after the DEAE cellulose column chromatography is eluted with distilled water, 0.1M NaCl solution and 0.3M NaCl solution in sequence, so that the Inonotus obliquus polysaccharide IOP-2 is obtained; When the supernatant is precipitated by the ethanol solution, the volume concentration of ethanol in the system is 80%.

2. The Inotodiol Acidic Polysaccharide IOP-2 according to claim 1, characterized in that, The distilled water is added in a solid-liquid ratio of 1g:50mL of fruiting body powder and distilled water.

3. The Inotodiolic acid polysaccharide IOP-2 according to claim 1, characterized in that, The high-speed centrifugation is performed at 9000rpm for 10min.

4. The Inotodiolic acid polysaccharide IOP-2 according to claim 1, characterized in that, The re-centrifugation is performed at 8000rpm for 15min.

5. Use of the Inonotus obliquus acidic polysaccharide IOP-2 in the preparation of a drug for treating thyroid cancer according to any one of claims 1-4.

6. A medicament for treating thyroid cancer, characterized by comprising the compound of claim 1. The active ingredient comprises the Inonotus obliquus acidic polysaccharide IOP-2 according to any one of claims 1-4.

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