Use of acetylated honeysuckle flower polysaccharide in preparation of a drug for treating thyroid cancer

Abstract

This invention relates to the field of thyroid cancer drug technology, specifically to the use of acetylated honeysuckle polysaccharide in the preparation of drugs for treating thyroid cancer. This method is based on chemical structural modification of naturally derived honeysuckle polysaccharide, adjusting its molecular structure by introducing acetyl groups. The resulting acetylated honeysuckle polysaccharide exhibits relatively ideal inhibitory activity against various types of thyroid cancer cells (including differentiated and undifferentiated types). Unacetylated honeysuckle polysaccharide has almost no inhibitory effect on the more aggressive and drug-resistant undifferentiated thyroid cancer cells; after acetylation, it not only enhances the inhibitory effect on differentiated thyroid cancer cells but also demonstrates effective killing ability against undifferentiated thyroid cancer cells. This technical solution can solve the technical problem of the lack of effective treatment methods for undifferentiated thyroid cancer in the prior art and has ideal prospects for widespread application.

Description

Technical Field

[0001] This invention relates to the field of thyroid cancer drug technology, specifically to the use of an acetylated honeysuckle polysaccharide in the preparation of drugs for treating thyroid cancer. Background Technology

[0002] Thyroid carcinoma (TC), the most common malignant tumor of the endocrine system, has seen a continuous rise in its incidence rate globally in recent years. According to the latest statistics, thyroid cancer ranked seventh globally in incidence in 2022, and fifth among female cancer patients, with women being three times more likely to be affected than men. Histologically, thyroid cancer is mainly classified into medullary thyroid carcinoma (MTC), undifferentiated thyroid carcinoma (ATC), and differentiated thyroid carcinoma (DTC). DTC is further subdivided into follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma (PTC), with PTC being the most common type, accounting for over 80%.

[0003] Although some patients with differentiated thyroid cancer can achieve good clinical outcomes and high 5-year survival rates with conventional treatments (such as thyroidectomy, thyroid-stimulating hormone suppression therapy, radioactive iodine therapy, and exogenous T4 supplementation), these treatment strategies are mainly applicable to differentiated thyroid cancer. Their effectiveness is extremely limited for the more malignant and aggressive undifferentiated thyroid cancer (ATC). Although undifferentiated thyroid cancer accounts for only a small fraction of all thyroid cancer cases, it causes more than 50% of thyroid cancer-related deaths, making it one of the deadliest subtypes of the disease.

[0004] Undifferentiated thyroid carcinoma (ATC) is a highly malignant tumor originating from thyroid follicular epithelial cells. It is characterized by extremely low or complete cell differentiation, rapid growth, and early local invasion and distant metastasis. Currently, treatment options for ATC are very limited. Traditional surgical resection is often difficult to achieve completely, radiotherapy and chemotherapy are ineffective, and the application of targeted therapy and immunotherapy is limited by tumor heterogeneity and drug resistance mechanisms. Furthermore, due to its rapid progression, most patients are already in an advanced stage or have distant metastases at diagnosis, further reducing treatment success rates. Therefore, there is an urgent need to explore new treatment strategies and technologies to overcome the bottlenecks in efficacy, safety, and applicable populations of existing therapies, thereby significantly improving the survival rate and quality of life for patients with ATC. Developing novel treatment methods is not only crucial for improving the prognosis of this highly malignant tumor but also brings new breakthroughs to the overall treatment system for thyroid cancer. Summary of the Invention

[0005] The purpose of this invention is to provide the use of acetylated honeysuckle polysaccharide in the preparation of a drug for treating thyroid cancer, in order to solve the technical problem of the lack of effective treatment methods for undifferentiated thyroid cancer (ATC) in the prior art.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The application of an acetylated honeysuckle polysaccharide in the preparation of a drug for treating thyroid cancer, wherein the acetylated honeysuckle polysaccharide is obtained by transferring an acetyl group to the hydroxyl group of the honeysuckle polysaccharide under the action of an acylation reagent and a catalyst.

[0008] Further, 45.5-50.5 mg: 0.9-1.1 mL: 39-41 mL: 4.9-5.1 mg of honeysuckle polysaccharide, water, acetic anhydride and 4-dimethylaminopyridine were mixed and reacted at 20-30℃ for 14-18 h to obtain acetylated honeysuckle polysaccharide.

[0009] Furthermore, the monosaccharide composition of the honeysuckle polysaccharide is as follows: galacturonic acid 53.4%-80.2%, glucose 8.0%-12.0%, galactose 11.7%-17.5%, arabinose 6.6%-9.8%, and mannose 0.3%-0.5%.

[0010] Furthermore, the main chain of the honeysuckle polysaccharide is 1,4-α-Gal p A repeating units; the main chain includes t-α-Ara f and t-α-Gal p Terminal fragment; side chain is 1,5-α-Ara f and 1,4-β-Glcp The repeating unit is connected to 1,2,4-α-Rha p The O-4 bits.

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

[0012] Preferably, 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 size exclusion 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 extraction, and the supernatant is collected; after concentration, removal of protein, and alcohol precipitation, the precipitate is collected; and after freeze-drying, the total sugar of honeysuckle is obtained.

[0015] S2: The aqueous solution of total sugars from honeysuckle was loaded into a DEAE-cellulose chromatography column. The DEAE-cellulose chromatography column was eluted sequentially with distilled water and NaCl solution. The fractions corresponding to the total sugar content peak and the uronic acid peak in the elution curve were taken from the fraction obtained by elution with NaCl solution. After concentration, dialysis and lyophilization, the polysaccharide fraction (WLJP-025) was obtained.

[0016] S3: Dissolve the polysaccharide fraction in sodium chloride solution, load it into a Sepharose CL-6B chromatography column, elute with NaCl solution, and take the eluent portion corresponding to the maximum total sugar content peak and the uronic acid peak of the elution curve; after concentration, dialysis and lyophilization, honeysuckle polysaccharide (WLJP-025p) is obtained.

