Application of honeysuckle polysaccharide in preparation of medicine for treating thyroid cancer

Abstract

The invention relates to the technical field of drugs for treating thyroid cancer, in particular to application of honeysuckle polysaccharide in preparation of drugs for treating thyroid cancer. According to the technical scheme, on the basis of separating and purifying the honeysuckle polysaccharide with relatively good uniformity, the honeysuckle polysaccharide is found to have no toxic effect on normal thyroid follicular cells, but selectively inhibits proliferation of recurrent metastatic papillary thyroid carcinoma cells, and has an inhibiting effect on transplanted tumors of nude mice inoculated with a recurrent metastatic papillary thyroid carcinoma cell line. According to the scheme, the honeysuckle flower polysaccharide is good in homogeneity, the molecular weight is 23 + / -4 kDa, the honeysuckle flower polysaccharide is mainly composed of five monosaccharides, and alpha-(1-> 4)-D-GalpA is mainly connected to form a main chain. The polysaccharide has no toxic effect on normal thyroid cells while effectively inhibiting the cell viability of IHH-4. According to the technical scheme, the technical problem that drugs for treating recurrent metastatic papillary thyroid carcinoma with small side effects and high specificity are lacked in the prior art can be solved, and the drug has an ideal application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of thyroid cancer drugs, and in particular to application of honeysuckle polysaccharide in preparing drugs for treating thyroid cancer. Background Art

[0002] Thyroid carcinoma (TC) is the most common malignant tumor of the endocrine system in the world. In the past 30 years, thyroid cancer has been the fastest growing type of cancer among all tumors and is expected to rank fourth in the world. Papillary thyroid carcinoma (PTC) is the most common subtype, but people do not pay enough attention to it because most patients have a good prognosis after conventional treatment. In fact, the recurrence rate of papillary thyroid carcinoma is high, and about 7-15% of them have distant metastasis. The mortality rate of patients with recurrent and metastatic papillary thyroid carcinoma is as high as 70%.

[0003] Recurrent and metastatic papillary thyroid cancer refers to the reappearance of papillary thyroid cancer that has been treated at the primary site (i.e., local recurrence), or the spread of cancer cells to other parts of the body (such as lymph nodes, etc.) to form new tumor lesions (i.e., distant metastasis). This usually occurs after initial surgical resection and / or radioactive iodine treatment. Early screening for the risk of recurrence and metastasis is very important for reducing patient mortality, but there is currently no recognized "gold standard" for assessing the risk of recurrence and metastasis of papillary thyroid cancer. Clinically, the risk of recurrence or metastasis of papillary thyroid cancer is mainly assessed by tumor size, lymph node metastasis characteristics, and degree of vascular invasion, but it has little effect on reducing mortality. Another tricky problem is that the treatment options for recurrent and metastatic papillary thyroid cancer are limited. Currently, radioactive iodine is the main treatment for recurrent and metastatic papillary thyroid cancer, but about half of the patients are insensitive to treatment, and the prognosis of these patients is even worse, with a 10-year survival rate of only 10%. When cancer is insensitive to radioactive iodine or is not suitable for use, targeted drugs (such as sorafenib) are also tried for the treatment of recurrent and metastatic papillary thyroid cancer. In some cases, although targeted therapies can prolong patients' survival, they may cause a series of side effects because they affect normal cellular processes, and over time, tumors may develop resistance to targeted therapies, which may lead to treatment failure. Further research on specific drugs for recurrent and metastatic papillary thyroid carcinoma is urgently needed in order to obtain drugs with fewer side effects and high specificity for the treatment of recurrent and metastatic papillary thyroid carcinoma to meet the needs of clinical application. Summary of the invention

[0004] The purpose of the present invention is to use honeysuckle polysaccharide in the preparation of drugs for treating thyroid cancer, so as to solve the technical problem that there is a lack of drugs with small side effects and high specificity for treating recurrent and metastatic thyroid papillary carcinoma in the prior art. This scheme provides a natural active substance that is highly efficient, low-toxic and selective in killing thyroid cancer tumor cells, which can inhibit the lack of effective therapeutic drugs for recurrent and metastatic thyroid cancer.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] Application of honeysuckle polysaccharide in preparing medicine for treating thyroid cancer.

[0007] Furthermore, the thyroid cancer is recurrent metastatic papillary thyroid cancer.

[0008] Furthermore, the monosaccharide composition of the honeysuckle polysaccharide is: galacturonic acid 53.4% ​​to 80.2%, glucose 8% to 12%, galactose 11.7% to 17.5%, arabinose 6.6% to 9.8%, and mannose 0.3% to 0.5%.

[0009] Furthermore, the honeysuckle polysaccharide is connected in sequence by α-(1→4)-D-GalpA to form a main chain.

[0010] Furthermore, the side chains of the honeysuckle polysaccharide include β-(1→4)-linked galactan, α-(1→5)-linked and (1→3,5)-linked arabinan / arabinogalactan, and α-(1→4)-linked glucan.

