Application of honeysuckle polysaccharide in preparing medicine for treating thyroid cancer

Abstract

The present invention relates to the technical field of thyroid cancer drugs, and specifically relates to the application of honeysuckle polysaccharide in the preparation of drugs for treating thyroid cancer. Based on the isolation and purification of honeysuckle polysaccharide with good homogeneity, it is found that it has no toxic effect on normal thyroid follicular cells, but selectively inhibits the proliferation of recurrent and metastatic papillary thyroid cancer cells, and has an inhibitory effect on the xenograft tumors of nude mice inoculated with recurrent and metastatic papillary thyroid cancer cell lines. The honeysuckle polysaccharide of this scheme has good homogeneity, a molecular weight of 23 ± 4 kDa, and is mainly composed of five monosaccharides, mainly connected by α-(1→4)-D-GalpA to form the main chain. While the polysaccharide effectively inhibits the cell viability of IHH-4, it has no toxic effect on normal thyroid cells. This technical scheme can solve the technical problem in the prior art of lacking drugs with small side effects and high specificity for treating recurrent and metastatic papillary thyroid cancer, and has an ideal application prospect.

Description

Technical Field

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

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

[0003] Recurrent and metastatic papillary thyroid carcinoma refers to the recurrence of papillary thyroid carcinoma that has been previously 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 foci (i.e., distant metastasis). This usually occurs after primary surgical resection and / or radioactive iodine treatment. Early screening for the risk of recurrence and metastasis is very important for reducing the mortality rate of patients, but there is currently no recognized "gold standard" for evaluating the risk of recurrence and metastasis of papillary thyroid carcinoma. Clinically, the risk of recurrence or metastasis of papillary thyroid carcinoma is mainly evaluated based on tumor size, lymph node metastasis characteristics, degree of vascular invasion, etc., but the effect on reducing the mortality rate is minimal. Another intractable problem is that the treatment methods for recurrent and metastatic papillary thyroid carcinoma are limited. Currently, radioactive iodine is the main treatment method for recurrent and metastatic papillary thyroid carcinoma, but about half of the patients are insensitive to the treatment. The prognosis of these patients is worse, and the 10-year survival rate is only 10%. When the cancer is insensitive to radioactive iodine or not suitable for its use, targeted drugs (such as sorafenib) are also tried for the treatment of recurrent and metastatic papillary thyroid carcinoma. In some cases, although targeted therapy can prolong the survival period of patients, since these drugs can affect normal cell processes, they may cause a series of side effects, and over time, the tumor may develop drug resistance to targeted therapy, which may lead to the failure of treatment. There is an urgent need to further study specific drugs for recurrent and metastatic papillary thyroid carcinoma in order to obtain drugs with small side effects and high specificity for treating recurrent and metastatic papillary thyroid carcinoma to meet the needs of clinical applications. Summary of the Invention

[0004] The object of the present invention is to provide the use of honeysuckle polysaccharide in the preparation of a medicament for treating thyroid cancer, so as to solve the technical problem in the prior art that there is a lack of a medicament with small side effects and high specificity for treating recurrent and metastatic papillary thyroid cancer. The present solution provides an effective therapeutic drug lacking for inhibiting recurrent and metastatic thyroid cancer, and is a natural active substance with high efficiency and low toxicity that selectively kills thyroid cancer tumor cells.

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

[0006] The use of honeysuckle polysaccharide in the preparation of a medicament for treating thyroid cancer.

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

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

[0009] Further, the honeysuckle polysaccharide is formed by sequentially connecting α-(1→4)-D-GalpA as the main chain.

[0010] Further, 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] Further, the relative average molecular weight of the honeysuckle polysaccharide is 10 - 40 kDa;

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

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

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

[0015] S2: Load the aqueous solution of total honeysuckle sugar onto a DEAE-cellulose chromatography column, and elute the DEAE-cellulose chromatography column successively with distilled water and 0.25 mol / L NaCl solution. Take the parts corresponding to the total sugar content peak and the uronic acid peak of the elution curve from the components eluted with 0.25 mol / L NaCl solution; then, after concentration, dialysis, and lyophilization, obtain the honeysuckle polysaccharide WLJP-025 fraction.

[0016] S3: Dissolve the honeysuckle polysaccharide WLJP-025 fraction in sodium chloride solution, load it onto a Sepharose CL-6B chromatography column, and elute it with 0.1 mol / L NaCl solution. Take the eluate part corresponding to the maximum total sugar content peak and the uronic acid peak of the elution curve; then, after concentration, dialysis, and lyophilization, obtain the honeysuckle polysaccharide WLJP-025p.