[0017] Furthermore, in step S2, the concentration of the NaCl solution is 0.23-0.27 mol / L; preferably, the concentration of the NaCl solution is 0.25 mol / L.

[0018] The DEAE-cellulose chromatography column was eluted with 2-3 column volumes of distilled water and NaCl solution at a flow rate of 9.5-10.5 mL / min; preferably, the distilled water volume was 2 column volumes and the elution flow rate was 10 mL / min.

[0019] In step S3, the concentration of the NaCl solution is 0.09-0.11 mol / L; preferably, the concentration of the NaCl solution is 0.1 mol / L.

[0020] The elution rate using NaCl solution is 0.08-0.12 mL / min; preferably, the flow rate is 0.1 mL / min.

[0021] Furthermore, the thyroid cancer includes undifferentiated thyroid cancer and differentiated thyroid cancer; the differentiated thyroid cancer includes papillary thyroid carcinoma.

[0022] The acetylated honeysuckle polysaccharide is used to inhibit the proliferation and migration of differentiated thyroid cancer cells; the differentiated thyroid cancer cells have a risk of metastasis or originate from thyroid cancer patients who have already metastasized; the acetylated honeysuckle polysaccharide is used to inhibit the proliferation and migration of undifferentiated thyroid cancer cells.

[0023] This technical solution also provides a method for preparing acetylated honeysuckle polysaccharide, comprising the following steps performed sequentially:

[0024] S1: After removing fat-soluble substances from honeysuckle, water is used as a solvent for decoction extraction, and the supernatant is collected; after concentration, removal of protein, and alcohol precipitation, the precipitate is collected; and after freeze-drying, the total sugar of honeysuckle is obtained.

[0025] S2: The aqueous solution of total sugars from honeysuckle was loaded into a DEAE-cellulose chromatography column. The DEAE-cellulose chromatography column was eluted sequentially with distilled water and NaCl solution. The fractions corresponding to the total sugar content peak and the uronic acid peak of the elution curve were taken from the fractions eluted with NaCl solution. After concentration, dialysis and lyophilization, the polysaccharide fractions were obtained.

[0026] S3: Dissolve the polysaccharide fraction in sodium chloride solution, load it into a Sepharose CL-6B chromatography column, elute with NaCl solution, and take the eluent portion corresponding to the maximum total sugar content peak and the uronic acid peak of the elution curve; after concentration, dialysis and lyophilization, honeysuckle polysaccharide is obtained.

[0027] S4: The acylation reagent transfers the acetyl group to the hydroxyl group of honeysuckle polysaccharide under the action of a catalyst, and then obtains acetylated honeysuckle polysaccharide (AC025p-0) after dialysis, concentration and freeze drying.

[0028] This technical solution also provides an acetylated honeysuckle polysaccharide prepared according to the aforementioned method for preparing acetylated honeysuckle polysaccharide.

[0029] The technical principle of this technical solution is as follows:

[0030] This invention relates to the use of acetylated honeysuckle polysaccharide in the preparation of drugs for treating thyroid cancer. This technology is based on chemical structural modification of naturally derived honeysuckle polysaccharides, adjusting its molecular structure by introducing acetyl groups (–COCH3). The resulting acetylated honeysuckle polysaccharide exhibits relatively ideal inhibitory activity against various types of thyroid cancer cells (including differentiated and undifferentiated types).

[0031] Honeysuckle polysaccharides are derived from honeysuckle, a plant belonging to the Caprifoliaceae family in traditional Chinese medicine. Lonicera japonica A type of pectin-type polysaccharide extracted from *Lonicera japonica* (Thunb.) exhibits good biocompatibility and certain antitumor potential. Acetylation is achieved by covalently attaching acetyl groups to the free hydroxyl groups (-OH) of the polysaccharide molecule using an acylation reagent (such as acetic anhydride), forming an ester bond (-O-CO-CH3). This alters the polysaccharide's spatial conformation, hydrophilicity / hydrophobicity, charge distribution, and its interaction with biomolecules. In this technical approach, acetylation modification not only alters the physicochemical properties of the polysaccharide but, more importantly, significantly enhances its biological activity. Experimental studies show that unacetylated *Lonicera japonica* polysaccharide (WLJP-025p) has a certain inhibitory effect on differentiated thyroid cancer cells (such as the IHH-4 cell model), but has almost no inhibitory effect on undifferentiated thyroid cancer cells (such as the 8305C cell model), which are more invasive and drug-resistant (considered to have a higher degree of malignancy in clinical classification). The acetylated honeysuckle polysaccharide (AC025p-0) obtained after acetylation treatment not only enhanced the inhibitory effect on differentiated thyroid cancer cells, but also showed for the first time an effective killing ability against undifferentiated thyroid cancer cells.

[0032] The beneficial effects of this technical solution are as follows:

[0033] (1) Enhances anti-cancer activity:

[0034] Experimental results showed that, compared with the unacetylated honeysuckle polysaccharide WLJP-025p, the acetylated honeysuckle polysaccharide AC025p-0 exhibited a stronger inhibitory effect on the proliferation of differentiated thyroid cancer cells (such as IHH-4) compared with the unacetylated honeysuckle polysaccharide; and for the first time, it showed a strong inhibitory effect on the proliferation of undifferentiated thyroid cancer cells (such as 8305C).

[0035] Experimental results showed that, compared with the unacetylated honeysuckle polysaccharide WLJP-025p, the acetylated honeysuckle polysaccharide AC025p-0 exhibited a stronger inhibitory effect on the migration of differentiated thyroid cancer cells (such as IHH-4) and undifferentiated thyroid cancer cells (such as 8305C).

[0036] (2) Expanding the scope of treatment: It is worth noting that the unacetylated WLJP-025p has almost no inhibitory effect on undifferentiated thyroid cancer cells 8305C, while AC025p-0 can effectively inhibit these highly malignant cell lines. This indicates that acetylation treatment greatly expands the application scope of honeysuckle polysaccharide, making it a potential new option for the treatment of thyroid cancer.