[0011] Furthermore, the relative average molecular weight of honeysuckle polysaccharide is 10-40 kDa;

[0012] Preferably, the relative average molecular weight of the obtained honeysuckle polysaccharide is determined to be 23±4 kDa using high performance liquid chromatography based on a TSKgel G4000 PWxl gel exclusion chromatography column.

[0013] Furthermore, the honeysuckle polysaccharide is prepared by the following method:

[0014] S1: After removing fat-soluble substances from honeysuckle, water is used as a solvent for decoction and extraction, and the supernatant is taken; after concentration, protein removal, and alcohol precipitation, the precipitate is collected; and after freeze-drying, the total sugar of honeysuckle is obtained;

[0015] S2: Load the aqueous solution of total sugars from honeysuckle onto a DEAE-cellulose chromatography column, and use distilled water and 0.25 mol / L NaCl solution to elute the DEAE-cellulose chromatography column in sequence, and take the portion corresponding to the total sugar content peak and the uronic acid peak of the elution curve from the components eluted with the 0.25 mol / L NaCl solution; and then concentrate, dialyze and freeze-dry to obtain the honeysuckle polysaccharide WLJP-025 component;

[0016] S3: The honeysuckle polysaccharide WLJP-025 component was dissolved in sodium chloride solution, loaded on a Sepharose CL-6B chromatography column, eluted with a 0.1 mol / L NaCl solution, and the eluate corresponding to the maximum total sugar content peak and uronic acid peak of the elution curve was taken; after concentration, dialyzation and freeze-drying, the honeysuckle polysaccharide WLJP-025p was obtained.

[0017] Further, in S2, the DEAE-cellulose chromatography column was eluted with 2 column volumes of distilled water and 0.25 mol / L NaCl solution, and the elution flow rate was 10 mL / min.

[0018] Furthermore, in S3, the flow rate of elution using a 0.1 mol / L NaCl solution was 0.1 mL / min.

[0019] Furthermore, the in vitro effective concentration of the honeysuckle polysaccharide on IHH-4 cells is 500-2000 μg / mL; in the nude mouse transplant tumor model, the dosage of the honeysuckle polysaccharide is 100 mg / kg.

[0020] The technical principle and beneficial effects of this technical solution are:

[0021] The technical scheme provides a Lonicera japonica polysaccharide (LJP) which has a significant inhibitory effect on the recurrent metastatic thyroid cancer cell line IHH4 and has no toxic effect on the normal thyroid cell line Nthy-ori3-1. It is a rare Chinese medicine active substance with high efficiency and low toxicity. Among them, IHH4 is a cell line derived from human papillary thyroid carcinoma, which is widely used to study the biological characteristics of thyroid cancer and develop new treatment methods. IHH4 cells have a weak ability to absorb radioactive iodine (RAI), which reflects the resistance of some recurrent and metastatic papillary thyroid carcinomas to radioactive iodine treatment in clinical practice. This cell line is often used in laboratory environments to simulate the behavior of recurrent and metastatic papillary thyroid carcinoma, including tumor growth, invasion and metastasis. The IHH4 cell line can be used to test the effects of new anticancer drugs or targeted therapies, especially when looking for new treatments for radioactive iodine-resistant thyroid cancer.

[0022] After the in vitro experiment, the model animal experiment was continued and found that honeysuckle polysaccharide had a significant inhibitory effect on papillary thyroid cancer-bearing animals (recurrent metastatic cell line IHH4), suggesting that honeysuckle polysaccharide may be an effective therapeutic drug. In-depth research on the effect of honeysuckle polysaccharide on recurrent metastatic papillary thyroid cancer is of great significance.

[0023] In summary, based on the separation and purification of honeysuckle polysaccharides with good uniformity, in-depth research found that it has no toxic effect on normal thyroid follicular cells, but selectively inhibits the proliferation of recurrent and metastatic thyroid papillary cancer cells and has an inhibitory effect on nude mouse transplanted tumors.

[0024] Thyroid cancer is a malignant tumor, especially recurrent and metastatic tumors, which have a poor prognosis and low survival rate. Thyroid hormone homeostasis imbalance is an important triggering factor of thyroid cancer, but there are no drugs targeting hormone metabolic enzymes. This study discovered a highly effective and low-toxic active substance that selectively kills thyroid cancer tumor cells and regulates thyroid hormone homeostasis, providing conditions for the development of drugs with few side effects and high specificity for the treatment of recurrent and metastatic papillary thyroid cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the elution curve of the total sugar from Flos Lonicerae separated and purified by DEAE cellulose column chromatography in Example 1 (490 nm: total sugar content; 525 nm: uronic acid content).

[0026] Figure 2This is the elution curve of the honeysuckle polysaccharide WLJP-025 of Example 1 on a Sepharose CL-6B chromatography column (490 nm: total sugar content; 525 nm: uronic acid content; Vo: external water volume; Vt: total volume).