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

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

[0019] Furthermore, the in vitro action concentration of the honeysuckle polysaccharide against IHH-4 cells is 500 - 2000 μg / mL; in the nude mouse xenograft tumor model, the administration dose of the honeysuckle polysaccharide is 100 mg / kg.

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

[0021] This technical solution provides that Lonicera japonica polysaccharides (LJP) have a significant inhibitory effect on the recurrent and metastatic thyroid cancer cell line IHH4, and have no toxic effect on the normal thyroid cell line Nthy-ori3-1. It is a rare traditional Chinese medicine active substance with the characteristics of high efficiency and low toxicity discovered so far. 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. The IHH4 cells have a weak ability to uptake radioactive iodine (RAI), which reflects the situation of resistance to radioactive iodine treatment in some recurrent and metastatic papillary thyroid carcinomas clinically. This cell line is often used in the laboratory environment to simulate the behavior of recurrent and metastatic papillary thyroid carcinoma, including processes such as tumor growth, invasion and metastasis. The IHH4 cell line can be used to test the effects of new anti-cancer drugs or targeted therapies, especially when searching for new therapies for radioiodine-resistant thyroid cancer.

[0022] After in vitro experiments, continued model animal experiments found that Lonicera japonica polysaccharides have a significant inhibitory effect on thyroid papillary carcinoma-bearing animals (recurrent and metastatic cell line IHH4), suggesting that Lonicera japonica polysaccharides may be an effective therapeutic drug. In-depth study of Lonicera japonica polysaccharides against recurrent and metastatic papillary thyroid carcinoma is of great significance.

[0023] To sum up, based on the isolation and purification of relatively homogeneous Lonicera japonica polysaccharides, this technical solution deeply studies and finds that it has no toxic effect on normal thyroid follicular cells, but selectively inhibits the proliferation of recurrent and metastatic papillary thyroid cancer cells and has an inhibitory effect on xenograft tumors in nude mice.

[0024] Thyroid cancer is a malignant tumor, especially for patients with recurrent and metastatic tumors, the prognosis is poor and the survival rate is low. The imbalance of thyroid hormone homeostasis is an important trigger factor for thyroid cancer, but there is no drug for hormone metabolism enzyme therapy yet. The research of this solution has discovered an active substance with high efficiency and low toxicity that selectively kills thyroid cancer tumor cells and regulates thyroid hormone homeostasis, providing conditions for developing drugs with less side effects and high specificity for the treatment of recurrent and metastatic papillary thyroid carcinoma. Description of the Drawings

[0025] Figure 1 Elution curve of the total sugar of Lonicera japonica purified by DEAE cellulose column chromatography in Example 1 (490 nm: total sugar content; 525 nm: uronic acid content).

[0026] Figure 2Elution curve of honeysuckle polysaccharide WLJP-025 from 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 Extraction and separation steps of honeysuckle polysaccharide from Example 1.

[0028] Figure 4 Detection results of honeysuckle polysaccharide WLJP-025p from Example 2 on a TSKgel G4000PWxl molecular sieve.

[0029] Figure 5 Monosaccharide composition detection results of honeysuckle polysaccharide WLJP-025p from 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 NMR detection spectrum of honeysuckle polysaccharide WLJP-025p from Example 2.

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

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

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

[0034] Figure 10Experimental results of the effect of Lonicera japonica polysaccharide (WLJP-025p) in Example 3 on the cell viability of normal thyroid cells (Nthy-ori3-1) and metastatic papillary thyroid cancer cells (IHH-4) (*P<0.05, **P<0.01, ***P<0.001 vs. medium group).

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

[0036] Figure 12 Experimental results of the colony formation assay in Example 4 to detect the effect of WLJP-025p on the proliferation ability of single metastatic papillary thyroid cancer cells (IHH-4).

[0037] Figure 13 Experimental results of the EdU assay in Example 4 to detect the effect of WLJP-025p on the proliferation ability of metastatic papillary thyroid cancer cells (IHH-4).

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

[0039] Figure 15 Flow cytometry in Example 5 to detect the apoptosis of metastatic papillary thyroid cancer cells (IHH-4) after treatment with WLJP-025p (***P<0.001 vs. medium group).

[0040] Figure 16 Cell cycle changes of metastatic papillary thyroid cancer cells (IHH-4) after treatment with WLJP-025p in Example 6 (**P<0.01, ***P<0.001 vs. medium group).