[0037] (3) Improved safety: In addition, the study also confirmed that neither WLJP-025p nor AC025p-0 has any toxic effect on normal thyroid epithelial cells Nthy-ori3-1 and human embryonic kidney cells HEK293, indicating that the two compounds have good biocompatibility and are suitable for further development into clinical drugs.

[0038] In summary, this invention provides a novel strategy to enhance the therapeutic potential of natural polysaccharides, particularly for undifferentiated thyroid cancer with short survival and high malignancy, offering new hope to patients. By rationally designing and regulating the chemical structure of polysaccharides, more effective anti-tumor drugs can be developed, which is of great significance for improving the survival rate and quality of life of cancer patients. Attached Figure Description

[0039] Figure 1 The pseudo-structural formula of honeysuckle polysaccharide WLJP-025p in Example 1 is (A: galacturonic acid; B: rhamnose; C: arabinofuranose; D: glucose; E: galactose; F: acetoxy; G: methoxy).

[0040] Figure 2 The infrared spectra of honeysuckle polysaccharide (WLJP-025p) and acetylated honeysuckle polysaccharide (AC025p-0) from Example 3 are shown.

[0041] Figure 3 The 1H NMR spectra of honeysuckle polysaccharide (WLJP-025p) and acetylated honeysuckle polysaccharide (AC025p-0) in Example 3 are shown (A: WLJP-025p; B: AC025p-0).

[0042] Figure 4 The results of the inhibitory activity of honeysuckle polysaccharide (WLJP-025p) and acetylated honeysuckle polysaccharide (AC025p-0) in Example 4 are shown in the figure (A: IHH-4 cells; B: 8305C cells; * indicates p < 0.05 compared with the control group; ** indicates p < 0.01 compared with the control group; *** indicates p < 0.001 compared with the control group).

[0043] Figure 5 The toxicity results of honeysuckle polysaccharide (WLJP-025p) and acetylated honeysuckle polysaccharide (AC025p-0) in Example 4 are shown in the figure (A: Nthy-ori3-1 cells; B: HEK-293 cells; * indicates p < 0.05 compared with the control group; ** indicates p < 0.01 compared with the control group; *** indicates p < 0.001 compared with the control group).

[0044] Figure 6 The results of the inhibitory activity of honeysuckle polysaccharide (WLJP-025p) and acetylated honeysuckle polysaccharide (AC025p-0) on cancer cell migration in Example 4 are shown in the figure (left: crystal violet stained cell photograph; right: statistical graph of the number of migrating cells; # indicates that at a concentration of 1000 μg / ml, there was a significant difference in the number of migrating cells between the WLJP-025p group and the AC025p-0 group, p < 0.05; ### indicates that at a concentration of 1000 μg / ml, there was a significant difference in the number of migrating cells between the WLJP-025p group and the AC025p-0 group, p < 0.001; compared with the control group, p < 0.05; & indicates that at a concentration of 2000 μg / ml, there was a significant difference in the number of migrating cells between the WLJP-025p group and the AC025p-0 group, p < 0.001). Detailed Implementation

[0045] The following detailed description illustrates the specific implementation method:

[0046] Example 1: Honeysuckle polysaccharide ( Lonicera japonica The structural composition of polysaccharides (LJP)

[0047] The preparation method of honeysuckle polysaccharide (WLJP-025p) used in this solution can be found in the applicant's prior patent CN119235904A (Application of honeysuckle polysaccharide in the preparation of drugs for treating thyroid cancer). All content related to WLJP-025p in patent CN119235904A is incorporated into this patent application. Based on WLJP-025p, this technical solution further explores its new applications by acetylation treatment.

[0048] (1) Composition and structure of honeysuckle polysaccharide WLJP-025p

[0049] Monosaccharide composition: galacturonic acid 53.4%-80.2%, glucose 8.0%-12.0%, galactose 11.7%-17.5%, arabinose 6.6%-9.8%, mannose 0.3%-0.5%.

[0050] The relative average molecular weight of honeysuckle polysaccharides is 10-40 kDa. Preferably, the relative average molecular weight of the obtained honeysuckle polysaccharides was determined to be 23±4 kDa using high performance liquid chromatography based on a TSKgel G4000PWxl gel size exclusion column. More detailed descriptions and experimental data can be found in patent CN119235904A.

[0051] Based on the above research results, the inventors conducted a more in-depth study on the structural composition of honeysuckle polysaccharide WLJP-025p, including further clarifying the glycosidic bond linkage through WLJP-025p methylation analysis, and resolving the polysaccharide structure through more in-depth nuclear magnetic resonance spectroscopy (NMR) analysis.

[0052] (1.1) Methylation analysis of WLJP-025p

[0053] Weigh a small amount of sample (approximately 5 mg), dissolve it in 1 ml of pure water, add 1 ml of 100 mg / ml 1-cyclohexyl-2-morpholinoethyl carbodiimide methyl p-toluenesulfonate, and react for 2 h. Add 1 ml of 2 M imidazole, divide the sample into two equal portions, add 1 ml of 30 mg / ml NaBH4 and 1 ml of 30 mg / ml NaBD4 (sodium borodeuteride) to each portion, and react for 3 h. Terminate the reaction by adding 100 μl of glacial acetic acid. Dialyze for 48 h, then freeze-dry the sample for further processing. Dissolve the sample in 500 μl of DMSO. Add 1 mg of NaOH and incubate for 30 min. Add 50 μl of iodomethane solution and react for 1 h. Add 1 ml of water and 2 ml of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the washing with water 3 times. Pipette the lower dichloromethane phase and dry it under nitrogen. Add 1 ml of 2 M imidazole, divide the sample into two equal portions, and add 1 ml of 30 mg / ml NaBH4 and 1 ml of 30 mg / ml NaBD4 to each portion, reacting for 3 h. Add 100 μl of 2 M TFA (trifluoroacetic acid), react at 121℃ for 90 min. Evaporate to dryness at 30℃. Add 50 μl of 2 M ammonia and 50 μl of 1 M NaBD4, mix well, and react at room temperature for 2.5 h. Add 20 μl of acetic acid to terminate the reaction, dry under nitrogen, wash twice with 250 μl of methanol, and dry under nitrogen. Add 250 μl of acetic anhydride, vortex to mix, and react at 100℃ for 2.5 h. Add 1 ml of water and let stand for 10 min. Add 500 μl of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and repeat the washing with water 3 times. Take the lower dichloromethane phase and analyze it by GC-MS.