[0027] Figure 3 This is the step of extracting and separating honeysuckle polysaccharides in Example 1.

[0028] Figure 4 This is the test result of honeysuckle polysaccharide WLJP-025p in Example 2 on TSKgel G4000PWxl molecular sieve.

[0029] Figure 5 The monosaccharide composition detection results of the honeysuckle polysaccharide WLJP-025p of Example 2 (Man: mannose; Rha: rhamnose; GlcA: glucuronic acid; GalA: galacturonic acid; Glc: glucose; Gal: galactose; Xyl,: xylose; Ara: arabinose; Fuc: fucose).

[0030] Figure 6 This is the NMR detection spectrum of the honeysuckle polysaccharide WLJP-025p of Example 2.

[0031] Figure 7 These are the experimental results of the effect of honeysuckle polysaccharide (WLJP) of Example 3 on the cell viability of normal thyroid cells (Nthy-ori3-1) and metastatic thyroid papillary carcinoma cells (IHH-4) (*P<0.05, **P<0.01, ***P<0.001 vs. medium group).

[0032] Figure 8 These are the experimental results of the effect of honeysuckle polysaccharide (WLJP-05) of Example 3 on the cell viability of normal thyroid cells (Nthy-ori3-1) and metastatic thyroid papillary carcinoma cells (IHH-4) (*P<0.05, **P<0.01, ***P<0.001 vs. medium group).

[0033] Fig. 9 These are the experimental results of the effect of honeysuckle polysaccharide (WLJP-N) of Example 3 on the cell viability of normal thyroid cells (Nthy-ori3-1) and metastatic thyroid papillary carcinoma cells (IHH-4) (*P<0.05, **P<0.01, ***P<0.001 vs. medium group).

[0034] Fig.10These are the experimental results of the effect of honeysuckle polysaccharide (WLJP-025p) of Example 3 on the cell viability of normal thyroid cells (Nthy-ori3-1) and metastatic thyroid papillary carcinoma cells (IHH-4) (*P<0.05, **P<0.01, ***P<0.001 vs. medium group).

[0035] Fig.11 The results of the scratch test in Example 4 were used to detect the effect of WLJP-025p on the proliferation and migration ability of metastatic thyroid papillary carcinoma cells (IHH-4) (**P<0.01, ***P<0.001 vs. medium group).

[0036] Fig.12 The colony assay in Example 4 is used to detect the effect of WLJP-025p on the proliferation ability of a single metastatic thyroid papillary carcinoma cell (IHH-4).

[0037] Fig.13 The results of the EdU experiment in Example 4 were used to detect the effect of WLJP-025p on the proliferation ability of metastatic thyroid papillary carcinoma cells (IHH-4).

[0038] Fig.14 This is the TUNEL staining of Example 5 to detect the effect of WLJP-025p on the apoptosis of metastatic thyroid papillary carcinoma cells (IHH-4).

[0039] Fig.15 This is the flow cytometry test of Example 5 to detect the apoptosis of metastatic thyroid papillary carcinoma cells (IHH-4) after the action of WLJP-025p (***P<0.001 vs. medium group).

[0040] Fig.16 The figure shows the changes in the cell cycle of metastatic thyroid papillary carcinoma cells (IHH-4) after the action of WLJP-025p in Example 6 (**P<0.01, ***P<0.001 vs. medium group).

[0041] Fig.17 The figure shows the effect of WLJP-025p in Example 7 on the tumor size and body weight of tumor-bearing mice (***P<0.001 vs. control group). DETAILED DESCRIPTION

[0042] The following is further described in detail through specific implementation methods:

[0043] Example 1: Preparation of Lonicera japonica polysaccharides (LJP)

[0044] The preparation method of honeysuckle polysaccharide (abbreviated as: WLJP-025p) used in this scheme can be found in the doctoral dissertation "Separation and purification of honeysuckle polysaccharide and its protective effect on allergic rhinitis / dermatitis" (Bai Xinyu, Yanbian University). This scheme mainly explores the new uses of honeysuckle polysaccharide to fully explore the application direction of honeysuckle polysaccharide and increase new treatment methods for thyroid cancer. Specifically, the preparation method of honeysuckle polysaccharide is as follows:

[0045] (1) Preparation of total sugar from honeysuckle

[0046] Add ethanol to dried honeysuckle (dried flower buds or flowers with initial opening of Lonicera japonica Thunb.; Lonicerae japonicae flos), reflux extract for 4 hours to remove fat-soluble substances; then add 10L of distilled water to 500g of the residue, boil and extract twice at 100°C (4 hours / time), combine the supernatants of the two boilings and concentrate to about 700mL, centrifuge for 15min (4500rpm / min, 15min), collect the supernatant, and treat with 15% trichloroacetic acid at 4°C for 4 hours to remove protein. After neutralization, add 3 times the volume of anhydrous ethanol to precipitate polysaccharides. The next day, centrifuge the alcohol precipitate (4500rpm / min, 10min), collect the precipitate and freeze-dry it in vacuum to obtain light brown honeysuckle total sugar powder (WLJP, yield of about 5.1%, w / w). Extracting total honeysuckle polysaccharides by water extraction and alcohol precipitation is a conventional form in the prior art. By appropriately adjusting the process parameters according to the above process, light brown total honeysuckle sugar powder can also be prepared.