[0041] Figure 17 Effect of WLJP-025p on the tumor size and body weight of tumor-bearing mice in Example 7 (***P<0.001 vs. control group). Detailed implementation manners

[0042] The following is a further detailed description through specific implementation manners:

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

[0044] For the preparation method of the honeysuckle polysaccharide (abbreviation: WLJP-025p) used in this solution, reference can be made to the doctoral dissertation "Research on the Isolation, Purification and Protective Effect of Honeysuckle Polysaccharide on Allergic Rhinitis / Dermatitis" (Bai Xinyu, Yanbian University). This solution mainly explores and studies the new uses of this honeysuckle polysaccharide to fully explore the application directions of honeysuckle polysaccharide and increase new treatment approaches for thyroid cancer. Specifically, the preparation method of honeysuckle polysaccharide is as follows:

[0045] (1) Preparation of total honeysuckle sugar

[0046] Add ethanol to dried honeysuckle (dried flower buds or flowers with initial blooming of Lonicera japonica Thunb.; LONICERAE JAPONICAE FLOS), and reflux for 4 hours to remove fat-soluble substances; then add 10 L of distilled water to 500 g of the residue, boil and extract twice at 100 °C (4 hours each time), combine the supernatants of the two extractions and concentrate to about 700 mL, centrifuge for 15 min (4500 rpm / min, 15 min), collect the supernatant, and treat with 15% trichloroacetic acid at 4 °C for 4 hours to remove proteins. After neutralization, add 3 times the volume of absolute ethanol to precipitate the polysaccharide. The next day, centrifuge the alcohol-precipitated product (4500 rpm / min, 10 min), collect the precipitate part, and vacuum freeze-dry to obtain light brown total honeysuckle sugar powder (WLJP, yield about 5.1%, w / w). The extraction of total honeysuckle polysaccharide 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 form brown products, which have an obvious absorption peak at 490 nm. The absorbance change at this wavelength is proportional to the sugar content within a certain range.

[0048] Standard curve: Take 1 mL of mixed standard solutions of glucose and galacturonic acid with different concentrations (0, 10, 20, 30, 40, 50, 60 μg / mL) in test tubes. Add 0.5 mL of 6% phenol solution (w / w) and 2.5 mL of concentrated sulfuric acid to each test tube, immediately shake and mix well, place in a boiling water bath for 10 min, cool, and measure the absorbance of each test tube at 490 nm. Plot the standard curve with the standard concentration C (μg / mL) as the abscissa and the absorbance at 490 nm as the ordinate.

[0049] Determination of sugar content in total sugar: Take 1 mL of honeysuckle total sugar solution with a concentration of 50 μg / mL, 0.5 mL of 6% phenol solution (w / w), and 2.5 mL of concentrated sulfuric acid. Conduct the remaining 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 sugar content of the total sugar.

[0050] After steps such as hot water extraction, concentration, protein removal, ethanol precipitation, and vacuum freeze-drying of dried honeysuckle, the water-extracted total sugar WLJP of honeysuckle is obtained, and the yield is approximately 5.1% (w / w). The purity of WLJP is analyzed by the phenol-sulfuric acid method to be approximately 93.7%.

[0051] (2) Isolation and purification of honeysuckle total sugar

[0052] Purification of honeysuckle total sugar by DEAE-cellulose

[0053] First, pre-treat the column packing of DEAE-cellulose (Shanghai Hengxin Chemical Reagent Co., Ltd., CAS: 9013-34-7). Immerse the DEAE-cellulose in distilled water for 3 - 5 days; wring out the water, then soak the cellulose in 0.5 mol / L NaOH for 1 hour, and wash it with distilled water until neutral; finally, soak the cellulose in 0.5 mol / L hydrochloric acid for 1 hour, and wash it with distilled water until neutral for use. The above treatment process of DEAE-cellulose is the conventional method of the existing technology. After completing the above pre-treatment, load the DEAE-cellulose into the column. After removing the bubbles from the treated DEAE-cellulose, slowly pack it into the chromatography column (50×7.5 cm, Cl-type), and wash it with 2 column volumes of distilled water, 2 M NaCl solution, and distilled water respectively for use.