[0054] Chromatographic conditions: The chromatographic system used was an Agilent GC-MS system (Agilent 7890A-5977B; Agilent Technologies, USA); the column was a BPX70 (30 m × 0.25 mm × 0.25 µm, SGE, Australia). The injection volume was 1 μl, the split ratio was 10:1, and the carrier gas was high-purity helium. The initial temperature of the column oven was 140℃, held for 2.0 min, and then programmed to increase to 230℃ at a rate of 3℃ / min, held for 3 min.

[0055] Mass spectrometry conditions: The mass spectrometry system used was an Agilent 5977B quadrupole mass spectrometer (Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. Electron impact ionization (EI) was used, and analytes were detected in full scan (SCAN) mode, with a mass scan range (m / z) of 50-350.

[0056] Methylation analysis is one of the most important and classic methods for studying glycosidic bond linkages. Fully methylated polysaccharides undergo hydrolysis, reduction, and acetylation. Gas chromatography-mass spectrometry (GC-MS) is then used to analyze the partially methylated sugar alcohol acetates produced during degradation, ultimately determining the type of glycosidic bond. By comparing these glycosidic bonds with the standard spectral library PMAA (partially methylated or partially acetylated alditol acetates), and considering the methylation and fragmentation patterns of different types of sugar alcohol acetates, they are assigned to their respective groups. PMAA ionization follows a pattern where two methylated carbon atoms are most prone to breakage, resulting in a higher abundance of the resulting ion. The next most common is a combination of one methylated and one acetylated carbon atom. Cation fragments containing methylated carbon atoms form preferentially, while breakage between two acetylated carbon atoms is less frequent, leading to a lower abundance of the resulting ion. Groups such as -CH3OH (m / z 32), -CH2CO (m / z 42), and -HOAc (m / z 60) detach from primary ions to form secondary ions, generating corresponding ion abundances. Terminal PMAAs are more prone to breakage at C2-C3 and C3-C4. After comparing the characteristic ion peaks of standard hexose and pentose PMAAs, and considering their retention times, PMAAs with terminal groups have the earliest retention times, followed by those with two linkage points. The more linkage points, the longer the retention time. Pentose PMAAs have shorter retention times than hexose PMAAs. Furthermore, methylation data from relevant literature were used to identify the resulting acetylated sugar alcohols. As shown in the table, a total of five sugar residues were identified: t-Ara f 1,5-Ara f t-Gal p 1,4-Gal p A, 1,4-Glc p The relative molar percentages were 9.07%, 9.18%, 6.91%, 70.56%, and 4.28%, respectively, as detailed in Table 1.

[0057] Table 1: Results of polysaccharide methylation analysis

[0058]

[0059] (1.2) Nuclear Magnetic Resonance Spectroscopy (NMR) Analysis

[0060] Dissolve an appropriate amount of purified polysaccharide thoroughly in D2O to prepare a polysaccharide solution with a concentration greater than or equal to 40 mg / mL. Transfer the dissolved solution to an NMR tube, adding 0.5 mL. Place the NMR tube in an NMR spectrometer to scan one-dimensional 1H and 13C spectra, and two-dimensional COSY, HSQC, HMBC, and NOESY spectra. Quantitative analysis of the target analyte was performed using a Bruker (Germany) 500 MHz NMR spectrometer at a scanning temperature of 25 °C. Liquid probe QXI 1H / 31P / 13C / 15N 5mm quad-resonance reverse detection probe (Z-gradient, ATM Acc) technical parameters: Signal-to-noise ratio (1H): 888; Resolution (Hz): 0.32 (rotating); BBFO1H-19F, 31P-15N, 1H decoupling / observe multi-core forward detection probe (Z-gradient, ATM) technical parameters: Signal-to-noise ratio (1H): 798; Resolution (Hz): 0.26 (rotating); Signal-to-noise ratio (13C): 328; Resolution (Hz): 0.1.

[0061] According to the 1D and 2D NMR spectra of WLJP-025p, most proton and carbon signals are distributed in the ranges of δ 3.0~5.7ppm and δ 60~110ppm, respectively, which are characteristic shifts of polysaccharides. The terminal proton signal ranges from δ 4.48~5.20ppm, and the terminal carbon signal ranges from δ 92ppm-107ppm. Generally, the chemical shift of the α-glycosidic bond terminal proton signal is greater than 5.0ppm, and the chemical shift of the β-glycosidic bond terminal proton signal is less than 5.0ppm. This indicates that the polysaccharide contains two different types of sugar units, α and β, which is consistent with the results of the infrared spectroscopy analysis. 13 The C-ray spectroscopy spectrum showed a uronic acid signal of 174.81 ppm, an esterified uronic acid signal of 170.89 ppm, a characteristic peak of methyl ester group of 52.81 ppm, and an acetyl group of 20.22 ppm. Therefore, it can be confirmed that WLJP-025p contains uronic acid groups and has methyl esterified and acetylated substituents. A distinct 1,4-α-Gal at 4.98 / 99.18 ppm is observed. p A-terminal signal. The signal at 16.65 ppm in the carbon spectrum indicates the presence of rhamnose residues 1,2,4-α-Rha. p The presence of this crossover signal (1.20 / 16.65) in the HSQC spectrum can be attributed to Rha. pThe H6 / C6 groups were identified as H2 and H4 based on literature, while the remaining signals were too weak to be assigned. Obvious cross signals (5.04 / 107.04 ppm and 5.00 / 107.37 ppm) were observed in the HSQC spectra, typical of furanose end-group signals, and were assigned to H2-H5 by the 1H-1H-COSY spectra. The corresponding C2-C5 groups were assigned sequentially by the HSQC spectra. Referring to the general chemical shifts of Araf, t-α-Ara... f Except for the terminal proton signal, no other residues showed significant shifts. The C1 and C5 signals showed a clear shift to the lower field, confirming the presence of 1,5-α-Ara. f The attribution of methylated sugar residues, combined with the composition and proportion of methylated sugar residues, indicates that the cross signal of 4.82 / 99.98 ppm in the HSQC spectrum can be attributed to t-α-Gal. p 4.50 / 96.08 ppm can be identified as 1,4-β-Glc p The residue t-α-Ara f 1,5-α-Ara f 1,4-α-Gal p A, 1,4-α-Gal p A(OMe), t-α-Gal p 1,4-β-Glc p 1,2,4-α-Rha p The residues were named A, B, C, D, E, F, and G in sequence. The connection order was determined based on the HMBC and NOESY spectra. In the HMBC spectrum, the correlation peak at 4.07 / 78.30 ppm indicates that residue G's O-2 is linked to residue C's C-4; the correlation peak at 3.96 / 107.31 ppm indicates that residue G's O-4 is linked to residue D's C-1. In the NOESY spectrum, the correlation signals (4.82, 4.37), (5.05, 4.36), (4.98, 4.37), and (5.00, 3.62) indicate that E-1 is linked to C-4, C-1 to C-4, and B-1 to B-5.