[0047] The phenol-sulfuric acid method is used to detect the sugar content of total sugar: concentrated sulfuric acid can hydrolyze and dehydrate polysaccharides to generate substances such as furfural or hydroxymethylfurfural. These substances can react with phenol to generate brown products. The product has an obvious absorption peak at 490nm, and the absorbance change at this wavelength is proportional to the sugar content within a certain range.

[0048] Standard curve: Take 1 mL of each mixed standard solution of glucose and galacturonic acid of different concentrations (0, 10, 20, 30, 40, 50, 60 μg / mL) in a test tube, add 0.5 mL of 6% phenol solution (w / w) and 2.5 mL of concentrated sulfuric acid to each test tube, shake and mix immediately, boil in a boiling water bath for 10 min, and after cooling, measure the absorbance of each test tube at 490 nm. Draw a standard curve with the standard concentration C (μg / mL) as the horizontal axis and the absorbance at 490 nm as the vertical axis.

[0049] Detection of total sugar content: Take 1 mL of 50 μg / mL honeysuckle total sugar solution, 0.5 mL of 6% phenol solution (w / w) and 2.5 mL of concentrated sulfuric acid, and perform the other operations as above. Measure the absorbance of the total sugar solution, substitute the absorbance into the standard curve equation to calculate the concentration, and divide this concentration by the sample concentration to obtain the total sugar content.

[0050] After the dried honeysuckle is boiled in hot water, concentrated, protein removed, ethanol precipitated, and freeze-dried in vacuum, the honeysuckle water-extracted total sugar WLJP is obtained, with a yield of about 5.1% (w / w). The purity of WLJP is about 93.7% when analyzed by phenol-sulfuric acid method.

[0051] (2) Separation and purification of total sugars from honeysuckle

[0052] Purification of Total Sugar from Honeysuckle by DEAE-cellulose

[0053] First, pretreat the DEAE-cellulose (Shanghai Hengxin Chemical Reagent Co., Ltd., CAS: 9013-34-7) column filler. First, soak the DEAE-cellulose in distilled water (3 to 5 days); after wringing out the water, soak the cellulose in 0.5 mol / L NaOH for 1 hour, and wash it with distilled water until it is neutral; finally, soak the cellulose in 0.5 mol / L hydrochloric acid for 1 hour, wash it with distilled water until it is neutral, and set aside. The above-mentioned DEAE-cellulose treatment processes are all conventional methods in the prior art. After completing the above-mentioned pretreatment, DEAE-cellulose is loaded into the column. After the treated DEAE-cellulose is debubbled, it is slowly loaded into a chromatography column (50×7.5 cm, Cl-type), and washed with 2 times the column volume of distilled water, 2M NaCl solution, and distilled water, respectively, and set aside.

[0054] After completing the above preparations, DEAE-cellulose is used to purify the total sugar of honeysuckle. Weigh the total sugar of honeysuckle and dissolve it in distilled water (about 40mg / mL). After centrifugation (4500rpm / min, 15min), all the supernatants are loaded on the treated DEAE-cellulose chromatography column, and gradient elution is performed with 2 column volumes of distilled water, 0.25mol / L NaCl solution and 0.5mol / L NaCl solution, respectively, at a flow rate of 10mL / min. In a specific embodiment, when collecting the eluate of the gradient elution, it is collected in order of elution time and about 130mL is used as one tube, and the sugar content and uronic acid content in each eluate are detected by phenol-sulfuric acid method and m-hydroxybiphenyl method, respectively. Among them, the parts corresponding to the total sugar content peak and uronic acid peak of the elution curve are taken, and the components in tubes 36-55 are used to prepare WLJP-025. In actual operation, the collected components for preparing WLJP-025 are not strictly limited to tubes 36-55. It is only necessary to collect the eluent in the collection tube corresponding to the total sugar content peak and the uronic acid peak according to the elution curve. The components corresponding to the total sugar content peak and the uronic acid peak of the elution curve obtained by eluting with 0.25 mol / L NaCl solution are sequentially subjected to conventional concentration, dialysis and vacuum freeze-drying steps to obtain an acidic sugar component WLJP-025.

[0055] like Figure 1 As shown, the total sugar WLJP of honeysuckle was loaded on DEAE-cellulose column chromatography. From the elution curve, it can be seen that a major polysaccharide component WLJP-025 was obtained by elution with 0.25 mol / L sodium chloride solution. The distribution peaks of sugar content and uronic acid content in its elution curve were consistent, indicating that the component had good charge uniformity.