[0054] After the above preparations, DEAE-cellulose was used to purify the total sugars from honeysuckle. Weigh the total sugars from honeysuckle and dissolve them in distilled water (about 40 mg / mL). After centrifugation (4500 rpm / min, 15 min), all the supernatant was loaded onto a pre-treated DEAE-cellulose chromatography column. Gradient elution was carried out with 2 column volumes of distilled water, 0.25 mol / L NaCl solution, and 0.5 mol / L NaCl solution, respectively, at a flow rate of 10 mL / min. In a specific embodiment, when collecting the eluates of gradient elution, they were collected in the order of elution time and about every 130 mL as a tube. The phenol-sulfuric acid method and the m-hydroxybiphenyl method were used to detect the sugar content and uronic acid content in each eluate. Among them, the components corresponding to the total sugar content peak and uronic acid peak of the elution curve, the components in the 36th - 55th tubes were used to prepare WLJP-025. In actual operation, the collected components used to prepare WLJP-025 are not strictly limited to the 36th - 55th tubes. Just according to the elution curve, collect the eluates in the tubes corresponding to the total sugar content peak and uronic acid peak. The components corresponding to the total sugar content peak and uronic acid peak of the elution curve, eluted with 0.25 mol / L NaCl solution, were successively subjected to conventional concentration, dialysis, and vacuum freeze-drying steps to obtain an acidic sugar component WLJP-025.

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

[0056] Sepharose CL-6B was used to further purify WLJP-025

[0057] Weigh 40 mg of the WLJP-025 component, dissolve it in 1 mL of 0.15 mol / L NaCl, and after centrifugation (12,000 rpm / min, 3 min), load the supernatant onto a Sepharose CL-6B molecular sieve column (GE Healthcare, CAS: 62610-50-8, 60 cm × 1.0 cm), elute with 0.1 mol / L NaCl solution at a flow rate of 0.1 mL / min, collect one fraction (tube) approximately every 12 min, detect the sugar content and uronic acid content in each eluate fraction, plot an elution curve with the number of collected fractions as the abscissa and the absorbance of each fraction as the ordinate, take the eluate part corresponding to the peak of the maximum total sugar content and the uronic acid peak in the elution curve for the preparation of WLJP-025p, where the components in tubes 10 - 35 are used for the preparation of WLJP-025p. In actual operation, the collected components used for the preparation of WLJP-025p are not strictly limited to tubes 10 - 35, and only the eluate in the collection tubes corresponding to the peaks of the total sugar content and the uronic acid peak need to be collected according to the elution curve. After the obtained components are successively subjected to conventional steps such as concentration, dialysis, and vacuum freeze-drying, a component WLJP-025p with good homogeneity is obtained, and the yield is approximately 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 Figure 2 shown. Two elution peaks, one large and one small, appeared for WLJP-025 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 part WLJP-025p.

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

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

[0061] (1) Detection of the homogeneity and molecular weight of WLJP-025p

[0062] The Shimadzu HPLC system (Shimadzu, Japan) and the Schambeck RI2000A refractive index detector (Schambeck SFD GmbH, Germany) were used. A TSKgel G4000 PWxl gel permeation chromatography column (7.8×300 mm, TOSOH, Japan) was connected in series. The detection temperature was 35 °C and the flow rate was 0.5 mL / min. 20 μL of the WLJP-025p solution at a concentration of 5 mg / mL was injected for detection. The standards were a series of dextrans with known molecular weights (2000 kDa, 800 kDa, 500 kDa, 100 kDa, 10 kDa). A standard curve for calculating the molecular weight was plotted with the logarithm of the molecular weight of each standard on the ordinate and the corresponding retention time on the abscissa. The molecular weight of WLJP-025p was calculated according to 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 high performance liquid chromatography. The results are as Figure 4 shown. WLJP-025p mainly showed one elution peak, indicating good homogeneity, which was consistent with the results of the 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 was calculated to be 23±4 kDa. If different measurement methods were used (for example, changing the chromatography column, TSKgel G3000 is also one of the commonly used chromatography columns for molecular weight detection), the measured molecular weight values would show certain differences, but generally they were all in the range of 10 - 40 kDa.

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

[0065] Hydrolysis of polysaccharides

[0066] 2 mg of the honeysuckle polysaccharide WLJP-025p was weighed and placed in an acid- and heat-resistant glass vial. 1 mL of 2 mol / L hydrochloric acid in methanol was added, sealed, and reacted at 80 °C for 16 hours. After drying, 1 mL of 2 mol / L trifluoroacetic acid solution was added, sealed, and reacted at 120 °C for 1 hour, and then the trifluoroacetic acid was removed and evaporated to dryness.