[0062] In conclusion, it can be deduced that WLJP-025p is a pectin-type polysaccharide with a main chain of 1,4-α-Gal p The repeating unit of A (from α-(1→4)-D-Gal) p (A is sequentially linked to form the main chain), containing numerous esterification groups and a small amount of acetylation groups, with 1,5-α-Ara side chains. f and 1,4-β-Glc p The repeating unit is connected to 1,2,4-α-Rha p The 0-4 bits. Also includes t-α-Ara f and t-α-Gal pEnd-base segment. See the structural schematic diagram of WLJP-025p. Figure 1 .

[0063] Table 2: Different residues of WLJP-025p sample 13 C and 1 Chemical shifts on 1H NMR (carbon-13 and hydrogen NMR) (C1-C6 represent the carbon atoms of sugar residues and their positions; -OAc represents acetyl; -OMe represents methoxy)

[0064]

[0065] Example 2: Preparation method of honeysuckle polysaccharide WLJP-025p

[0066] For details of the preparation method, please refer to the applicant's prior patent CN119235904A (Application of honeysuckle polysaccharide in the preparation of drugs for treating thyroid cancer), and the process flow is as follows:

[0067] S1: After removing fat-soluble substances from honeysuckle, water is used as a solvent for decoction extraction, and the supernatant is collected; after concentration, removal of protein, and alcohol precipitation, the precipitate is collected; and after freeze-drying, the total sugar of honeysuckle is obtained.

[0068] S2: The aqueous solution of total sugars from honeysuckle was loaded into a DEAE-cellulose chromatography column. The DEAE-cellulose chromatography column was eluted sequentially with distilled water and NaCl solution. The fractions corresponding to the total sugar content peak and the uronic acid peak in the elution curve were taken from the fraction obtained by elution with NaCl solution. After concentration, dialysis and lyophilization, the honeysuckle polysaccharide WLJP-025 fraction (polysaccharide separation fraction) was obtained.

[0069] S3: Dissolve the honeysuckle polysaccharide WLJP-025 fraction in sodium chloride solution, load it into a Sepharose CL-6B chromatography column, elute with NaCl solution, and take the eluent portion corresponding to the maximum total sugar content peak and the uronic acid peak of the elution curve; after concentration, dialysis and lyophilization, honeysuckle polysaccharide (WLJP-025p) is obtained.

[0070] Preferably, in step S2, the concentration of the NaCl solution is 0.23-0.27 mol / L, more preferably 0.25 mol / L.

[0071] In step S3, the concentration of the NaCl solution is 0.09-0.11 mol / L, more preferably 0.1 mol / L.

[0072] Preferably, in S2, the DEAE-cellulose chromatography column is eluted with 2-3 column volumes (preferably 2 volumes) of distilled water and NaCl solution, and the elution flow rate is 9.5-10.5 mL / min, more preferably 10 mL / min.

[0073] Preferably, in S3, the elution rate using NaCl solution is 0.08-0.12 mL / min, more preferably 0.1 mL / min.

[0074] The honeysuckle polysaccharide WLJP-025p described in Example 1 can be prepared by following the above method.

[0075] Example 3: Preparation of acetylated honeysuckle polysaccharide

[0076] The WLJP-025p prepared above was mixed thoroughly with polysaccharide:water:acetic anhydride:DMAP (4-dimethylaminopyridine) in a ratio of 50 mg:1 mL:40 mL:5 mg (material ratio optional range: 45.5-50.5 mg:0.9-1.1 mL:39-41 mL:4.9-5.1 mg). The mixture was then placed on a magnetic stirrer (stirring speed 100-200 rpm) and reacted at room temperature (25℃±5℃) for 16 h (reaction time optional range 14-18 h). The resulting solution was then transferred to a dialysis bag with a molecular weight cutoff of MW3500. Pure water was used as the dialysate (external fluid) for dialysis. The pure water in the external dialysis fluid was replaced after the first 2 h, and thereafter every 4 h, for a total of 3 days of dialysis (overnight dialysis does not require water replacement). During the dialysis process, the pure water in the container was stirred at 150 rpm using a magnetic stirrer arm. After dialysis, collect the fluid from the dialysis bag, concentrate it to 20-50 mL in a 70°C water bath, and centrifuge at 4500 rpm for 10 minutes. After centrifugation, collect the supernatant and freeze-dry it to obtain AC025p-0.