[0056] Further purification of WLJP-025 by Sepharose CL-6B

[0057] 40 mg of WLJP-025 component was weighed and dissolved in 1 mL of 0.15 mol / L NaCl. After centrifugation (12000 rpm / min, 3 min), the supernatant was loaded on a Sepharose CL-6B molecular sieve column (GE Healthcare, CAS: 62610-50-8, 60 cm×1.0 cm), and eluted with 0.1 mol / L NaCl solution at a flow rate of 0.1 mL / min. One portion (tube) was collected approximately every 12 min, and the sugar content and uronic acid content in each eluate were detected. The elution curve was plotted with the number of collected portions as the abscissa and the absorbance of each portion as the ordinate. The eluate portion corresponding to the maximum total sugar content peak and uronic acid peak of the elution curve was taken for the preparation of WLJP-025p, wherein the components in tubes 10-35 were used to prepare WLJP-025p. In actual operation, the collected components for preparing WLJP-025p are not strictly limited to tubes 10-35. It is only necessary to collect the eluate in the collection tubes corresponding to the total sugar content peak and the uronic acid peak according to the elution curve. After the obtained components are sequentially subjected to conventional concentration, dialysis and vacuum freeze-drying steps, a component WLJP-025p with good uniformity is obtained, and the yield is about 1.9% (w / w).

[0058] After the honeysuckle polysaccharide component WLJP-025 was separated and purified by Sepharose CL-6B column chromatography, the elution curve was as follows: Figure 2 As shown in the figure, WLJP-025 showed two elution peaks, one large and one small, in Sepharose CL-6B column chromatography, and the total sugar distribution was basically consistent with the uronic acid distribution. As shown in the figure, we collected the relatively rich polysaccharide portion WLJP-025p.

[0059] In addition, the overall process of separation and purification of total sugars from honeysuckle can be seen Figure 3 From the total sugar WLJP of honeysuckle, WLJP-N (fraction collected by elution with distilled water), WLJP-025 (fraction collected by elution with 0.25 mol / L NaCl solution), and WLJP-05 (fraction collected by elution with 0.5 mol / L NaCl solution) can be obtained by DEAE-cellulose chromatography. WLJP-025 is then chromatographed by Sepharose CL-6B molecular sieve to obtain WLJP-025p.

[0060] Example 2: Characterization of polysaccharide WLJP-025p

[0061] (1) Homogeneity and molecular weight detection of WLJP-025p

[0062] A Shimadzu HPLC system (Shimadzu, Japan) and a Schambeck RI2000A differential refractometer (Schambeck SFD GmbH, Germany) were used, connected in series with a TSKgel G4000 PWxl gel exclusion chromatography column (7.8×300mm, TOSOH, Japan), with a detection temperature of 35°C and a flow rate of 0.5mL / min. 20μL of 5mg / mL WLJP-025p solution was taken for detection. The standard products were a series of dextran with known molecular weights (2000kDa, 800kDa, 500kDa, 100kDa, 10kDa). The logarithm of the molecular weight of each standard product was used as the ordinate and the corresponding retention time was used as the abscissa to draw a standard curve for molecular weight calculation. The molecular weight can be calculated based on the regression equation of the standard curve and the retention time of WLJP-025p.

[0063] The molecular weight and homogeneity of WLJP-025p were analyzed by HPLC. Figure 4 As shown, WLJP-025p mainly presents one elution peak, indicating that its uniformity is good, which is consistent with the results of Sepharose CL-6B chromatography column. According to the molecular weight standard curve and the elution time of WLJP-025p, the relative average molecular weight of WLJP-025p is calculated to be 23±4kDa. If a different measurement method is used (for example, changing the chromatographic column, TSKgel G3000 is also one of the commonly used chromatographic columns for molecular weight detection), the measured molecular weight values ​​will show certain differences, but are generally within the range of 10-40kDa.

[0064] (2) Detection of monosaccharide composition of WLJP-025p

[0065] Hydrolysis of polysaccharides

[0066] Weigh 2 mg of honeysuckle polysaccharide WLJP-025p, put it into an acid-resistant and high-temperature resistant glass vial, add 1 mL of 2 mol / L hydrochloric acid methanol, seal it and react at 80°C for 16 hours; after blowing dry, add 1 mL of 2 mol / L trifluoroacetic acid solution, seal it and react at 120°C for 1 hour, remove the trifluoroacetic acid and evaporate it to dryness.

[0067] Derivatization of hydrolysates

[0068] First, add 0.5mL of 0.5mol / L PMP and 0.5mL of 0.3mol / L NaOH solution to the hydrolyzate evaporated in the "polysaccharide hydrolysis" experiment, mix thoroughly and react at 70°C for 30min. Then, add 0.3mol / L HCl solution to the reaction solution for neutralization; finally, extract and remove free PMP with chloroform, filter with 0.22μm filter membrane, and wait for HPLC monitoring.