[0067] Derivatization of the hydrolysis product

[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) NMR Detection of WLJP-025p

[0076] Dissolve WLJP-025p (20 mg) in D2O (1 mL, 99.8%) and stir overnight at room temperature. Obtain one-dimensional NMR spectra ( 1 1H NMR, 13 13C NMR) and two-dimensional NMR spectra (HSQC, HMBC) on a Bruker AV600 spectrometer.

[0077] Analyze the fine structure of WLJP-025p using one-dimensional and two-dimensional NMR spectra. The results are as Figure 6 shown, and the chemical shift assignments of each peak are listed in Table 2. The NMR spectra of the HG-rich domain-containing pectin polysaccharide WLJP-025p. In WLJP-025p, some of the GalA residues are methyl-esterified, and the chemical shifts are at 99.75 / 5.01 ppm, 70.81 / 3.97 ppm, 71.03 / 4.15 ppm, 79.27 / 4.47 ppm, 73.38 / 3.74 ppm, and 173.88 / -ppm, corresponding to the C1 / H1 to C6 / H6 groups in methyl-esterified GalA. In natural pectin polysaccharides, C-2 or C-3 of the GalA residue is partially acetylated. In the NMR results of WLJP-025p, the chemical shift of -CH3COO in O-2 or O-3 of the GalA residue is 22.95 ppm, indicating that there is also partial acetylation in WLJP-025p. In addition, for the Gal monosaccharide residue, the chemical shifts at 103.30 / 4.61 ppm and 102.32 / 4.65 ppm represent (1→4)-linked and terminal β-D-Galp residues, respectively; for the Ara monosaccharide residue, the chemical shifts at 107.46 / 5.11 ppm, 110.03 / 5.09 ppm, and 110.13 / 5.16 ppm represent (1→3,5)-linked, (1→5)-linked, and terminal α-Araf residues; for the Glc monosaccharide residue, the chemical shifts at 99.04 / 5.10 ppm and 99.37 / 4.86 ppm correspond to (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 to form 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: Chemical Shifts of WLJP-025p in 13 13C and 1 1H NMR

[0079]

[0080]

[0081] Example 3: Screening of honeysuckle polysaccharide

[0082] In vitro experimental studies were carried out on the four kinds of honeysuckle polysaccharides (LJP) prepared in Example 1. The four kinds of honeysuckle polysaccharides (LJP) were: WLJP, WLJP-N, WLJP-05 and WLJP-025p. The drug targets were two cell lines: normal thyroid cells (Nthy-ori3-1) and metastatic papillary thyroid cancer cells (IHH-4). After the cells arrived, they were cultured in a 5% CO2, 37 °C incubator. When the density reached 80-90%, subculture was carried out, and they were inoculated into 96 / 24 / 12 / 6-well plates or 60 mm culture dishes according to experimental requirements for experiments. IHH-4 complete medium: RPMI-1640 + 10% FBS + 1% P / S; Nthy-ori-3 complete medium: RPMI-1640 + 10% FBS + 1% P / S. The above media are conventional media in the prior art and will not be elaborated here. The CCK-8 method was used to evaluate the effect of the drug on cell viability. The Cell Counting Kit-8 (CCK-8) cell proliferation detection reagent can indirectly reflect the number of living cells according to its correlation and has been widely used in anti-tumor drug screening, cell proliferation tests, cytotoxicity tests, etc. The cells were inoculated into 96-well plates at 2×10 3 cells / well, and LJP at 250, 500, 1000, 2000 μg / mL was added respectively. After acting for 12, 24, 48 h, the CCK-8 method was used to detect the cell survival rate. 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 shown in Figure 7 , Figure 8 , Figure 9 and Figure 10As shown, it can be seen from the experimental results 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. Therefore, WLJP-025p was selected for subsequent research. Thus, it can be seen that although polysaccharide substances are all extracted from honeysuckle, 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 having toxic effects on normal thyroid cells. It is an ideal potential drug with low side effects and strong specificity for recurrent and metastatic papillary thyroid cancer. 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 papillary thyroid cancer. The honeysuckle polysaccharide WLJP-025p (monosaccharide composition, molecular weight, main chain, side chain, etc.) described in Example 2 all have 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] Cell damage was simulated by scratching, and the initial degree of damage was recorded. The effect of WLJP-025p on the proliferation and migration ability of IHH-4 cells was evaluated according to the scratch recovery speed and degree. The results are as Figure 11 shown. After the action of WLJP-025p, the scratch recovery speed slowed down, indicating an inhibitory effect on 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 formation assay and EdU staining. The proliferation ability of single cells can be judged by the number of colonies formed. The results are as Figure 12 shown. After the action of WLJP-025p, the number of colonies decreased significantly. In addition, in the EdU experiment, the cell proliferation rate was detected by the number of labeled cells. The more cells labeled with green fluorescence, the faster the proliferation. The results are as Figure 13 shown. After the action of WLJP-025p, the green fluorescence decreased significantly, and the number of live cells also decreased significantly. 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 the apoptosis of IHH-4