[0077] The acetylated honeysuckle polysaccharide was characterized by infrared spectroscopy and proton nuclear magnetic resonance spectroscopy. The results are as follows: Figure 2 , Figure 3 As shown, the overall waveforms of AC025p-0 and WLJP-025p are similar in the infrared spectrum, with a wavelength of 1731 cm⁻¹. -1 The peak at 1731 cm⁻¹ is attributed to the stretching vibration of the C=O bond; the introduction of the acetyl group leads to AC₀25p⁻O at 1731 cm⁻¹. -1 The absorption at the position was significantly enhanced, and the chemical shift of AC025p-0 in the 1H NMR spectrum changed significantly between 1.8 and 2.2 ppm, which also indicates that the acetylation modification of AC025p-0 was successful.

[0078] Example 4: Study on the effects of acetylated honeysuckle polysaccharide AC025p-0

[0079] The specific details of the acetylated honeysuckle polysaccharide AC025p-0 used in this embodiment are as follows: Honeysuckle WLJP-025p was prepared according to the method of Example 1 of the prior patent CN119235904A, and then acetylated honeysuckle polysaccharide AC025p-0 was obtained by using the optimal method of Example 3 of this application.

[0080] The cell models used in this embodiment include: IHH-4 cells (human papillary thyroid carcinoma cell line, belonging to differentiated thyroid carcinoma cell line), 8305C cells (human undifferentiated thyroid carcinoma cell line), Nthy-ori3-1 cells (human normal thyroid epithelial cell line), and HEK-293 cells (human embryonic kidney 293 cell line).

[0081] Undifferentiated thyroid carcinoma is highly malignant, with short survival and high mortality. 8305C cells provide an important tool for studying this disease. 8305C cells exhibit diverse morphologies, including spindle-shaped and irregular shapes, with significant size variations, and grow loosely adherently. 8305C cells possess highly malignant biological characteristics, proliferating rapidly and invasively, capable of forming large tumor colonies in a short time. These cells do not express or express low levels of thyroid-specific proteins, losing some differentiation characteristics of thyroid cells, while simultaneously highly expressing genes associated with tumor malignant progression, facilitating tumor cell invasion and metastasis.

[0082] Cells were cultured in a 5% CO2 incubator at 37°C. When the cell density reached 80-90%, they were passaged and seeded into 96-well plates according to experimental requirements. IHH-4, 8305C, and Nthy-ori-3 were cultured in RPMI-1640 medium containing 10% (v / v) FBS (fetal bovine serum) and 1% (v / v) P / S (penicillin / streptomycin antibiotics, stock solution containing 10,000 U / mL penicillin and 10,000 μg / mL streptomycin). HEK-293 was cultured in DMEM medium containing 10% (v / v) FBS and 1% (v / v) P / S (penicillin / streptomycin antibiotics, stock solution containing 10,000 U / mL penicillin and 10,000 μg / mL streptomycin). These media are standard media in existing technology and will not be described in detail here. The CCK-8 assay was used to evaluate the effect of drugs on cell viability. The CCK-8 (Cell Counting Kit-8) cell proliferation assay can indirectly reflect the number of viable cells based on its correlation and has been widely used in anti-tumor drug screening, cell proliferation assays, and cytotoxicity assays. Cells are incubated at 2 × 10⁻⁶ cells / day. 3 Cells / well (IHH-4 and 8305C cells) or 5 × 10⁶ cells / well 3Nthy-ori3-1 and HEK-293 cells were seeded per well in 96-well plates. After treatment with 500, 1000, and 2000 μg / mL of honeysuckle polysaccharide WLJP-025p or acetylated honeysuckle polysaccharide AC025p-0 for 48 h, cell viability was assessed using the CCK-8 assay. The calculation formula is as follows: Cell viability (%) = [(OD value of experimental wells - OD value of blank wells) / (OD value of control wells - OD value of blank wells)] × 100% to detect cell proliferation. Transwell assays are widely used in cell migration studies, especially for assessing the migration ability of tumor cells. Cells were seeded at 1 × 10⁻⁶ cells / well. 4 Each cell / well was seeded in the upper chamber of the small chamber, and WLJP-025p or AC025p-0 was added to a final concentration of 1000 or 2000 μg / mL, respectively. After incubation for 48 h, crystal violet was used for staining and photography was performed. The images were then processed and counted using Imagej.

[0083] The results of the cell proliferation activity experiment are shown in Tables 3 and 4. Figure 4 As shown, the experimental data revealed the effect of acetylated honeysuckle polysaccharide in inhibiting thyroid cancer cells, specifically demonstrating inhibitory activity and a significant improvement in inhibitory effect on thyroid cancer cells of different differentiation stages.

[0084] First, acetylation significantly enhanced the inhibitory effect of honeysuckle polysaccharides on differentiated thyroid cancer cells (IHH-4). While the unacetylated honeysuckle polysaccharide WLJP-025p exhibited some inhibitory activity against IHH-4 cells (approximately 76% inhibition rate after 48 hours of administration at 2000 μg / mL), the acetylated AC025p-0 showed a significantly enhanced inhibitory effect: after 48 hours of administration at the same concentration (2000 μg / mL), the inhibition rate against IHH-4 cells reached 94%, an increase of 18 percentage points compared to WLJP-025p. This significant improvement far exceeded the expected effect of conventional structural modifications. Although acetylation of polysaccharides theoretically reduces their immunogenicity, the immunogenicity of polysaccharides is not directly related to their inhibitory effect on cancer cells. Current technology cannot determine whether acetylation of WLJP-025p can effectively enhance the drug's inhibitory effect on differentiated thyroid cancer cells while simultaneously ensuring the drug's safety for normal cells.