[0069] HPLC on-machine monitoring

[0070] A Waters e2695 high performance liquid chromatography system and a Waters 2489 UV detector (detection wavelength of 245 nm) were used, and the brand and model of the chromatographic column was Dikma Platisil ODS (4.6×250 mm, 5 μm). The mobile phase was acetonitrile: PBS (0.1 mol / L, pH 7.0) = 18:81 (V / V), the flow rate was 1.0 mL / min, the injection volume was 20 μL, and the column temperature was 35°C.

[0071] Test results

[0072] The monosaccharide composition of honeysuckle polysaccharide component WLJP-025p is as follows Figure 5 As shown in Table 1, WLJP-025p is mainly composed of GalA, Ara, Glc, Gal and Man. After multiple tests, the content of several polysaccharides ranges from: GalA (galacturonic acid): 53.4% ​​to 80.2% (66.8±13.4%); Ara (arabinose): 6.6% to 9.8% (8.2±1.6%); Gal (galactose): 11.7% to 17.5% (14.6±2.9%); Glc (glucose): 8%-12% (10±2%); Man (mannose): 0.3% to 0.5% (0.4±0.2%). Since WLJP-025p contains more GalA residues, it is speculated that WLJP-025p is a pectin polysaccharide. In addition, WLJP-025p contains some neutral monosaccharide residues, such as Gal, Ara and Glc residues, and the content of Gal residues is higher than that of Ara residues, indicating that WLJP-025p may contain sugar structural units of galactan, arabinan, glucan and / or arabinogalactan. Therefore, WLJP-025p may be a pectin polysaccharide rich in HG domains, containing neutral sugar side chains of different structural types.

[0073] Table 1: Monosaccharide composition of WLJP-025p

[0074]

[0075] (3) Nuclear magnetic resonance detection of WLJP-025p

[0076] WLJP-025p (20 mg) was dissolved in D2O (1 mL, 99.8%) and stirred at room temperature overnight. One-dimensional NMR spectra were obtained on a Bruker AV600 spectrometer ( 1 H NMR, 13 C NMR) and two-dimensional NMR spectroscopy (HSQC, HMBC).

[0077] The fine structure of WLJP-025p was analyzed by one-dimensional and two-dimensional NMR spectroscopy. Figure 6 As shown, the chemical shifts of each peak are listed in Table 2. The NMR spectrum of the HG-type domain-rich pectin polysaccharide WLJP-025p shows that the GalA residues in WLJP-025p are partially methylated, with chemical shifts at 99.75 / 5.01ppm, 70.81 / 3.97ppm, 71.03 / 4.15ppm, 79.27 / 4.47ppm, 73.38 / 3.74ppm and 173.88 / -ppm, corresponding to the C1 / H1 to C6 / H6 groups in the methylated GalA. The C-2 or C-3 of the GalA residue is easily partially acetylated in natural pectin polysaccharides. In the WLJP-025p NMR results, the chemical shift of -CH3COO in the O-2 or O-3 of the GalA residue is 22.95ppm, indicating that WLJP-025p is also partially acetylated. In addition, for Gal monosaccharide residues, the chemical shifts at 103.30 / 4.61ppm and 102.32 / 4.65ppm represent the (1→4)-linked and terminal β-D-Galp residues, respectively; for Ara monosaccharide residues, the chemical shifts at 107.46 / 5.11ppm, 110.03 / 5.09ppm and 110.13 / 5.16ppm represent the (1→3,5)-linked, (1→5)-linked, and terminal α-Araf residues; for Glc monosaccharide residues, the chemical shifts at 99.04 / 5.10ppm and 99.37 / 4.86ppm correspond to the (1→4)-linked, (1→5)-linked and terminal α-Glcp residues, respectively. In summary, WLJP-025p is mainly composed of α-(1→4)-D-GalpA connected in sequence as the main chain, and the side chains are mainly composed of β-(1→4)-galactan, α-(1→5)-linked and (1→3,5)-linked arabinan / arabinogalactan, and α-(1→4)-linked glucan.

[0078] Table 2: WLJP-025p 13 C and 1 Chemical shifts in H NMR

[0079]

[0080]