[0088] Detection of apoptosis by TUNEL method

[0089] The TUNEL staining solution can label the TdT enzyme produced by apoptotic cells, and the degree of apoptosis is judged according to the amount / intensity of red fluorescence. As Figure 14 shown, WLJP-025p promotes apoptosis of IHH-4 cells after 24 h of treatment.

[0090] Detection of apoptosis by flow cytometry

[0091] As Figure 15 shown, WLJP-025p increases the apoptosis rate of IHH-4 cells after 24 h of treatment, affecting the normal growth and reproduction of cells.

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

[0093] According to the above experimental results, the conditions of treating with 500 and 1000 μg / mL of WLJP-025p for 24 h were selected for subsequent experiments. As Figure 16 shown, after treatment with WLJP-025p, the number of S-phase cells decreased, and the number of G2 / M-phase cells increased, enhancing cell cycle arrest and inhibiting cell proliferation and differentiation.

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

[0095] First, a nude mouse xenograft model was constructed: Cells in the logarithmic growth phase were taken to prepare a cell suspension with a cell concentration of 1×10 7 / mL. 200 μL of the cell suspension was subcutaneously injected into the right axilla of nude mice per mouse, and the growth of the xenografts was observed daily. Nude mice with uniform tumor formation were randomly divided into a tumor group and an LJP group, with 4 mice in each group. According to the body weight of the mice, intragastric administration was given, and 0.1 mL of LJP (100 mg / kg) was given per 10 g. The tumor group was given an equal amount of normal saline. After 10 days of administration, samples (serum, tumor) were taken from the eyeballs. During the administration period, the body weight of the mice and the changes in the length (L) and width (W) of the tumor were recorded; the formula for calculating the tumor volume was: tumor volume = 0.52×L×W 2 .

[0096] To further verify the efficacy of WLJP-025p, this study investigated the effect of WLJP-025p on tumor growth in vivo through a nude mouse xenograft model. Nude mice with relatively uniform tumor sizes were randomly divided into a control group and a WLJP-025p group, with 4 mice in each group. According to the body weight of the mice, intragastric administration was given, and 0.1 mL of WLJP-025p (100 mg / kg) was given per 10 g. The control group was given an equal amount of normal saline by gavage. The results were as Figure 17 shown, WLJP-025p could inhibit the growth of nude mouse xenografts and had no significant effect on the body weight of nude mice.

[0097] The above are only embodiments of the present invention, and common knowledge such as specific structures and characteristics known in the art are not described in detail herein. It should be noted that for those skilled in the art, without departing from the structure of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope claimed in this application shall be subject to the content of its claims, and the specific implementation manners and the like recorded in the specification can be used to interpret the content of the claims.

Claims

1. The use of honeysuckle polysaccharide in the preparation of a drug for treating thyroid cancer, characterized in that: The thyroid cancer is papillary thyroid cancer; The honeysuckle polysaccharide is composed of α-(1→4)-D-Gal p A are connected in sequence to form a main chain; 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; The monosaccharide composition of the honeysuckle polysaccharide is: galacturonic acid 53.4%-80.2%, glucose 8%-12%, galactose 11.7%-17.5%, arabinose 6.6%-9.8%, mannose 0.3%-0.5%; 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. From the components eluted with 0.25 mol / L NaCl solution, take the parts corresponding to the total sugar content peak and the uronic acid peak of the elution curve; 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 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.

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 relative average molecular weight of honeysuckle polysaccharide is 10-40 kDa.

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

5. The use of the honeysuckle polysaccharide according to claim 4 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.

6. The use of the honeysuckle polysaccharide according to claim 5 in the preparation of a medicament for treating thyroid cancer, characterized in that: In S3, elution was performed using a 0.1 mol / L NaCl solution at a flow rate of 0.1 mL / min.

7. Use of the honeysuckle polysaccharide according to claim 6 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.

Images