[0085] Secondly, after acetylation modification, the inhibitory activity of honeysuckle polysaccharide against undifferentiated thyroid cancer cells 8305C changed from non-existent to significant. Undifferentiated thyroid cancer is a highly malignant tumor type, and its clinical treatment is extremely difficult. Experimental results showed that the unacetylated WLJP-025p had no inhibitory activity against 8305C cells, and there was no significant difference in cell survival rate between the treatment group and the control group (p>0.05), proving that it could not be used for the treatment of undifferentiated thyroid cancer. However, the acetylated AC025p-0 showed an unexpected inhibitory effect: after administration of 2000 μg / mL for 48 h, the inhibition rate against 8305C cells was close to 80%, and it showed a significant concentration dependence (significant inhibition was already shown at 500 μg / mL, and extremely significant at 2000 μg / mL). This change from "ineffective" to "highly effective inhibition" was something the inventors had not anticipated. Based on the mechanism of action of honeysuckle polysaccharide WLJP-025p, the inventors originally believed that AC025p-0 had no effect or only a slight effect on undifferentiated thyroid cancer cells. Experimental data show that acetylation modification endows honeysuckle polysaccharide with inhibitory ability against highly malignant undifferentiated cancer cells, demonstrating its promising application potential in this type of tumor.

[0086] Acetylation of honeysuckle polysaccharides can broaden their application scope, expanding their indications from differentiated thyroid cancer to undifferentiated thyroid cancer—an expansion that was unexpected by those skilled in the art. Furthermore, acetylated honeysuckle polysaccharide AC025p-0 exhibits better inhibitory effects on undifferentiated thyroid cancer cells than on differentiated thyroid cancer cells, suggesting that acetylated honeysuckle polysaccharide AC025p-0 holds promise for development as a drug specifically for the treatment of undifferentiated thyroid cancer, overcoming the current lack of treatment options for this condition. Therefore, acetylation modification not only enhances the inhibitory efficacy of honeysuckle polysaccharides against differentiated thyroid cancer cells but also endows them with significant inhibitory activity against undifferentiated thyroid cancer cells—a "double enhancement" far exceeding the expectations of conventional modifications.

[0087] In addition to their killing effect on thyroid cancer cells, WLJP-025p and AC025p-0 also exhibited good safety characteristics against normal cells. Experimental results showed that within a dose range of 500-2000 μg / mL, both polysaccharides had no significant toxic effects on normal human thyroid cells (Nthy-ori3-1 cell line) and human embryonic kidney cells (HEK293 cell line). Specifically, there was no statistically significant difference in cell viability between the treated group and the control group, indicating that WLJP-025p and AC025p-0 at this concentration range did not significantly affect the survival of normal cells. This selective killing of tumor cells and low toxicity to normal cells provides important safety evidence for their subsequent clinical application. Detailed experimental data can be found in Tables 5 and 6. Figure 5 .

[0088] Table 3: CCK-8 experimental results of IHH-4 cells (n=3, mean±SD)

[0089]

[0090] Table 4: CCK-8 assay results in 8305C cells (n=3, mean±SD)

[0091]

[0092] Table 5: CCK-8 assay results of AC025p-0 on human thyroid cells (Nthy-ori3-1 cell line) (n=3, mean±SD)

[0093]

[0094] Table 6: CCK-8 assay results of AC025p-0 on human embryonic kidney cells (HEK293 cell line) (n=3, mean±SD)

[0095]

[0096] For detailed results of the tumor cell migration ability experiment, please refer to... Figure 6The experimental results show that acetylated honeysuckle polysaccharide AC025p-0 has a stronger inhibitory effect on the migration of cancer cells (including IHH4 cells and 8305C cells) compared to honeysuckle polysaccharide WLJP-025p. At a dose of 1000 μg / mL, acetylated honeysuckle polysaccharide AC025p-0 significantly enhanced the inhibitory effect on the migration of IHH4 cells and 8305C cells compared to honeysuckle polysaccharide WLJP-025p (indicated by the symbol "#" in the bar chart); at a dose of 2000 μg / mL, acetylated honeysuckle polysaccharide AC025p-0 significantly enhanced the inhibitory effect on the migration of IHH4 cells and 8305C cells compared to honeysuckle polysaccharide WLJP-025p (indicated by the symbol "&" in the bar chart).

[0097] Tumor cell proliferation refers to the process by which tumor cells increase in number through division, and is the core reason for increased tumor size and burden. Its essence is cell cycle disorder, leading to uncontrolled and continuous cell division, manifested as increased cell density and tumor enlargement within the tumor tissue. Tumor cell migration refers to the process by which tumor cells detach from the primary site and move to distant sites through invasion of surrounding tissue spaces or blood vessels and lymphatic vessels; it is a key step in tumor metastasis. Proliferation focuses on increasing the number of tumor cells, determining the size of the primary tumor; migration focuses on locational movement, determining the tumor's invasive and metastatic capabilities. These are two independent but interconnected processes in tumor progression. Proliferation and migration are the twin engines that enable tumor cells to adapt to their environment and achieve malignant progression. Proliferation expands the tumor population, while migration expands its survival space, jointly determining the degree of tumor malignancy. Therefore, an ideal tumor suppressor drug must possess the dual ability to inhibit both tumor cell proliferation and migration. Tumor cell proliferation leads to the continuous expansion of the primary lesion, increasing the tumor burden, while migration is the core link in tumor metastasis. Once tumor cells successfully migrate and form metastatic lesions in distant organs, it greatly increases the difficulty of treatment and the patient's risk of death. Only by targeting both processes simultaneously can tumor progression be more comprehensively controlled and patient prognosis effectively improved.

[0098] Based on the above data, AC025p-0 exhibits excellent dual inhibitory efficacy. It not only significantly inhibits the proliferation of thyroid cancer cells, reduces the number of tumor cells, and curbs the expansion of the primary lesion, but also effectively inhibits tumor cell migration, hindering their invasion of surrounding tissues and distant metastasis, thus controlling tumor development at multiple stages. In contrast, the effect of WLJP-025p is relatively limited. When facing undifferentiated thyroid cancer cells, it performs poorly in inhibiting proliferation (almost no inhibitory effect), making it difficult to effectively control the increase in the number of tumor cells, and only able to inhibit cancer cell migration to a certain extent. This single and limited effect makes it far less effective than AC025p-0 in treating highly malignant undifferentiated thyroid cancer. Therefore, compared to unacetylated WLJP-025p, acetylated honeysuckle polysaccharide AC025p-0, with its dual ability to simultaneously inhibit tumor cell proliferation and migration, demonstrates a more prominent advantage in combating various thyroid tumors, especially highly malignant undifferentiated thyroid cancer, making it a more ideal therapeutic drug.