[0081] Example 3: Screening of Honeysuckle Polysaccharide

[0082] The four kinds of honeysuckle polysaccharides (LJP) prepared in Example 1 were subjected to in vitro experimental studies. The four kinds of honeysuckle polysaccharides (LJP) were: WLJP, WLJP-N, WLJP-05 and WLJP-025p. The drug targets two cell lines: normal thyroid cells (Nthy-ori3-1) and metastatic thyroid papillary carcinoma cells (IHH-4). After the cells arrived, they were cultured in a 5% CO2, 37°C incubator. When the density reached 80-90%, they were subcultured and inoculated into 96 / 24 / 12 / 6-well plates or 60mm culture dishes for experiments according to experimental requirements. IHH-4 complete culture medium: RPMI-1640+10% FBS+1% P / S; Nthy-ori-3 complete culture medium: RPMI-1640+10% FBS+1% P / S. The above culture medium is a conventional culture medium in the prior art and will not be described here. The CCK-8 method was used to evaluate the effect of the drug on cell activity. Cell Counting Kit-8 (CCK-8) cell proliferation detection reagent can indirectly reflect the number of living cells based on its correlation, and has been widely used in anti-tumor drug screening, cell proliferation assays, cytotoxicity assays, etc. 3 Cells were inoculated in 96-well plates and 250, 500, 1000, and 2000 μg / mL of LJP were added for 12, 24, and 48 h, respectively. The cell survival rate was detected by CCK-8 method. The calculation formula was as follows: cell survival rate (%) = (OD value of experimental well / OD value of control well) × 100% to detect cell proliferation activity.

[0083] The experimental results are as follows Figure 7 , Figure 8 , Fig. 9 and Fig.10As shown, the experimental results show that: although WLJP, WLJP-N and WLJP-05 can inhibit the cell viability of IHH-4, they are cytotoxic to normal thyroid cells (Nthy-ori-3); WLJP-025p not only inhibits the cell viability of IHH-4, but also has no cytotoxicity to Nthy-ori-3, so WLJP-025p was selected for subsequent research. It can be seen that the same polysaccharide extracted from honeysuckle, the polysaccharides with different compositions and structures obtained by different extraction methods, have completely different effects on thyroid (cancer) cells. Only WLJP-025p can effectively inhibit the cell viability of IHH-4 while not producing toxic effects on normal thyroid cells. It is an ideal potential drug with small side effects and strong specificity specifically for recurrent and metastatic thyroid papillary carcinoma. Compared with other types of honeysuckle polysaccharides, WLJP-025p has achieved technical effects that the researchers did not expect in the treatment of recurrent and metastatic thyroid papillary carcinoma. The honeysuckle polysaccharide WLJP-025p described in Example 2 (monosaccharide composition, molecular weight, main chain and side chain, etc.) has the efficacy of WLJP-025p described in Example 3.

[0084] Example 4: Effect of WLJP-025p on the proliferation and migration ability of IHH-4 cells

[0085] The scratches were used to simulate cell damage, and the initial damage layer was recorded. The effect of WLJP-025p on the proliferation and migration ability of IHH-4 cells was evaluated according to the recovery speed and degree of the scratches. The results are as follows: Fig.11 As shown, WLJP-025p slowed down the recovery of scratches and inhibited the proliferation and migration of IHH-4 cells.

[0086] The effect of WLJP-025p on the proliferation of IHH-4 cells was detected by colony assay and EdU staining. The proliferation capacity of a single cell can be determined by the number of colonies formed. The results are shown in Fig.12 As shown in Figure 2, the number of colonies significantly decreased after WLJP-025p was applied. In addition, the EdU experiment detected the cell proliferation rate by the number of labeled cells. The more cells were labeled with green fluorescence, the faster the proliferation. The results are shown in Figure 2. Fig.13 As shown in the figure, after the action of WLJP-025p, the green fluorescence was significantly reduced, and the number of living cells was also significantly reduced. Therefore, it can be seen that WLJP-025p inhibits the proliferation of IHH-4 cells in a dose-dependent manner.

[0087] Example 5: Effect of WLJP-025p on IHH-4 apoptosis

[0088] TUNEL assay for apoptosis

[0089] TUNEL stain can mark the TdT enzyme produced by apoptotic cells. The degree of apoptosis can be determined based on the amount and strength of red fluorescence. Fig.14 As shown, WLJP-025p promoted the apoptosis of IHH-4 cells after 24 h of treatment.

[0090] Flow cytometry to detect apoptosis

[0091] like Fig.15 As shown, WLJP-025p increased the apoptosis rate of IHH-4 cells after 24 hours of treatment, affecting the normal growth and reproduction of cells.

[0092] Example 6: Effect of WLJP-025p on IHH-4 cell cycle

[0093] Based on the above experimental results, the conditions of WLJP-025p 500 and 1000 μg / mL for 24 h were selected for subsequent experiments. Fig.16 As shown, after the action of WLJP-025p, the number of cells in S phase decreased, the number of cells in G2 / M phase increased, the cell cycle arrest was enhanced, and cell proliferation and differentiation were inhibited.

[0094] Example 7: Effect of WLJP-025p on tumor growth in nude mouse transplant tumor model

[0095] First, a nude mouse transplant tumor model was constructed: cells in the logarithmic growth phase were taken and prepared to a cell concentration of 1×10 7 / mL of cell suspension, 200μL / mouse of the cell suspension was subcutaneously injected into the right armpit of nude mice, and the growth of the transplanted tumor was observed daily. Nude mice with uniform tumors were randomly divided into a tumor group and an LJP group, with 4 mice in each group. The mice were gavaged according to their body weight, 0.1mL LJP (100mg / kg) was given for every 10g, and the tumor group was given an equal amount of normal saline. Eyeball samples (serum, tumor) were collected 10 days after administration. The changes in mouse weight and the length (L) and width (W) of the tumor were recorded during the administration period; the tumor volume calculation formula: tumor volume = 0.52×L×W 2 .