[0099] In conclusion, acetylated honeysuckle polysaccharide AC025p-0 has the potential to be further developed into a highly effective and low-toxic functional food, health product, or medicine for the treatment or prevention of thyroid cancer.

[0100] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The application of an acetylated honeysuckle polysaccharide in the preparation of a drug for treating thyroid cancer, characterized in that: The thyroid cancer is undifferentiated thyroid cancer; the acetylated honeysuckle polysaccharide is obtained by mixing 45.5-50.5 mg: 0.9-1.1 mL: 39-41 mL: 4.9-5.1 mg of honeysuckle polysaccharide, water, acetic anhydride and 4-dimethylaminopyridine, and reacting at 20-30℃ for 14-18 h to obtain acetylated honeysuckle polysaccharide; The monosaccharide composition of the honeysuckle polysaccharide is as follows: galacturonic acid 53.4%-80.2%, glucose 8.0%-12.0%, galactose 11.7%-17.5%, arabinose 6.6%-9.8%, and mannose 0.3%-0.5%. The main chain of the honeysuckle polysaccharide is a repeating unit of 1,4-α-GalpA; the main chain includes t-α-Araf and t-α-Galp terminal segments; the side chains are repeating units of 1,5-α-Araf and 1,4-β-Glcp, which are linked to the O-4 positions of 1,2,4-α-Rhap. The honeysuckle polysaccharide was prepared by the following method: S1: After removing fat-soluble substances from honeysuckle, water is used as a solvent for decoction extraction, and the supernatant is collected; after concentration, removal of protein, 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 into a DEAE-cellulose chromatography column. Elute the DEAE-cellulose column with 2-3 column volumes of distilled water, followed by elution with a 0.23-0.27 mol / L NaCl solution. The elution flow rate of the distilled water and NaCl solution is 9.5-10.5 mL / min. Take the fractions corresponding to the total sugar content peak and uronic acid peak from the elution curve obtained from the NaCl solution elution. After concentration, dialysis, and lyophilization, the polysaccharide fraction is obtained. S3: Dissolve the polysaccharide fraction in sodium chloride solution, load it onto a Sepharose CL-6B chromatography column, and elute with NaCl solution at a concentration of 0.09-0.11 mol / L at a flow rate of 0.08-0.12 mL / min. Take the eluent portion corresponding to the maximum total sugar content peak and the uronic acid peak of the elution curve; after concentration, dialysis and lyophilization, honeysuckle polysaccharide is obtained.

2. The application of the acetylated honeysuckle polysaccharide according to claim 1 in the preparation of a drug for treating thyroid cancer, characterized in that: The relative average molecular weight of the honeysuckle polysaccharide is 10-40 kDa.

3. The application of the acetylated honeysuckle polysaccharide according to claim 2 in the preparation of a drug for treating thyroid cancer, characterized in that: The relative average molecular weight of the honeysuckle polysaccharide was determined to be 23 ± 4 kDa using high performance liquid chromatography based on a TSKgel G4000 PWxl gel size exclusion column.

4. The application of the acetylated honeysuckle polysaccharide according to claim 1 in the preparation of a drug for treating thyroid cancer, characterized in that: In step S2, the concentration of NaCl solution was 0.25 mol / L; the DEAE-cellulose chromatography column was eluted with 2 column volumes of distilled water and NaCl solution at a flow rate of 10 mL / min.

5. The application of the acetylated honeysuckle polysaccharide according to claim 1 in the preparation of a drug for treating thyroid cancer, characterized in that: In step S3, the concentration of the NaCl solution is 0.1 mol / L; the flow rate of elution using the NaCl solution is 0.1 mL / min.

6. A method for preparing acetylated honeysuckle polysaccharide, characterized in that: The following steps are performed sequentially: S1: After removing fat-soluble substances from honeysuckle, water is used as a solvent for decoction extraction, and the supernatant is collected; after concentration, removal of protein, 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 into a DEAE-cellulose chromatography column. Elute the DEAE-cellulose column with 2-3 column volumes of distilled water, followed by elution with a 0.23-0.27 mol / L NaCl solution. The elution flow rate of the distilled water and NaCl solution is 9.5-10.5 mL / min. Take the fractions corresponding to the total sugar content peak and uronic acid peak from the elution curve obtained from the NaCl solution elution. After concentration, dialysis, and lyophilization, the polysaccharide fraction is obtained. S3: The polysaccharide fraction was dissolved in sodium chloride solution and loaded into a Sepharose CL-6B chromatography column. Elution was performed using a 0.09-0.11 mol / L NaCl solution at a flow rate of 0.08-0.12 mL / min. The eluent fraction corresponding to the maximum total sugar content peak and the uronic acid peak of the elution curve was collected. After concentration, dialysis, and lyophilization, honeysuckle polysaccharide was obtained. The monosaccharide composition of the honeysuckle polysaccharide is as follows: galacturonic acid 53.4%-80.2%, glucose 8.0%-12.0%, galactose 11.7%-17.5%, arabinose 6.6%-9.8%, and mannose 0.3%-0.5%. The main chain of the honeysuckle polysaccharide is a repeating unit of 1,4-α-GalpA; the main chain includes t-α-Araf and t-α-Galp terminal segments; the side chains are repeating units of 1,5-α-Araf and 1,4-β-Glcp, which are linked to the O-4 positions of 1,2,4-α-Rhap. S4: Mix 45.5-50.5 mg: 0.9-1.1 mL: 39-41 mL: 4.9-5.1 mg of honeysuckle polysaccharide, water, acetic anhydride and 4-dimethylaminopyridine, and react at 20-30℃ for 14-18 h to obtain acetylated honeysuckle polysaccharide.

7. The acetylated honeysuckle polysaccharide prepared according to the method described in claim 6.

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