[0096] In order to further verify the efficacy of WLJP-025p, this study used a nude mouse transplant tumor model to study the effect of WLJP-025p on tumor growth in vivo. Nude mice with relatively uniform tumor size were selected and randomly divided into a control group and a WLJP-025p group, with 4 mice in each group. The mice were given 0.1 ml of WLJP-025p (100 mg / kg) per 10 g by gavage, and the control group was given an equal amount of normal saline by gavage. The results are shown in the figure. Fig.17 As shown, WLJP-025p can inhibit the growth of xenograft tumors in nude mice and has no significant effect on the body weight of nude mice.

[0097] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Application of honeysuckle polysaccharide in the preparation of drugs for the treatment of thyroid cancer.

2. The use of the honeysuckle polysaccharide according to claim 1 in the preparation of a medicament for treating thyroid cancer, characterized in that: The thyroid cancer is recurrent metastatic papillary thyroid cancer.

3. The use of the honeysuckle polysaccharide according to claim 1 in the preparation of a medicament for treating thyroid cancer, characterized in that: The monosaccharide composition of the honeysuckle polysaccharide is: 53.4% ​​to 80.2% of galacturonic acid, 8% to 12% of glucose, 11.7% to 17.5% of galactose, 6.6% to 9.8% of arabinose, and 0.3% to 0.5% of mannose.

4. The use of the honeysuckle polysaccharide according to claim 3 in the preparation of a medicament for treating thyroid cancer, characterized in that: The honeysuckle polysaccharide is connected in sequence by α-(1→4)-D-GalpA to form a main chain.

5. The use of honeysuckle polysaccharide according to claim 4 in the preparation of a medicament for treating thyroid cancer, characterized in that: The side chains of the honeysuckle polysaccharide include β-(1→4)-linked galactan, α-(1→5)-linked and (1→3,5)-linked arabinan / arabinogalactan, and α-(1→4)-linked glucan.

6. Use of the honeysuckle polysaccharide according to claim 5 in the preparation of a medicament for treating thyroid cancer, characterized in that: The relative average molecular weight of honeysuckle polysaccharide is 10-40 kDa; Preferably, the relative average molecular weight of the obtained honeysuckle polysaccharide is determined to be 23±4 kDa using high performance liquid chromatography based on a TSKgel G4000 PWxl gel exclusion chromatography column.

7. The use of the honeysuckle polysaccharide according to claim 1 in the preparation of a medicament for treating thyroid cancer, characterized in that: The honeysuckle polysaccharide is prepared by the following method: S1: After removing fat-soluble substances from honeysuckle, water is used as a solvent for decoction and extraction, and the supernatant is taken; after concentration, protein removal, and alcohol precipitation, the precipitate is collected; and after freeze-drying, the total sugar of honeysuckle is obtained; S2: Load the aqueous solution of total sugars from honeysuckle onto a DEAE-cellulose chromatography column, and use distilled water and 0.25 mol / L NaCl solution to elute the DEAE-cellulose chromatography column in sequence, and take the portion corresponding to the total sugar content peak and the uronic acid peak of the elution curve from the components eluted with the 0.25 mol / L NaCl solution; and then concentrate, dialyze and freeze-dry to obtain the honeysuckle polysaccharide WLJP-025 component; S3: The honeysuckle polysaccharide WLJP-025 component was dissolved in sodium chloride solution, loaded on a Sepharose CL-6B chromatography column, eluted with a 0.1 mol / L NaCl solution, and the eluate corresponding to the maximum total sugar content peak and uronic acid peak of the elution curve was taken; after concentration, dialyzation and freeze-drying, the honeysuckle polysaccharide WLJP-025p was obtained.

8. The use of honeysuckle polysaccharide according to claim 7 in the preparation of a medicament for treating thyroid cancer, characterized in that: In S2, the DEAE-cellulose chromatography column was eluted with 2 column volumes of distilled water and 0.25 mol / L NaCl solution, and the elution flow rate was 10 mL / min.

9. The use of the honeysuckle polysaccharide according to claim 7 in the preparation of a medicament for treating thyroid cancer, characterized in that: In S3, the elution was performed using a 0.1 mol / L NaCl solution at a flow rate of 0.1 mL / min.

10. The use of the honeysuckle polysaccharide according to claim 2 in the preparation of a medicament for treating thyroid cancer, characterized in that: The in vitro action concentration of the honeysuckle polysaccharide on IHH-4 cells is 500-2000 μg / mL; in the nude mouse transplant tumor model, the dosage of the honeysuckle polysaccharide is 100 mg / kg.

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